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

PubMed Central

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

2016-01-01

SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between −175 to −60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo. PMID:27173006

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2

PubMed Central

Reinke, Lennart Michel; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated. PMID:28636671

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

PubMed

Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

2017-01-01

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

Identification, synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors.

PubMed

Kumar, Vathan; Tan, Kian-Pin; Wang, Ying-Ming; Lin, Sheng-Wei; Liang, Po-Huang

2016-07-01

Severe acute respiratory syndrome (SARS) led to a life-threatening form of atypical pneumonia in late 2002. Following that, Middle East Respiratory Syndrome (MERS-CoV) has recently emerged, killing about 36% of patients infected globally, mainly in Saudi Arabia and South Korea. Based on a scaffold we reported for inhibiting neuraminidase (NA), we synthesized the analogues and identified compounds with low micromolar inhibitory activity against 3CL(pro) of SARS-CoV and MERS-CoV. Docking studies show that a carboxylate present at either R(1) or R(4) destabilizes the oxyanion hole in the 3CL(pro). Interestingly, 3f, 3g and 3m could inhibit both NA and 3CL(pro) and serve as a starting point to develop broad-spectrum antiviral agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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

2017-05-02

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xue, Xiaoyu; Yu, Hongwei; Yang, Haitao

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{supmore » 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}.« less

[Cell entry mechanisms of coronaviruses].

PubMed

Taguchi, Fumihiro; Matsuyama, Shutoku

2009-12-01

Enveloped viruses enter into cells via fusion of their envelope and cellular membrane. Spike (S) protein of coronavirus (CoV) is responsible for entry events. We studied the cell entry mechanisms of two different CoVs, murine coronavirus mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV). MHV-JHM that induces syncytia in infected cells entered directly from cell surface, i.e., fusion of envelope and plasma membrane, whereas SARS-CoV and MHV-2 that fail to induce syncytia entered via endosome in a protease-dependent fashion, i.e., fusion of envelope and endosomal membrane. The latter viruses entered directly from cell surface, when receptor-bound viruses were treated with proteases that activate fusion activity of their S proteins. The entry pathway of SARS-CoV could influence the severity of the disease. It was also reveled that a highly neurovirulent JHM spread in a receptor-independent fashion, which could result in a high neuropathogenicity of the virus.

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

USDA-ARS?s Scientific Manuscript database

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...

Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: structure-activity relationship study.

PubMed

Thanigaimalai, Pillaiyar; Konno, Sho; Yamamoto, Takehito; Koiwai, Yuji; Taguchi, Akihiro; Takayama, Kentaro; Yakushiji, Fumika; Akaji, Kenichi; Kiso, Yoshiaki; Kawasaki, Yuko; Chen, Shen-En; Naser-Tavakolian, Aurash; Schön, Arne; Freire, Ernesto; Hayashi, Yoshio

2013-07-01

This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (8; Ki = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL(pro). In particular, compounds 26m and 26n exhibited good inhibitory activities with Ki values of 0.39 and 0.33 μM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1' position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin.

PubMed

Ren, Wuze; Qu, Xiuxia; Li, Wendong; Han, Zhenggang; Yu, Meng; Zhou, Peng; Zhang, Shu-Yi; Wang, Lin-Fa; Deng, Hongkui; Shi, Zhengli

2008-02-01

Severe acute respiratory syndrome (SARS) is caused by the SARS-associated coronavirus (SARS-CoV), which uses angiotensin-converting enzyme 2 (ACE2) as its receptor for cell entry. A group of SARS-like CoVs (SL-CoVs) has been identified in horseshoe bats. SL-CoVs and SARS-CoVs share identical genome organizations and high sequence identities, with the main exception of the N terminus of the spike protein (S), known to be responsible for receptor binding in CoVs. In this study, we investigated the receptor usage of the SL-CoV S by combining a human immunodeficiency virus-based pseudovirus system with cell lines expressing the ACE2 molecules of human, civet, or horseshoe bat. In addition to full-length S of SL-CoV and SARS-CoV, a series of S chimeras was constructed by inserting different sequences of the SARS-CoV S into the SL-CoV S backbone. Several important observations were made from this study. First, the SL-CoV S was unable to use any of the three ACE2 molecules as its receptor. Second, the SARS-CoV S failed to enter cells expressing the bat ACE2. Third, the chimeric S covering the previously defined receptor-binding domain gained its ability to enter cells via human ACE2, albeit with different efficiencies for different constructs. Fourth, a minimal insert region (amino acids 310 to 518) was found to be sufficient to convert the SL-CoV S from non-ACE2 binding to human ACE2 binding, indicating that the SL-CoV S is largely compatible with SARS-CoV S protein both in structure and in function. The significance of these findings in relation to virus origin, virus recombination, and host switching is discussed.

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

PubMed

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

2014-12-07

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simmons, Graham, E-mail: gsimmons@bloodsystems.or; Bertram, Stephanie; Glowacka, Ilona

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacha,U.; Barilla, J.; Gabelli, S.

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 3CLpromore » 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.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Graziano,V.; McGrath, W.; Yang, L.

The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, K{sub D}, have varied more than 650000-fold, from <1 nM to more than 200 {mu}M. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimermore » equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded K{sub D} values of 5.8 {+-} 0.8 {mu}M (obtained from the entire scattering curve), 6.5 {+-} 2.2 {mu}M (obtained from the radii of gyration), and 6.8 {+-} 1.5 {mu}M (obtained from the forward scattering). The K{sub D} from chemical cross-linking was 12.7 {+-} 1.1 {mu}M, and from enzyme kinetics, it was 5.2 {+-} 0.4 {mu}M. While each of these three techniques can present different, potential limitations, they all yielded similar K{sub D} values.« less

Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response▿

PubMed Central

Glowacka, Ilona; Bertram, Stephanie; Müller, Marcel A.; Allen, Paul; Soilleux, Elizabeth; Pfefferle, Susanne; Steffen, Imke; Tsegaye, Theodros Solomon; He, Yuxian; Gnirss, Kerstin; Niemeyer, Daniela; Schneider, Heike; Drosten, Christian; Pöhlmann, Stefan

2011-01-01

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion. PMID:21325420

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 dimermore » 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.« less

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

PubMed

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

2013-05-01

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

Inhibitors of SARS-3CLpro: Virtual Screening, Biological Evaluation and Molecular Dynamics Simulation Studies

PubMed Central

Mukherjee, Prasenjit; Shah, Falgun; Desai, Prashant; Avery, Mitchell

2011-01-01

SARS-CoV from the coronaviridae family has been identified as the etiological agent of Severe Acute Respiratory Syndrome (SARS), a highly contagious upper respiratory disease that reached epidemic status in 2002. SARS-3CLpro, a cysteine protease indispensible to the viral life cycle, has been identified as one of the key therapeutic target against SARS. A combined ligand and structure based virtual screening was carried out against the Asinex Platinum collection. Multiple low micromolar inhibitors of the enzyme were identified through this search, one of which also showed activity against SARS-CoV in a whole cell CPE assay. Furthermore, multi nanosecond explicit solvent simulations were carried out using the docking poses of the identified hits to study the overall stability of the binding site interactions as well as identify important changes in the interaction profile that were not apparent from the docking study. Cumulative analysis of the evaluated compounds and the simulation studies led to the identification of certain protein-ligand interaction patterns which would be useful in further structure based design efforts. PMID:21604711

Surface vimentin is critical for the cell entry of SARS-CoV.

PubMed

Yu, Yvonne Ting-Chun; Chien, Ssu-Chia; Chen, I-Yin; Lai, Chia-Tsen; Tsay, Yeou-Guang; Chang, Shin C; Chang, Ming-Fu

2016-01-22

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused a global panic due to its high morbidity and mortality during 2002 and 2003. Soon after the deadly disease outbreak, the angiotensin-converting enzyme 2 (ACE2) was identified as a functional cellular receptor in vitro and in vivo for SARS-CoV spike protein. However, ACE2 solely is not sufficient to allow host cells to become susceptible to SARS-CoV infection, and other host factors may be involved in SARS-CoV spike protein-ACE2 complex. A host intracellular filamentous cytoskeletal protein vimentin was identified by immunoprecipitation and LC-MS/MS analysis following chemical cross-linking on Vero E6 cells that were pre-incubated with the SARS-CoV spike protein. Moreover, flow cytometry data demonstrated an increase of the cell surface vimentin level by 16.5 % after SARS-CoV permissive Vero E6 cells were treated with SARS-CoV virus-like particles (VLPs). A direct interaction between SARS-CoV spike protein and host surface vimentin was further confirmed by far-Western blotting. In addition, antibody neutralization assay and shRNA knockdown experiments indicated a vital role of vimentin in cell binding and uptake of SARS-CoV VLPs and the viral spike protein. A direct interaction between vimentin and SARS-CoV spike protein during viral entry was observed. Vimentin is a putative anti-viral drug target for preventing/reducing the susceptibility to SARS-CoV infection.

Lack of innate interferon responses during SARS coronavirus infection in a vaccination and reinfection ferret model.

PubMed

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

2012-01-01

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

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

PubMed

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

2013-11-01

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

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

PubMed Central

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

2013-01-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. PMID:23911968

Serology of severe acute respiratory syndrome: implications for surveillance and outcome.

PubMed

Chen, Xinchun; Zhou, Boping; Li, Meizhong; Liang, Xiaorong; Wang, Huosheng; Yang, Guilin; Wang, Hui; Le, Xiaohua

2004-04-01

Severe acute respiratory syndrome (SARS) is a novel infectious disease. No information is currently available on host-specific immunity against the SARS coronavirus (CoV), and detailed characteristics of the epidemiology of SARS CoV infection have not been identified. ELISA was used to detect antibody to SARS CoV. Reverse-transcriptase polymerase chain reaction was used to detect SARS CoV RNA. T cells in peripheral blood of patients were quantified by flow cytometry. Of 36 patients with probable SARS CoV infection, 30 (83.3%) were positive for IgG antibody to SARS CoV; in contrast, only 3 of 48 patients with suspected SARS CoV infection, 0 of 112 patients with fever but without SARS, and 0 of 96 healthy control individuals were positive for it. IgG antibody to SARS CoV was first detected between day 5 and day 47 after onset of illness (mean +/- SD, 18.7+/-10.4). Detection of antibody to SARS CoV is useful in the diagnosis of SARS; however, at the incubation and initial phases of the illness, serological assay is of little value, because of late seroconversion in most patients.

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

PubMed

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

2014-01-01

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

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

PubMed

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

2014-08-22

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

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

PubMed

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

2017-01-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Ting; Ooi, Amy; Lee, Hooi Chen

An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pHmore » 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.« less

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus

PubMed Central

Ge, Xing-Yi; Zhang, Wei; Li, Bei; Xie, Jia-Zheng; Shen, Xu-Rui; Zhang, Yun-Zhi; Wang, Ning; Luo, Dong-Sheng; Zheng, Xiao-Shuang; Wang, Mei-Niang; Wang, Lin-Fa

2017-01-01

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases. PMID:29190287

Real-Time Reverse Transcription–Polymerase Chain Reaction Assay for SARS-associated Coronavirus

PubMed Central

Emery, Shannon L.; Bowen, Michael D.; Newton, Bruce R.; Winchell, Jonas M.; Meyer, Richard F.; Tong, Suxiang; Cook, Byron T.; Holloway, Brian P.; McCaustland, Karen A.; Rota, Paul A.; Bankamp, Bettina; Lowe, Luis E.; Ksiazek, Tom G.; Bellini, William J.; Anderson, Larry J.

2004-01-01

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection. PMID:15030703

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

PubMed

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

2014-04-01

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

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus.

PubMed

Hu, Ben; Zeng, Lei-Ping; Yang, Xing-Lou; Ge, Xing-Yi; Zhang, Wei; Li, Bei; Xie, Jia-Zheng; Shen, Xu-Rui; Zhang, Yun-Zhi; Wang, Ning; Luo, Dong-Sheng; Zheng, Xiao-Shuang; Wang, Mei-Niang; Daszak, Peter; Wang, Lin-Fa; Cui, Jie; Shi, Zheng-Li

2017-11-01

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

PubMed

Lee, Hyun; Mittal, Anuradha; Patel, Kavankumar; Gatuz, Joseph L; Truong, Lena; Torres, Jaime; Mulhearn, Debbie C; Johnson, Michael E

2014-01-01

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development. Copyright © 2013. Published by Elsevier Ltd.

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

PubMed Central

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

Identification of a broad-spectrum antiviral small molecule against severe acute respiratory syndrome coronavirus and Ebola, Hendra, and Nipah viruses by using a novel high-throughput screening assay.

PubMed

Elshabrawy, Hatem A; Fan, Jilao; Haddad, Christine S; Ratia, Kiira; Broder, Christopher C; Caffrey, Michael; Prabhakar, Bellur S

2014-04-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) and Ebola, Hendra, and Nipah viruses are members of different viral families and are known causative agents of fatal viral diseases. These viruses depend on cathepsin L for entry into their target cells. The viral glycoproteins need to be primed by protease cleavage, rendering them active for fusion with the host cell membrane. In this study, we developed a novel high-throughput screening assay based on peptides, derived from the glycoproteins of the aforementioned viruses, which contain the cathepsin L cleavage site. We screened a library of 5,000 small molecules and discovered a small molecule that can inhibit the cathepsin L cleavage of all viral peptides with minimal inhibition of cleavage of a host protein-derived peptide (pro-neuropeptide Y). The small molecule inhibited the entry of all pseudotyped viruses in vitro and the cleavage of SARS-CoV spike glycoprotein in an in vitro cleavage assay. In addition, the Hendra and Nipah virus fusion glycoproteins were not cleaved in the presence of the small molecule in a cell-based cleavage assay. Furthermore, we demonstrate that the small molecule is a mixed inhibitor of cathepsin L. Our broad-spectrum antiviral small molecule appears to be an ideal candidate for future optimization and development into a potent antiviral against SARS-CoV and Ebola, Hendra, and Nipah viruses. We developed a novel high-throughput screening assay to identify small molecules that can prevent cathepsin L cleavage of viral glycoproteins derived from SARS-CoV and Ebola, Hendra, and Nipah viruses that are required for their entry into the host cell. We identified a novel broad-spectrum small molecule that could block cathepsin L-mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from SARS-CoV or Ebola, Hendra, or Nipah virus. The small molecule can be further optimized and developed into a potent broad-spectrum antiviral drug.

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

PubMed Central

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

2011-01-01

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

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus.

PubMed

Kitabatake, Masahiro; Inoue, Shingo; Yasui, Fumihiko; Yokochi, Shoji; Arai, Masaaki; Morita, Kouichi; Shida, Hisatoshi; Kidokoro, Minoru; Murai, Fukashi; Le, Mai Quynh; Mizuno, Kyosuke; Matsushima, Kouji; Kohara, Michinori

2007-01-08

A vaccine for severe acute respiratory syndrome (SARS) is being intensively pursued against its re-emergence. We generated a SARS coronavirus (SARS-CoV) spike protein-expressing recombinant vaccinia virus (RVV-S) using highly attenuated strain LC16m8. Intradermal administration of RVV-S into rabbits induced neutralizing (NT) antibodies against SARS-CoV 1 week after administration and the NT titer reached 1:1000 after boost immunization with RVV-S. Significantly, NT antibodies against SARS-CoV were induced by administration of RVV-S to rabbits that had been pre-immunized with LC16m8. RVV-S can induce NT antibodies against SARS-CoV despite the presence of NT antibodies against VV. These results suggest that RVV-S may be a powerful SARS vaccine, including in patients previously immunized with the smallpox vaccine.

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

PubMed

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

2013-08-01

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

SARS-CoV infection in a restaurant from palm civet.

PubMed

Wang, Ming; Yan, Meiying; Xu, Huifang; Liang, Weili; Kan, Biao; Zheng, Bojian; Chen, Honglin; Zheng, Han; Xu, Yanmei; Zhang, Enmin; Wang, Hongxia; Ye, Jingrong; Li, Guichang; Li, Machao; Cui, Zhigang; Liu, Yu-Fei; Guo, Rong-Tong; Liu, Xiao-Ning; Zhan, Liu-Hua; Zhou, Duan-Hua; Zhao, Ailan; Hai, Rong; Yu, Dongzhen; Guan, Yi; Xu, Jianguo

2005-12-01

Epidemiologic investigations showed that 2 of 4 patients with severe acute respiratory syndrome (SARS) identified in the winter of 2003-2004 were a waitresss at a restaurant in Guangzhou, China, that served palm civets as food and a customer who ate in the restaurant ashort distance from animal cages. All 6 palm civets at the restaurant were positive for SARS-associated coronavirus (SARS-CoV). Partial spike (S) gene sequences of SARS-CoV from the 2 patients were identical to 4 of 5 Sgene viral sequences from palm civets. Phylogenetic analysis showed that SARS-CoV from palm civets in the restaurant was most closely related to animal isolates. SARS cases at the restaurant were the result of recent interspecies transfer from the putative palm civet reservoir, and not the result of continued circulation of SARS-CoV in the human population.

Severe Acute Respiratory Syndrome–associated Coronavirus Infection

PubMed Central

Ip, Margaret; Ng, KC; Wu, Alan; Lee, Nelson; Rainer, Timothy H.; Joynt, Gavin M.; Sung, Joseph J. Y.; Tam, John S.

2003-01-01

Whether severe acute respiratory syndrome–associated coronavirus (SARS-CoV) infection can be asymptomatic is unclear. We examined the seroprevalence of SARS-CoV among 674 healthcare workers from a hospital in which a SARS outbreak had occurred. A total of 353 (52%) experienced mild self-limiting illnesses, and 321 (48%) were asymptomatic throughout the course of these observations. None of these healthcare workers had antibody to SARS CoV, indicating that subclinical or mild infection attributable to SARS CoV in adults is rare. PMID:14718090

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

PubMed

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.

SARS-CoV Infection in a Restaurant from Palm Civet

PubMed Central

Wang, Ming; Yan, Meiying; Xu, Huifang; Liang, Weili; Kan, Biao; Zheng, Bojian; Chen, Honglin; Zheng, Han; Xu, Yanmei; Zhang, Enmin; Wang, Hongxia; Ye, Jingrong; Li, Guichang; Li, Machao; Cui, Zhigang; Liu, Yu-Fei; Guo, Rong-Tong; Liu, Xiao-Ning; Zhan, Liu-Hua; Zhou, Duan-Hua; Zhao, Ailan; Hai, Rong; Yu, Dongzhen; Guan, Yi

2005-01-01

Epidemiologic investigations showed that 2 of 4 patients with severe acute respiratory syndrome (SARS) identified in the winter of 2003–2004 were a waitress at a restaurant in Guangzhou, China, that served palm civets as food and a customer who ate in the restaurant a short distance from animal cages. All 6 palm civets at the restaurant were positive for SARS-associated coronavirus (SARS-CoV). Partial spike (S) gene sequences of SARS-CoV from the 2 patients were identical to 4 of 5 S gene viral sequences from palm civets. Phylogenetic analysis showed that SARS-CoV from palm civets in the restaurant was most closely related to animal isolates. SARS cases at the restaurant were the result of recent interspecies transfer from the putative palm civet reservoir, and not the result of continued circulation of SARS-CoV in the human population. PMID:16485471

Cynomolgus Macaque as an Animal Model for Severe Acute Respiratory Syndrome

PubMed Central

Lawler, James V; Endy, Timothy P; Hensley, Lisa E; Garrison, Aura; Fritz, Elizabeth A; Lesar, May; Baric, Ralph S; Kulesh, David A; Norwood, David A; Wasieloski, Leonard P; Ulrich, Melanie P; Slezak, Tom R; Vitalis, Elizabeth; Huggins, John W; Jahrling, Peter B; Paragas, Jason

2006-01-01

Background The emergence of severe acute respiratory syndrome (SARS) in 2002 and 2003 affected global health and caused major economic disruption. Adequate animal models are required to study the underlying pathogenesis of SARS-associated coronavirus (SARS-CoV) infection and to develop effective vaccines and therapeutics. We report the first findings of measurable clinical disease in nonhuman primates (NHPs) infected with SARS-CoV. Methods and Findings In order to characterize clinically relevant parameters of SARS-CoV infection in NHPs, we infected cynomolgus macaques with SARS-CoV in three groups: Group I was infected in the nares and bronchus, group II in the nares and conjunctiva, and group III intravenously. Nonhuman primates in groups I and II developed mild to moderate symptomatic illness. All NHPs demonstrated evidence of viral replication and developed neutralizing antibodies. Chest radiographs from several animals in groups I and II revealed unifocal or multifocal pneumonia that peaked between days 8 and 10 postinfection. Clinical laboratory tests were not significantly changed. Overall, inoculation by a mucosal route produced more prominent disease than did intravenous inoculation. Half of the group I animals were infected with a recombinant infectious clone SARS-CoV derived from the SARS-CoV Urbani strain. This infectious clone produced disease indistinguishable from wild-type Urbani strain. Conclusions SARS-CoV infection of cynomolgus macaques did not reproduce the severe illness seen in the majority of adult human cases of SARS; however, our results suggest similarities to the milder syndrome of SARS-CoV infection characteristically seen in young children. PMID:16605302

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

PubMed

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.

Association of RANTES with the replication of severe acute respiratory syndrome coronavirus in THP-1 cells.

PubMed

Li, D; Wu, N; Yao, H; Bader, A; Brockmeyer, Norbert H; Altmeyer, P

2005-03-29

Severe acute respiratory syndrome (SARS) is a novel infectious disease which is characterized by an overaggressive immune response. Chemokines are important inflammatory mediators and regulate disease due to viral infection. In previous study, we found that SARS-CoV has the ability to replicate in mononuclear cells. In present work, we sought to characterize the replication of SARS-CoV at the presence of RANTES in THP-1 cells. To determine whether RANTES play an role in the process of SARS, THP-1 cells were incubated with heat-inactivated SARS-CoV and ELISA was used to test RANTES levels in the supernatants; Then the effect of dexamethasone on the induced secretion was evaluated. Real-time PCR was used to investigate the effort of RANTES on the replication of SARS-CoV in vitro. Macrophages, induced by THP-1 cells, were used as cell model. Inactive SARS-CoV could induce THP-1 cells secret RANTES and this increase effect could not be suppressed by DXM. RANTES itself could inhibit the replication of SARS-CoV in THP-1 cells when it was added into the culture before or at the same time with the virus; No inhibition effect was shown when RANTES were added into the culture after SARS-CoV infected the cells.

Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies.

PubMed

Zhang, Hong; Wang, Guangwen; Li, Jian; Nie, Yuchun; Shi, Xuanling; Lian, Gewei; Wang, Wei; Yin, Xiaolei; Zhao, Yang; Qu, Xiuxia; Ding, Mingxiao; Deng, Hongkui

2004-07-01

Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a newly identified coronavirus (CoV), SARS-CoV. The spike (S) glycoprotein of CoV is the major structural protein responsible for induction of host immune response and virus neutralization by antibodies. Hence, knowledge of neutralization determinants on the S protein is helpful for designing protective vaccines. To analyze the antigenic structure of the SARS-CoV S2 domain, the carboxyl-terminal half of the S protein, we first used sera from convalescent SARS patients to test the antigenicity of 12 overlapping fragments spanning the entire S2 and identified two antigenic determinants (Leu 803 to Ala 828 and Pro 1061 to Ser 1093). To determine whether neutralizing antibodies can be elicited by these two determinants, we immunized animals and found that both of them could induce the S2-specific antisera. In some animals, however, only one determinant (Leu 803 to Ala 828) was able to induce the antisera with the binding ability to the native S protein and the neutralizing activity to the SARS-CoV pseudovirus. This determinant is highly conserved across different SARS-CoV isolates. Identification of a conserved antigenic determinant on the S2 domain of the SARS-CoV S protein, which has the potential for inducing neutralizing antibodies, has implications in the development of effective vaccines against SARS-CoV.

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

PubMed

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

2015-12-01

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

SARS-CoV Regulates Immune Function-Related Gene Expression in Human Monocytic Cells

PubMed Central

Hu, Wanchung; Yen, Yu-Ting; Singh, Sher; Kao, Chuan-Liang

2012-01-01

Abstract Severe acute respiratory syndrome (SARS) is characterized by acute respiratory distress syndrome (ARDS) and pulmonary fibrosis, and monocytes/macrophages are the key players in the pathogenesis of SARS. In this study, we compared the transcriptional profiles of SARS coronavirus (SARS-CoV)-infected monocytic cells against that infected by coronavirus 229E (CoV-229E). Total RNA was extracted from infected DC-SIGN-transfected monocytes (THP-1-DC-SIGN) at 6 and 24 h after infection, and the gene expression was profiled in oligonucleotide-based microarrays. Analysis of immune-related gene expression profiles showed that at 24 h after SARS-CoV infection: (1) IFN-α/β-inducible and cathepsin/proteasome genes were downregulated; (2) hypoxia/hyperoxia-related genes were upregulated; and (3) TLR/TLR-signaling, cytokine/cytokine receptor-related, chemokine/chemokine receptor-related, lysosome-related, MHC/chaperon-related, and fibrosis-related genes were differentially regulated. These results elucidate that SARS-CoV infection regulates immune-related genes in monocytes/macrophages, which may be important to the pathogenesis of SARS. PMID:22876772

CORONAVIRUS VIRULENCE GENES WITH MAIN FOCUS ON SARS-CoV ENVELOPE GENE

PubMed Central

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

2014-01-01

Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and 7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal models and in humans. The modification or deletion of different motifs within E protein, including the transmembrane domain that harbors an ion channel activity, small sequences within the middle region of the carboxy-terminus of E protein, and its most carboxy-terminal end, which contains a PDZ domain-binding motif (PBM) is sufficient to attenuate the virus. Interestingly, a comprehensive collection of SARS-CoVs in which these motifs have been modified elicited full and long-term protection even in old mice, making those deletion mutants promising vaccine candidates. These data indicate that despite its small size, E protein drastically influences the replication of CoVs and their pathogenicity. Although E protein is not essential for CoV genome replication or subgenomic mRNA synthesis, it affects virus morphogenesis, budding, assembly, intracellular trafficking, and virulence. In fact, E protein is responsible in a significant proportion of the inflammasome activation and the associated inflammation elicited by SARS-CoV in the lung parenchyma. This exacerbated inflammation causes edema accumulation leading to acute respiratory distress syndrome (ARDS) and, frequently, to the death of infected animal models or human patients. PMID:25093995

Attenuation and Restoration of Severe Acute Respiratory Syndrome Coronavirus Mutant Lacking 2′-O-Methyltransferase Activity

PubMed Central

Menachery, Vineet D.; Yount, Boyd L.; Josset, Laurence; Gralinski, Lisa E.; Scobey, Trevor; Agnihothram, Sudhakar; Katze, Michael G.

2014-01-01

ABSTRACT The sudden emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and, more recently, Middle Eastern respiratory syndrome CoV (MERS-CoV) underscores the importance of understanding critical aspects of CoV infection and pathogenesis. Despite significant insights into CoV cross-species transmission, replication, and virus-host interactions, successful therapeutic options for CoVs do not yet exist. Recent identification of SARS-CoV NSP16 as a viral 2′-O-methyltransferase (2′-O-MTase) led to the possibility of utilizing this pathway to both attenuate SARS-CoV infection and develop novel therapeutic treatment options. Mutations were introduced into SARS-CoV NSP16 within the conserved KDKE motif and effectively attenuated the resulting SARS-CoV mutant viruses both in vitro and in vivo. While viruses lacking 2′-O-MTase activity had enhanced sensitivity to type I interferon (IFN), they were not completely restored in their absence in vivo. However, the absence of either MDA5 or IFIT1, IFN-responsive genes that recognize unmethylated 2′-O RNA, resulted in restored replication and virulence of the dNSP16 mutant virus. Finally, using the mutant as a live-attenuated vaccine showed significant promise for possible therapeutic development against SARS-CoV. Together, the data underscore the necessity of 2′-O-MTase activity for SARS-CoV pathogenesis and identify host immune pathways that mediate this attenuation. In addition, we describe novel treatment avenues that exploit this pathway and could potentially be used against a diverse range of viral pathogens that utilize 2′-O-MTase activity to subvert the immune system. IMPORTANCE Preventing recognition by the host immune response represents a critical aspect necessary for successful viral infection. Several viruses, including SARS-CoV, utilize virally encoded 2′-O-MTases to camouflage and obscure their viral RNA from host cell sensing machinery, thus preventing recognition and activation of cell intrinsic defense pathways. For SARS-CoV, the absence of this 2′-O-MTase activity results in significant attenuation characterized by decreased viral replication, reduced weight loss, and limited breathing dysfunction in mice. The results indicate that both MDA5, a recognition molecule, and the IFIT family play an important role in mediating this attenuation with restored virulence observed in their absence. Understanding this virus-host interaction provided an opportunity to design a successful live-attenuated vaccine for SARS-CoV and opens avenues for treatment and prevention of emerging CoVs and other RNA virus infections. PMID:24478444

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

PubMed

Sutton, Troy C; Subbarao, Kanta

2015-05-01

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

Development of Animal Models Against Emerging Coronaviruses: From SARS to MERS coronavirus

PubMed Central

Sutton, Troy C; Subbarao, Kanta

2016-01-01

Two novel coronaviruses have emerged to cause severe disease in humans. While bats may be the primary reservoir for both viruses, SARS coronavirus (SARS-CoV) likely crossed into humans from civets in China, and MERS coronavirus (MERS-CoV) has been transmitted from camels in the Middle East. Unlike SARS-CoV that resolved within a year, continued introductions of MERS-CoV present an on-going public health threat. Animal models are needed to evaluate countermeasures against emerging viruses. With SARS-CoV, several animal species were permissive to infection. In contrast, most laboratory animals are refractory or only semi-permissive to infection with MERS-CoV. This host-range restriction is largely determined by sequence heterogeneity in the MERS-CoV receptor. We describe animal models developed to study coronaviruses, with a focus on host-range restriction at the level of the viral receptor and discuss approaches to consider in developing a model to evaluate countermeasures against MERS-CoV. PMID:25791336

Evaluation of Inapparent Nosocomial Severe Acute Respiratory Syndrome Coronavirus Infection in Vietnam by Use of Highly Specific Recombinant Truncated Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

PubMed Central

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Thai, Hong Thi Cam; Hasebe, Futoshi; del Carmen Parquet, Maria; Morita, Kouichi

2005-01-01

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N′ protein and NΔ121 protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N′ protein-based ELISA showed a highly nonspecific reaction. The NΔ121 protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N′ protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV NΔ121 protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the NΔ121 protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist. PMID:16002634

T-cell-mediated immune response to respiratory coronaviruses

PubMed Central

Channappanavar, Rudragouda; Zhao, Jincun; Perlman, Stanley

2014-01-01

Emerging respiratory coronaviruses such as the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV) pose potential biological threats to humans. SARS and MERS are manifested as severe atypical pneumonia associated with high morbidity and mortality in humans. The majority of studies carried out in SARS-CoV-infected humans and animals attribute a dysregulated/exuberant innate response as a leading contributor to SARS-CoV-mediated pathology. A decade after the 2002–2003 SARS epidemic, we do not have any approved preventive or therapeutic agents available in case of re-emergence of SARS-CoV or other related viruses. A strong neutralizing antibody response generated against the spike (S) glycoprotein of SARS-CoV is completely protective in the susceptible host. However, neutralizing antibody titers and the memory B cell response are short-lived in SARS-recovered patients and the antibody will target primary homologous strain. Interestingly, the acute phase of SARS in humans is associated with a severe reduction in the number of T cells in the blood. Surprisingly, only a limited number of studies have explored the role of the T cell-mediated adaptive immune response in respiratory coronavirus pathogenesis. In this review, we discuss the role of anti-virus CD4 and CD8 T cells during respiratory coronavirus infections with a special emphasis on emerging coronaviruses. PMID:24845462

Effects of Toll-Like Receptor Stimulation on Eosinophilic Infiltration in Lungs of BALB/c Mice Immunized with UV-Inactivated Severe Acute Respiratory Syndrome-Related Coronavirus Vaccine

PubMed Central

Iwata-Yoshikawa, Naoko; Uda, Akihiko; Suzuki, Tadaki; Tsunetsugu-Yokota, Yasuko; Sato, Yuko; Morikawa, Shigeru; Tashiro, Masato; Sata, Tetsutaro; Hasegawa, Hideki

2014-01-01

ABSTRACT Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-month-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from lethal doses of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was observed. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U), and poly(I·C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Additionally, microarray analysis showed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+ cells in the lungs of UV-V-immunized mice showed the upregulation of genes associated with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathology in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants. IMPORTANCE Inactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathology in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray analysis revealed that genes associated with chemotaxis, eosinophil migration, eosinophilia, and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine. PMID:24850731

Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection.

PubMed

Ng, Oi-Wing; Chia, Adeline; Tan, Anthony T; Jadi, Ramesh S; Leong, Hoe Nam; Bertoletti, Antonio; Tan, Yee-Joo

2016-04-12

Severe acute respiratory syndrome (SARS) is a highly contagious infectious disease which first emerged in late 2002, caused by a then novel human coronavirus, SARS coronavirus (SARS-CoV). The virus is believed to have originated from bats and transmitted to human through intermediate animals such as civet cats. The re-emergence of SARS-CoV remains a valid concern due to the continual persistence of zoonotic SARS-CoVs and SARS-like CoVs (SL-CoVs) in bat reservoirs. In this study, the screening for the presence of SARS-specific T cells in a cohort of three SARS-recovered individuals at 9 and 11 years post-infection was carried out, and all memory T cell responses detected target the SARS-CoV structural proteins. Two CD8(+) T cell responses targeting the SARS-CoV membrane (M) and nucleocapsid (N) proteins were characterized by determining their HLA restriction and minimal T cell epitope regions. Furthermore, these responses were found to persist up to 11 years post-infection. An absence of cross-reactivity of these CD8(+) T cell responses against the newly-emerged Middle East respiratory syndrome coronavirus (MERS-CoV) was also demonstrated. The knowledge of the persistence of SARS-specific celullar immunity targeting the viral structural proteins in SARS-recovered individuals is important in the design and development of SARS vaccines, which are currently unavailable. Copyright © 2016 Elsevier Ltd. All rights reserved.

42 CFR 73.0 - Applicability and related requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... and entities that possess SARS-CoV, Lujo virus, or Chapare virus must provide notice to CDC regarding their possession of SARS-CoV, Lujo virus, or Chapare virus on or before December 4, 2012. Currently registered individuals and entities possessing SARS-CoV, Lujo virus, or Chapare virus must meet all the...

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

PubMed

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

2017-03-08

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

Evaluation of inapparent nosocomial severe acute respiratory syndrome coronavirus infection in Vietnam by use of highly specific recombinant truncated nucleocapsid protein-based enzyme-linked immunosorbent assay.

PubMed

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Thai, Hong Thi Cam; Hasebe, Futoshi; Del Carmen Parquet, Maria; Morita, Kouichi

2005-07-01

Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N' protein and (N)Delta(121) protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N' protein-based ELISA showed a highly nonspecific reaction. The (N)Delta(121) protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N' protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV (N)Delta(121) protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the (N)Delta(121) protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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 generalmore » mechanism for coronaviruses to prevent IFN induction.« less

Abelson Kinase Inhibitors Are Potent Inhibitors of Severe Acute Respiratory Syndrome Coronavirus and Middle East Respiratory Syndrome Coronavirus Fusion

PubMed Central

Coleman, Christopher M.; Sisk, Jeanne M.; Mingo, Rebecca M.; Nelson, Elizabeth A.; White, Judith M.

2016-01-01

ABSTRACT The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) cause significant morbidity and morality. There is currently no approved therapeutic for highly pathogenic coronaviruses, even as MERS-CoV is spreading throughout the Middle East. We previously screened a library of FDA-approved drugs for inhibitors of coronavirus replication in which we identified Abelson (Abl) kinase inhibitors, including the anticancer drug imatinib, as inhibitors of both SARS-CoV and MERS-CoV in vitro. Here we show that the anti-CoV activity of imatinib occurs at the early stages of infection, after internalization and endosomal trafficking, by inhibiting fusion of the virions at the endosomal membrane. We specifically identified the imatinib target, Abelson tyrosine-protein kinase 2 (Abl2), as required for efficient SARS-CoV and MERS-CoV replication in vitro. These data demonstrate that specific approved drugs can be characterized in vitro for their anticoronavirus activity and used to identify host proteins required for coronavirus replication. This type of study is an important step in the repurposing of approved drugs for treatment of emerging coronaviruses. IMPORTANCE Both SARS-CoV and MERS-CoV are zoonotic infections, with bats as the primary source. The 2003 SARS-CoV outbreak began in Guangdong Province in China and spread to humans via civet cats and raccoon dogs in the wet markets before spreading to 37 countries. The virus caused 8,096 confirmed cases of SARS and 774 deaths (a case fatality rate of ∼10%). The MERS-CoV outbreak began in Saudi Arabia and has spread to 27 countries. MERS-CoV is believed to have emerged from bats and passed into humans via camels. The ongoing outbreak of MERS-CoV has resulted in 1,791 cases of MERS and 640 deaths (a case fatality rate of 36%). The emergence of SARS-CoV and MERS-CoV provides evidence that coronaviruses are currently spreading from zoonotic sources and can be highly pathogenic, causing serious morbidity and mortality in humans. Treatment of SARS-CoV and MERS-CoV infection is limited to providing supportive therapy consistent with any serious lung disease, as no specific drugs have been approved as therapeutics. Highly pathogenic coronaviruses are rare and appear to emerge and disappear within just a few years. Currently, MERS-CoV is still spreading, as new infections continue to be reported. The outbreaks of SARS-CoV and MERS-CoV and the continuing diagnosis of new MERS cases highlight the need for finding therapeutics for these diseases and potential future coronavirus outbreaks. Screening FDA-approved drugs streamlines the pipeline for this process, as these drugs have already been tested for safety in humans. PMID:27466418

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

PubMed

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

2012-08-01

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

Identification of a Broad-Spectrum Antiviral Small Molecule against Severe Acute Respiratory Syndrome Coronavirus and Ebola, Hendra, and Nipah Viruses by Using a Novel High-Throughput Screening Assay

PubMed Central

Elshabrawy, Hatem A.; Fan, Jilao; Haddad, Christine S.; Ratia, Kiira; Broder, Christopher C.; Caffrey, Michael

2014-01-01

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) and Ebola, Hendra, and Nipah viruses are members of different viral families and are known causative agents of fatal viral diseases. These viruses depend on cathepsin L for entry into their target cells. The viral glycoproteins need to be primed by protease cleavage, rendering them active for fusion with the host cell membrane. In this study, we developed a novel high-throughput screening assay based on peptides, derived from the glycoproteins of the aforementioned viruses, which contain the cathepsin L cleavage site. We screened a library of 5,000 small molecules and discovered a small molecule that can inhibit the cathepsin L cleavage of all viral peptides with minimal inhibition of cleavage of a host protein-derived peptide (pro-neuropeptide Y). The small molecule inhibited the entry of all pseudotyped viruses in vitro and the cleavage of SARS-CoV spike glycoprotein in an in vitro cleavage assay. In addition, the Hendra and Nipah virus fusion glycoproteins were not cleaved in the presence of the small molecule in a cell-based cleavage assay. Furthermore, we demonstrate that the small molecule is a mixed inhibitor of cathepsin L. Our broad-spectrum antiviral small molecule appears to be an ideal candidate for future optimization and development into a potent antiviral against SARS-CoV and Ebola, Hendra, and Nipah viruses. IMPORTANCE We developed a novel high-throughput screening assay to identify small molecules that can prevent cathepsin L cleavage of viral glycoproteins derived from SARS-CoV and Ebola, Hendra, and Nipah viruses that are required for their entry into the host cell. We identified a novel broad-spectrum small molecule that could block cathepsin L-mediated cleavage and thus inhibit the entry of pseudotypes bearing the glycoprotein derived from SARS-CoV or Ebola, Hendra, or Nipah virus. The small molecule can be further optimized and developed into a potent broad-spectrum antiviral drug. PMID:24501399

Receptor recognition and cross-species infections of SARS coronavirus

PubMed Central

Li, Fang

2013-01-01

Receptor recognition is a major determinant of the host range, cross-species infections, and pathogenesis of the severe acute respiratory syndrome coronavirus (SARS-CoV). A defined receptor-binding domain (RBD) in the SARS-CoV spike protein specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2). This article reviews the latest knowledge about how RBDs from different SARS-CoV strains interact with ACE2 from several animal species. Detailed research on these RBD/ACE2 interactions has established important principles on host receptor adaptations, cross-species infections, and future evolution of SARS-CoV. These principles may apply to other emerging animal viruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV). This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.” PMID:23994189

Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes.

PubMed

Lin, Min-Han; Moses, David C; Hsieh, Chih-Hua; Cheng, Shu-Chun; Chen, Yau-Hung; Sun, Chiao-Yin; Chou, Chi-Yuan

2018-02-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in southern China in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. Ten years later, a second highly pathogenic human CoV, MERS-CoV, emerged in the Middle East and has spread to other countries in Europe, North Africa, North America and Asia. As of November 2017, MERS-CoV had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against MERS-CoV. Here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (PL pro s) of MERS-CoV and SARS-CoV. Our findings suggest that disulfiram acts as an allosteric inhibitor of MERS-CoV PL pro but as a competitive (or mixed) inhibitor of SARS-CoV PL pro . The phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of SARS-CoV PL pro by disulfiram, while synergistic inhibition of MERS-CoV PL pro by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination

PubMed Central

Hu, Yong; Li, Wei; Gao, Ting; Cui, Yan; Jin, Yanwen; Li, Ping; Ma, Qingjun

2017-01-01

ABSTRACT Severe acute respiratory syndrome (SARS) is a respiratory disease, caused by a coronavirus (SARS-CoV), that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I·C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by overexpression of the full-length N protein but not N amino acids 1 to 361, which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein. IMPORTANCE The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the Middle East respiratory syndrome CoV (MERS-CoV) N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response. PMID:28148787

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

PubMed Central

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

2016-01-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Fang

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 civetmore » 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.« less

42 CFR 73.0 - Applicability and related requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... and entities that possess SARS-CoV, Lujo virus, or Chapare virus must provide notice to CDC regarding their possession of SARS-CoV, Lujo virus, or Chapare virus on or before December 4, 2012. Currently registered individuals and entities possessing SARS-CoV, Lujo virus, or Chapare virus must meet all the...

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I Interferon Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination.

PubMed

Hu, Yong; Li, Wei; Gao, Ting; Cui, Yan; Jin, Yanwen; Li, Ping; Ma, Qingjun; Liu, Xuan; Cao, Cheng

2017-04-15

Severe acute respiratory syndrome (SARS) is a respiratory disease, caused by a coronavirus (SARS-CoV), that is characterized by atypical pneumonia. The nucleocapsid protein (N protein) of SARS-CoV plays an important role in inhibition of type I interferon (IFN) production via an unknown mechanism. In this study, the SARS-CoV N protein was found to bind to the SPRY domain of the tripartite motif protein 25 (TRIM25) E3 ubiquitin ligase, thereby interfering with the association between TRIM25 and retinoic acid-inducible gene I (RIG-I) and inhibiting TRIM25-mediated RIG-I ubiquitination and activation. Type I IFN production induced by poly I·C or Sendai virus (SeV) was suppressed by the SARS-CoV N protein. SARS-CoV replication was increased by overexpression of the full-length N protein but not N amino acids 1 to 361, which could not interact with TRIM25. These findings provide an insightful interpretation of the SARS-CoV-mediated host innate immune suppression caused by the N protein. IMPORTANCE The SARS-CoV N protein is essential for the viral life cycle and plays a key role in the virus-host interaction. We demonstrated that the interaction between the C terminus of the N protein and the SPRY domain of TRIM25 inhibited TRIM25-mediated RIG-I ubiquitination, which resulted in the inhibition of IFN production. We also found that the Middle East respiratory syndrome CoV (MERS-CoV) N protein interacted with TRIM25 and inhibited RIG-I signaling. The outcomes of these findings indicate the function of the coronavirus N protein in modulating the host's initial innate immune response. Copyright © 2017 American Society for Microbiology.

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

PubMed

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

2014-12-19

Coronavirus (CoV) infection is usually detected by cellular sensors, which trigger the activation of the innate immune system. Nevertheless, CoVs have evolved viral proteins that target different signaling pathways to counteract innate immune responses. Some CoV proteins act as antagonists of interferon (IFN) by inhibiting IFN production or signaling, aspects that are briefly addressed in this review. After CoV infection, potent cytokines relevant in controlling virus infections and priming adaptive immune responses are also generated. However, an uncontrolled induction of these proinflammatory cytokines can lead to pathogenesis and disease severity as described for SARS-CoV and MERS-CoV. The cellular pathways mediated by interferon regulatory factor (IRF)-3 and -7, activating transcription factor (ATF)-2/jun, activator protein (AP)-1, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and nuclear factor of activated T cells (NF-AT), are the main drivers of the inflammatory response triggered after viral infections, with NF-κB pathway the most frequently activated. Key CoV proteins involved in the regulation of these pathways and the proinflammatory immune response are revisited in this manuscript. It has been shown that the envelope (E) protein plays a variable role in CoV morphogenesis, depending on the CoV genus, being absolutely essential in some cases (genus α CoVs such as TGEV, and genus β CoVs such as MERS-CoV), but not in others (genus β CoVs such as MHV or SARS-CoV). A comprehensive accumulation of data has shown that the relatively small E protein elicits a strong influence on the interaction of SARS-CoV with the host. In fact, after infection with viruses in which this protein has been deleted, increased cellular stress and unfolded protein responses, apoptosis, and augmented host immune responses were observed. In contrast, the presence of E protein activated a pathogenic inflammatory response that may cause death in animal models and in humans. The modification or deletion of different motifs within E protein, including the transmembrane domain that harbors an ion channel activity, small sequences within the middle region of the carboxy-terminus of E protein, and its most carboxy-terminal end, which contains a PDZ domain-binding motif (PBM), is sufficient to attenuate the virus. Interestingly, a comprehensive collection of SARS-CoVs in which these motifs have been modified elicited full and long-term protection even in old mice, making those deletion mutants promising vaccine candidates. These data indicate that despite its small size, E protein drastically influences the replication of CoVs and their pathogenicity. Although E protein is not essential for CoV genome replication or subgenomic mRNA synthesis, it affects virus morphogenesis, budding, assembly, intracellular trafficking, and virulence. In fact, E protein is responsible in a significant proportion of the inflammasome activation and the associated inflammation elicited by SARS-CoV in the lung parenchyma. This exacerbated inflammation causes edema accumulation leading to acute respiratory distress syndrome (ARDS) and, frequently, to the death of infected animal models or human patients. Copyright © 2014 Elsevier B.V. All rights reserved.

Receptor recognition and cross-species infections of SARS coronavirus.

PubMed

Li, Fang

2013-10-01

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

Understanding the T cell immune response in SARS coronavirus infection.

PubMed

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

2012-09-01

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

SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production.

PubMed

Tseng, Ying-Tzu; Wang, Shiu-Mei; Huang, Kuo-Jung; Wang, Chin-Tien

2014-04-27

Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.

Broad-Spectrum In Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against Emerging Viruses of the Family Coronaviridae▿

PubMed Central

O'Keefe, Barry R.; Giomarelli, Barbara; Barnard, Dale L.; Shenoy, Shilpa R.; Chan, Paul K. S.; McMahon, James B.; Palmer, Kenneth E.; Barnett, Brian W.; Meyerholz, David K.; Wohlford-Lenane, Christine L.; McCray, Paul B.

2010-01-01

Viruses of the family Coronaviridae have recently emerged through zoonotic transmission to become serious human pathogens. The pathogenic agent responsible for severe acute respiratory syndrome (SARS), the SARS coronavirus (SARS-CoV), is a member of this large family of positive-strand RNA viruses that cause a spectrum of disease in humans, other mammals, and birds. Since the publicized outbreaks of SARS in China and Canada in 2002-2003, significant efforts successfully identified the causative agent, host cell receptor(s), and many of the pathogenic mechanisms underlying SARS. With this greater understanding of SARS-CoV biology, many researchers have sought to identify agents for the treatment of SARS. Here we report the utility of the potent antiviral protein griffithsin (GRFT) in the prevention of SARS-CoV infection both in vitro and in vivo. We also show that GRFT specifically binds to the SARS-CoV spike glycoprotein and inhibits viral entry. In addition, we report the activity of GRFT against a variety of additional coronaviruses that infect humans, other mammals, and birds. Finally, we show that GRFT treatment has a positive effect on morbidity and mortality in a lethal infection model using a mouse-adapted SARS-CoV and also specifically inhibits deleterious aspects of the host immunological response to SARS infection in mammals. PMID:20032190

Understanding the T cell immune response in SARS coronavirus infection

PubMed Central

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

2012-01-01

The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development. PMID:26038429

Overactive Epidermal Growth Factor Receptor Signaling Leads to Increased Fibrosis after Severe Acute Respiratory Syndrome Coronavirus Infection

PubMed Central

Venkataraman, Thiagarajan; Coleman, Christopher M.

2017-01-01

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly pathogenic respiratory virus that causes morbidity and mortality in humans. After infection with SARS-CoV, the acute lung injury caused by the virus must be repaired to regain lung function. A dysregulation in this wound healing process leads to fibrosis. Many survivors of SARS-CoV infection develop pulmonary fibrosis (PF), with higher prevalence in older patients. Using mouse models of SARS-CoV pathogenesis, we have identified that the wound repair pathway, controlled by the epidermal growth factor receptor (EGFR), is critical to recovery from SARS-CoV-induced tissue damage. In mice with constitutively active EGFR [EGFR(DSK5) mice], we find that SARS-CoV infection causes enhanced lung disease. Importantly, we show that during infection, the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF) are upregulated, and exogenous addition of these ligands during infection leads to enhanced lung disease and altered wound healing dynamics. Our data demonstrate a key role of EGFR in the host response to SARS-CoV and how it may be implicated in lung disease induced by other highly pathogenic respiratory viruses. IMPORTANCE PF has many causative triggers, including severe respiratory viruses such as SARS-CoV. Currently there are no treatments to prevent the onset or limit the progression of PF, and the molecular pathways underlying the development of PF are not well understood. In this study, we identified a role for the balanced control of EGFR signaling as a key factor in progression to PF. These data demonstrate that therapeutic treatment modulating EGFR activation could protect against PF development caused by severe respiratory virus infection. PMID:28404843

Overactive Epidermal Growth Factor Receptor Signaling Leads to Increased Fibrosis after Severe Acute Respiratory Syndrome Coronavirus Infection.

PubMed

Venkataraman, Thiagarajan; Coleman, Christopher M; Frieman, Matthew B

2017-06-15

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly pathogenic respiratory virus that causes morbidity and mortality in humans. After infection with SARS-CoV, the acute lung injury caused by the virus must be repaired to regain lung function. A dysregulation in this wound healing process leads to fibrosis. Many survivors of SARS-CoV infection develop pulmonary fibrosis (PF), with higher prevalence in older patients. Using mouse models of SARS-CoV pathogenesis, we have identified that the wound repair pathway, controlled by the epidermal growth factor receptor (EGFR), is critical to recovery from SARS-CoV-induced tissue damage. In mice with constitutively active EGFR [EGFR(DSK5) mice], we find that SARS-CoV infection causes enhanced lung disease. Importantly, we show that during infection, the EGFR ligands amphiregulin and heparin-binding EGF-like growth factor (HB-EGF) are upregulated, and exogenous addition of these ligands during infection leads to enhanced lung disease and altered wound healing dynamics. Our data demonstrate a key role of EGFR in the host response to SARS-CoV and how it may be implicated in lung disease induced by other highly pathogenic respiratory viruses. IMPORTANCE PF has many causative triggers, including severe respiratory viruses such as SARS-CoV. Currently there are no treatments to prevent the onset or limit the progression of PF, and the molecular pathways underlying the development of PF are not well understood. In this study, we identified a role for the balanced control of EGFR signaling as a key factor in progression to PF. These data demonstrate that therapeutic treatment modulating EGFR activation could protect against PF development caused by severe respiratory virus infection. Copyright © 2017 American Society for Microbiology.

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

PubMed

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

2012-12-11

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

McBride, Corrin E., E-mail: cmcbrid5@jhmi.ed; Machamer, Carolyn E., E-mail: machamer@jhmi.ed

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 ofmore » 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.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pak, J.; Sharon, C; Satkunarajah, M

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 firstmore » 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.« less

Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies

PubMed Central

Zhu, Zhongyu; Chakraborti, Samitabh; He, Yuxian; Roberts, Anjeanette; Sheahan, Tim; Xiao, Xiaodong; Hensley, Lisa E.; Prabakaran, Ponraj; Rockx, Barry; Sidorov, Igor A.; Corti, Davide; Vogel, Leatrice; Feng, Yang; Kim, Jae-Ouk; Wang, Lin-Fa; Baric, Ralph; Lanzavecchia, Antonio; Curtis, Kristopher M.; Nabel, Gary J.; Subbarao, Kanta; Jiang, Shibo; Dimitrov, Dimiter S.

2007-01-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003/2004 by an independent animal-to-human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the human monoclonal antibodies (hmAbs) 80R and S3.1, which can potently neutralize isolates from the first outbreak. Here we report that two hmAbs, m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani or recombinant viruses bearing the GD03 and SZ16 spike (S) glycoproteins. Both antibodies competed with the SARS-CoV receptor, ACE2, for binding to the receptor-binding domain (RBD), suggesting a mechanism of neutralization that involves interference with the SARS-CoV–ACE2 interaction. Two putative hot-spot residues in the RBD (Ile-489 and Tyr-491) were identified within the SARS-CoV spike that likely contribute to most of the m396-binding energy. Residues Ile-489 and Tyr-491 are highly conserved within the SARS-CoV spike, indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS-CoV isolates with known sequences, except strains derived from bats. These antibodies exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets and could have potential applications for diagnosis, prophylaxis, and treatment of SARS-CoV infections. PMID:17620608

Inhibitors of SARS-CoV Entry - Identification using an Internally-Controlled Dual Envelope Pseudovirion Assay

PubMed Central

Zhou, Yanchen; Agudelo, Juliet; Lu, Kai; Goetz, David H.; Hansell, Elizabeth; Chen, Yen Ting; Roush, William R.; McKerrow, James; Craik, Charles S.; Amberg, Sean M.; Simmons, Graham

2011-01-01

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, Dual Envelope Pseudovirion (DEP) Assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes. PMID:21820471

SARS-associated Coronavirus Transmitted from Human to Pig

PubMed Central

Chen, Weijun; Yan, Minghua; Yang, Ling; Ding, Boliang; He, Bo; Wang, Yingzhen; Liu, Xiuli; Liu, Chenhui; Zhu, Hui; You, Bo; Huang, Shengyong; Zhang, Jiangguo; Mu, Feng; Xiang, Zhao; Feng, Xiaoli; Wen, Jie; Fang, Jianqiu; Yu, Jun; Yang, Huanming

2005-01-01

Severe acute respiratory syndrome–associated coronavirus (SARS-CoV) was isolated from a pig during a survey for possible routes of viral transmission after a SARS epidemic. Sequence and epidemiology analyses suggested that the pig was infected by a SARS-CoV of human origin. PMID:15757562

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

PubMed Central

de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

2015-01-01

ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses. PMID:26041291

Screening and identification of linear B-cell epitopes and entry-blocking peptide of severe acute respiratory syndrome (SARS)-associated coronavirus using synthetic overlapping peptide library.

PubMed

Hu, Hongbo; Li, Li; Kao, Richard Y; Kou, Binbin; Wang, Zhanguo; Zhang, Liang; Zhang, Huiyuan; Hao, Zhiyong; Tsui, Wayne H; Ni, Anping; Cui, Lianxian; Fan, Baoxing; Guo, Feng; Rao, Shuan; Jiang, Chengyu; Li, Qian; Sun, Manji; He, Wei; Liu, Gang

2005-01-01

A 10-mer overlapping peptide library has been synthesized for screening and identification of linear B-cell epitopes of severe acute respiratory syndrome associated coronavirus (SARS-CoV), which spanned the major structural proteins of SARS-CoV. One hundred and eleven candidate peptides were positive according to the result of PEPscan, which were assembled into 22 longer peptides. Five of these peptides showed high cross-immunoreactivities (approximately 66.7 to 90.5%) to SARS convalescent patients' sera from the severest epidemic regions of the China mainland. Most interestingly, S(471-503), a peptide located at the receptor binding domain (RBD) of SARS-CoV, could specifically block the binding between the RBD and angiotensin-converting enzyme 2, resulting in the inhibition of SARS-CoV entrance into host cells in vitro. The study demonstrated that S(471-503) peptide was a potential immunoantigen for the development of peptide-based vaccine or a candidate for further drug evaluation against the SARS-CoV virus-cell fusion.

Phosphatidylinositol 4-Kinase IIIβ Is Required for Severe Acute Respiratory Syndrome Coronavirus Spike-mediated Cell Entry*

PubMed Central

Yang, Ning; Ma, Ping; Lang, Jianshe; Zhang, Yanli; Deng, Jiejie; Ju, Xiangwu; Zhang, Gongyi; Jiang, Chengyu

2012-01-01

Phosphatidylinositol kinases (PI kinases) play an important role in the life cycle of several viruses after infection. Using gene knockdown technology, we demonstrate that phosphatidylinositol 4-kinase IIIβ (PI4KB) is required for cellular entry by pseudoviruses bearing the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike protein and that the cell entry mediated by SARS-CoV spike protein is strongly inhibited by knockdown of PI4KB. Consistent with this observation, pharmacological inhibitors of PI4KB blocked entry of SARS pseudovirions. Further research suggested that PI4P plays an essential role in SARS-CoV spike-mediated entry, which is regulated by the PI4P lipid microenvironment. We further demonstrate that PI4KB does not affect virus entry at the SARS-CoV S-ACE2 binding interface or at the stage of virus internalization but rather at or before virus fusion. Taken together, these results indicate a new function for PI4KB and suggest a new drug target for preventing SARS-CoV infection. PMID:22253445

Vaccine Efficacy in Senescent Mice Challenged with Recombinant SARS-CoV Bearing Epidemic and Zoonotic Spike Variants

PubMed Central

Deming, Damon; Sheahan, Timothy; Heise, Mark; Yount, Boyd; Davis, Nancy; Sims, Amy; Suthar, Mehul; Harkema, Jack; Whitmore, Alan; Pickles, Raymond; West, Ande; Donaldson, Eric; Curtis, Kristopher; Johnston, Robert; Baric, Ralph

2006-01-01

Background In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as the etiological agent of severe acute respiratory syndrome, a disease characterized by severe pneumonia that sometimes results in death. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely originating from bats or from other species including civets, raccoon dogs, domestic cats, swine, and rodents. A SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. We report the comprehensive investigation of SARS vaccine efficacy in young and senescent mice following homologous and heterologous challenge. Methods and Findings Using Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N), we demonstrate that VRP-S, but not VRP-N vaccines provide complete short- and long-term protection against homologous strain challenge in young and senescent mice. To test VRP vaccine efficacy against a heterologous SARS-CoV, we used phylogenetic analyses, synthetic biology, and reverse genetics to construct a chimeric virus (icGDO3-S) encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV. icGD03-S replicated efficiently in human airway epithelial cells and in the lungs of young and senescent mice, and was highly resistant to neutralization with antisera directed against the Urbani strain. Although VRP-S vaccines provided complete short-term protection against heterologous icGD03-S challenge in young mice, only limited protection was seen in vaccinated senescent animals. VRP-N vaccines not only failed to protect from homologous or heterologous challenge, but resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of SARS-CoV–challenged mice. VRP-N–induced pathology presented at day 4, peaked around day 7, and persisted through day 14, and was likely mediated by cellular immune responses. Conclusions This study identifies gaps and challenges in vaccine design for controlling future SARS-CoV zoonosis, especially in vulnerable elderly populations. The availability of a SARS-CoV virus bearing heterologous S glycoproteins provides a robust challenge inoculum for evaluating vaccine efficacy against zoonotic strains, the most likely source of future outbreaks. PMID:17194199

SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production

PubMed Central

2014-01-01

Background Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. Results SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. Conclusions The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly. PMID:24766657

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

PubMed

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

2011-04-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p < 0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p < 0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. Copyright © 2011 Elsevier B.V. All rights reserved.

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

PubMed

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

2013-10-01

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

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

PubMed

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

2013-02-01

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

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

PubMed

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

2013-04-30

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

Antigen Production in Plant to Tackle Infectious Diseases Flare Up: The Case of SARS.

PubMed

Demurtas, Olivia C; Massa, Silvia; Illiano, Elena; De Martinis, Domenico; Chan, Paul K S; Di Bonito, Paola; Franconi, Rosella

2016-01-01

Severe acute respiratory syndrome (SARS) is a dangerous infection with pandemic potential. It emerged in 2002 and its aetiological agent, the SARS Coronavirus (SARS-CoV), crossed the species barrier to infect humans, showing high morbidity and mortality rates. No vaccines are currently licensed for SARS-CoV and important efforts have been performed during the first outbreak to develop diagnostic tools. Here we demonstrate the transient expression in Nicotiana benthamiana of two important antigenic determinants of the SARS-CoV, the nucleocapsid protein (N) and the membrane protein (M) using a virus-derived vector or agro-infiltration, respectively. For the M protein, this is the first description of production in plants, while for plant-derived N protein we demonstrate that it is recognized by sera of patients from the SARS outbreak in Hong Kong in 2003. The availability of recombinant N and M proteins from plants opens the way to further evaluation of their potential utility for the development of diagnostic and protection/therapy tools to be quickly manufactured, at low cost and with minimal risk, to face potential new highly infectious SARS-CoV outbreaks.

X-ray Structural and Biological Evaluation of a Series of Potent and Highly Selective Inhibitors of Human Coronavirus Papain-like Proteases

PubMed Central

2015-01-01

Structure-guided design was used to generate a series of noncovalent inhibitors with nanomolar potency against the papain-like protease (PLpro) from the SARS coronavirus (CoV). A number of inhibitors exhibit antiviral activity against SARS-CoV infected Vero E6 cells and broadened specificity toward the homologous PLP2 enzyme from the human coronavirus NL63. Selectivity and cytotoxicity studies established a more than 100-fold preference for the coronaviral enzyme over homologous human deubiquitinating enzymes (DUBs), and no significant cytotoxicity in Vero E6 and HEK293 cell lines is observed. X-ray structural analyses of inhibitor-bound crystal structures revealed subtle differences between binding modes of the initial benzodioxolane lead (15g) and the most potent analogues 3k and 3j, featuring a monofluoro substitution at para and meta positions of the benzyl ring, respectively. Finally, the less lipophilic bis(amide) 3e and methoxypyridine 5c exhibit significantly improved metabolic stability and are viable candidates for advancing to in vivo studies. PMID:24568342

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

PubMed

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

2014-05-01

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

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

PubMed Central

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

2014-01-01

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. PMID:24519901

Challenges presented by MERS corona virus, and SARS corona virus to global health.

PubMed

Al-Hazmi, Ali

2016-07-01

Numerous viral infections have arisen and affected global healthcare facilities. Millions of people are at severe risk of acquiring several evolving viral infections through several factors. In the present article we have described about risk factors, chance of infection, and prevention methods of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV), human coronaviruses (CoVs) frequently cause a normal cold which is mild and self-restricting. Zoonotic transmission of CoVs such as the newly discovered MERS-CoV and SARS-CoV, may be associated with severe lower respiratory tract infection. The present review provides the recent clinical and pathological information on MERS and SARS. The task is to transform these discoveries about MERS and SARS pathogenesis and to develop intervention methods that will eventually allow the effective control of these recently arising severe viral infections. Global health sector has learnt many lessons through the recent outbreak of MERS and SARS, but the need for identifying new antiviral treatment was not learned. In the present article we have reviewed the literature on the several facets like transmission, precautions and effectiveness of treatments used in patients with MERS-CoV and SARS infections.

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

PubMed

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.

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

PubMed Central

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

2011-01-01

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 µg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 hours before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p<0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p<0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells. PMID:21338626

Cell Host Response to Infection with Novel Human Coronavirus EMC Predicts Potential Antivirals and Important Differences with SARS Coronavirus

PubMed Central

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

2013-01-01

ABSTRACT A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was able to replicate as efficiently as SARS-CoV in Calu-3 cells and similarly induced minimal transcriptomic changes before 12 h postinfection. Later in infection, HCoV-EMC induced a massive dysregulation of the host transcriptome, to a much greater extent than SARS-CoV. Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 (IL-17) pathway, but HCoV-EMC specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type I and II major histocompatibility complex (MHC) genes. This could have an important impact on the ability of the host to mount an adaptive host response. A unique set of 207 genes was dysregulated early and permanently throughout infection with HCoV-EMC, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. Overall, HCoV-EMC and SARS-CoV elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus. PMID:23631916

Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease

PubMed Central

Ratia, Kiira; Kilianski, Andrew; Baez-Santos, Yahira M.; Baker, Susan C.; Mesecar, Andrew

2014-01-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response. PMID:24854014

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Purnima; Gunalan, Vithiagaran; Liu Boping

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 tomore » 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.« less

From SARS coronavirus to novel animal and human coronaviruses.

PubMed

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

2013-08-01

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

The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

PubMed Central

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

2014-01-01

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation. PMID:25122212

Identification of Diverse Alphacoronaviruses and Genomic Characterization of a Novel Severe Acute Respiratory Syndrome-Like Coronavirus from Bats in China

PubMed Central

He, Biao; Zhang, Yuzhen; Xu, Lin; Yang, Weihong; Yang, Fanli; Feng, Yun; Xia, Lele; Zhou, Jihua; Zhen, Weibin; Feng, Ye; Guo, Huancheng

2014-01-01

ABSTRACT Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats in China, Europe, and Africa, most have a genetic organization significantly distinct from human/civet SARS CoVs in the receptor-binding domain (RBD), which mediates receptor binding and determines the host spectrum, resulting in their failure to cause human infections and making them unlikely progenitors of human/civet SARS CoVs. Here, a viral metagenomic analysis of 268 bat rectal swabs collected from four counties in Yunnan Province has identified hundreds of sequences relating to alpha- and betacoronaviruses. Phylogenetic analysis based on a conserved region of the RNA-dependent RNA polymerase gene revealed that alphacoronaviruses had diversities with some obvious differences from those reported previously. Full genomic analysis of a new SARS-like CoV from Baoshan (LYRa11) showed that it was 29,805 nucleotides (nt) in length with 13 open reading frames (ORFs), sharing 91% nucleotide identity with human/civet SARS CoVs and the most recently reported SARS-like CoV Rs3367, while sharing 89% with other bat SARS-like CoVs. Notably, it showed the highest sequence identity with the S gene of SARS CoVs and Rs3367, especially in the RBD region. Antigenic analysis showed that the S1 domain of LYRa11 could be efficiently recognized by SARS-convalescent human serum, indicating that LYRa11 is a novel virus antigenically close to SARS CoV. Recombination analyses indicate that LYRa11 is likely a recombinant descended from parental lineages that had evolved into a number of bat SARS-like CoVs. IMPORTANCE Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been discovered in bats worldwide, there are significant different genic structures, particularly in the S1 domain, which are responsible for host tropism determination, between bat SARS-like CoVs and human SARS CoVs, indicating that most reported bat SARS-like CoVs are not the progenitors of human SARS CoV. We have identified diverse alphacoronaviruses and a close relative (LYRa11) to SARS CoV in bats collected in Yunnan, China. Further analysis showed that alpha- and betacoronaviruses have different circulation and transmission dynamics in bat populations. Notably, full genomic sequencing and antigenic study demonstrated that LYRa11 is phylogenetically and antigenically closely related to SARS CoV. Recombination analyses indicate that LYRa11 is a recombinant from certain bat SARS-like CoVs circulating in Yunnan Province. PMID:24719429

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

PubMed

Ng, Oi-Wing; Tan, Yee-Joo

2017-01-02

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

Synthetic peptides coupled to the surface of liposomes effectively induce SARS coronavirus-specific cytotoxic T lymphocytes and viral clearance in HLA-A*0201 transgenic mice.

PubMed

Ohno, Satoshi; Kohyama, Shunsuke; Taneichi, Maiko; Moriya, Osamu; Hayashi, Hidenori; Oda, Hiroshi; Mori, Masahito; Kobayashi, Akiharu; Akatsuka, Toshitaka; Uchida, Tetsuya; Matsui, Masanori

2009-06-12

We investigated whether the surface-linked liposomal peptide was applicable to a vaccine based on cytotoxic T lymphocytes (CTLs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We first identified four HLA-A*0201-restricted CTL epitopes derived from SARS-CoV using HLA-A*0201 transgenic mice and recombinant adenovirus expressing predicted epitopes. These peptides were coupled to the surface of liposomes, and inoculated into mice. Two of the liposomal peptides were effective for peptide-specific CTL induction, and one of them was efficient for the clearance of vaccinia virus expressing epitopes of SARS-CoV, suggesting that the surface-linked liposomal peptide might offer an effective CTL-based vaccine against SARS.

Toll-Like Receptor 3 Signaling via TRIF Contributes to a Protective Innate Immune Response to Severe Acute Respiratory Syndrome Coronavirus Infection

PubMed Central

Totura, Allison L.; Whitmore, Alan; Agnihothram, Sudhakar; Schäfer, Alexandra; Katze, Michael G.; Heise, Mark T.

2015-01-01

ABSTRACT Toll-like receptors (TLRs) are sensors that recognize molecular patterns from viruses, bacteria, and fungi to initiate innate immune responses to invading pathogens. The emergence of highly pathogenic coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) is a concern for global public health, as there is a lack of efficacious vaccine platforms and antiviral therapeutic strategies. Previously, it was shown that MyD88, an adaptor protein necessary for signaling by multiple TLRs, is a required component of the innate immune response to mouse-adapted SARS-CoV infection in vivo. Here, we demonstrate that TLR3−/−, TLR4−/−, and TRAM−/− mice are more susceptible to SARS-CoV than wild-type mice but experience only transient weight loss with no mortality in response to infection. In contrast, mice deficient in the TLR3/TLR4 adaptor TRIF are highly susceptible to SARS-CoV infection, showing increased weight loss, mortality, reduced lung function, increased lung pathology, and higher viral titers. Distinct alterations in inflammation were present in TRIF−/− mice infected with SARS-CoV, including excess infiltration of neutrophils and inflammatory cell types that correlate with increased pathology of other known causes of acute respiratory distress syndrome (ARDS), including influenza virus infections. Aberrant proinflammatory cytokine, chemokine, and interferon-stimulated gene (ISG) signaling programs were also noted following infection of TRIF−/− mice that were similar to those seen in human patients with poor disease outcome following SARS-CoV or MERS-CoV infection. These findings highlight the importance of TLR adaptor signaling in generating a balanced protective innate immune response to highly pathogenic coronavirus infections. PMID:26015500

Three-Dimensional Human Bronchial-Tracheal Epithelial Tissue-Like Assemblies (TLAs) as Hosts for Severe Acute Respiratory Syndrome (SARS)-CoV Infection

NASA Technical Reports Server (NTRS)

Suderman, M. T.; McCarthy, M.; Mossell, E.; Watts, D. M.; Peters, C. J.; Shope, R.; Goodwin, T. J.

2006-01-01

A three-dimensional (3-D) tissue-like assembly (TLA) of human bronchial-tracheal mesenchymal (HBTC) cells with an overlay of human bronchial epithelial (BEAS-2B) cells was constructed using a NASA Bioreactor to survey the infectivity of SARS-CoV. This TLA was inoculated with a low passage number Urbani strain of SARS-CoV. At selected intervals over a 10-day period, media and cell aliquots of the 3-D TLA were harvested for viral titer assay and for light and electron microscopy examination. All viral titer assays were negative in both BEAS-2B two-dimensional monolayer and TLA. Light microscopy immunohistochemistry demonstrated antigen-antibody reactivity with anti-SARS-CoV polyclonal antibody to spike and nuclear proteins on cell membranes and cytoplasm. Coronavirus Group 2 cross-reactivity was demonstrated by positive reaction to anti-FIPV 1 and anti-FIPV 1 and 2 antibodies. TLA examination by transmission electron microscopy indicated increasing cytoplasmic vacuolation with numerous electron-dense bodies measuring 45 to 270 nm from days 4 through 10. There was no evidence of membrane blebbing, membrane duplication, or fragmentation of organelles in the TLAs. However, progressive disruption of endoplasmic reticulum was observed throughout the cells. Antibody response to SARS-CoV specific spike and nucleocapsid glycoproteins, cross-reactivity with FIPV antibodies, and the cytoplasmic pathology suggests this HBTE TLA model is permissive to SARS-CoV infection.

SARS and MERS: recent insights into emerging coronaviruses.

PubMed

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

2016-08-01

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

SARS-CoV and Emergent Coronaviruses: Viral Determinants of Interspecies Transmission

PubMed Central

Bolles, Meagan; Donaldson, Eric; Baric, Ralph

2011-01-01

Most new emerging viruses are derived from strains circulating in zoonotic reservoirs. Coronaviruses, which had an established potential for cross-species transmission within domesticated animals, suddenly became relevant with the unexpected emergence of the highly pathogenic human SARS-CoV strain from zoonotic reservoirs in 2002. SARS-CoV infected approximately 8000 people worldwide before public health measures halted the epidemic. Supported by robust time-ordered sequence variation, structural biology, well-characterized patient pools, and biological data, the emergence of SARS-CoV represents one of the best studied natural models of viral disease emergence from zoonotic sources. This review article summarizes previous and more recent advances into the molecular and structural characteristics, with particular emphasis on host-receptor interactions, that drove this remarkable virus disease outbreak in human populations. PMID:22180768

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding.

PubMed

Gui, Miao; Song, Wenfei; Zhou, Haixia; Xu, Jingwei; Chen, Silian; Xiang, Ye; Wang, Xinquan

2017-01-01

The global outbreak of SARS in 2002-2003 was caused by the infection of a new human coronavirus SARS-CoV. The infection of SARS-CoV is mediated mainly through the viral surface glycoproteins, which consist of S1 and S2 subunits and form trimer spikes on the envelope of the virions. Here we report the ectodomain structures of the SARS-CoV surface spike trimer in different conformational states determined by single-particle cryo-electron microscopy. The conformation 1 determined at 4.3 Å resolution is three-fold symmetric and has all the three receptor-binding C-terminal domain 1 (CTD1s) of the S1 subunits in "down" positions. The binding of the "down" CTD1s to the SARS-CoV receptor ACE2 is not possible due to steric clashes, suggesting that the conformation 1 represents a receptor-binding inactive state. Conformations 2-4 determined at 7.3, 5.7 and 6.8 Å resolutions are all asymmetric, in which one RBD rotates away from the "down" position by different angles to an "up" position. The "up" CTD1 exposes the receptor-binding site for ACE2 engagement, suggesting that the conformations 2-4 represent a receptor-binding active state. This conformational change is also required for the binding of SARS-CoV neutralizing antibodies targeting the CTD1. This phenomenon could be extended to other betacoronaviruses utilizing CTD1 of the S1 subunit for receptor binding, which provides new insights into the intermediate states of coronavirus pre-fusion spike trimer during infection.

The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner.

PubMed

Lai, Alex L; Millet, Jean K; Daniel, Susan; Freed, Jack H; Whittaker, Gary R

2017-12-08

Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2' cleavage site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca 2+ -dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform." Copyright © 2017 Elsevier Ltd. All rights reserved.

Recombinant truncated nucleocapsid protein as antigen in a novel immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of severe acute respiratory syndrome coronavirus infection.

PubMed

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Hasebe, Futoshi; Parquet, Maria del Carmen; Morikawa, Shigeru; Morita, Kouichi

2007-02-01

We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection.

The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

PubMed Central

Friedel, Caroline C.; Müller, Marcel A.; Carbajo-Lozoya, Javier; Stellberger, Thorsten; von Dall’Armi, Ekatarina; Herzog, Petra; Kallies, Stefan; Niemeyer, Daniela; Ditt, Vanessa; Kuri, Thomas; Züst, Roland; Pumpor, Ksenia; Hilgenfeld, Rolf; Schwarz, Frank; Zimmer, Ralf; Steffen, Imke; Weber, Friedemann; Thiel, Volker; Herrler, Georg; Thiel, Heinz-Jürgen; Schwegmann-Weßels, Christel; Pöhlmann, Stefan; Haas, Jürgen; Drosten, Christian; von Brunn, Albrecht

2011-01-01

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. PMID:22046132

Amino Acids 270 to 510 of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Are Required for Interaction with Receptor

PubMed Central

Babcock, Gregory J.; Esshaki, Diana J.; Thomas, William D.; Ambrosino, Donna M.

2004-01-01

A novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), has recently been identified as the causative agent of severe acute respiratory syndrome (SARS). SARS-CoV appears similar to other coronaviruses in both virion structure and genome organization. It is known for other coronaviruses that the spike (S) glycoprotein is required for both viral attachment to permissive cells and for fusion of the viral envelope with the host cell membrane. Here we describe the construction and expression of a soluble codon-optimized SARS-CoV S glycoprotein comprising the first 1,190 amino acids of the native S glycoprotein (S1190). The codon-optimized and native S glycoproteins exhibit similar molecular weight as determined by Western blot analysis, indicating that synthetic S glycoprotein is modified correctly in a mammalian expression system. S1190 binds to the surface of Vero E6 cells, a cell permissive to infection, as demonstrated by fluorescence-activated cell sorter analysis, suggesting that S1190 maintains the biologic activity present in native S glycoprotein. This interaction is blocked with serum obtained from recovering SARS patients, indicating that the binding is specific. In an effort to map the ligand-binding domain of the SARS-CoV S glycoprotein, carboxy- and amino-terminal truncations of the S1190 glycoprotein were constructed. Amino acids 270 to 510 were the minimal receptor-binding region of the SARS-CoV S glycoprotein as determined by flow cytometry. We speculate that amino acids 1 to 510 of the SARS-CoV S glycoprotein represent a unique domain containing the receptor-binding site (amino acids 270 to 510), analogous to the S1 subunit of other coronavirus S glycoproteins. PMID:15078936

Identification of residues of SARS-CoV nsp1 that differentially affect inhibition of gene expression and antiviral signaling.

PubMed

Jauregui, Andrew R; Savalia, Dhruti; Lowry, Virginia K; Farrell, Cara M; Wathelet, Marc G

2013-01-01

An epidemic of Severe Acute Respiratory Syndrome (SARS) led to the identification of an associated coronavirus, SARS-CoV. This virus evades the host innate immune response in part through the expression of its non-structural protein (nsp) 1, which inhibits both host gene expression and virus- and interferon (IFN)-dependent signaling. Thus, nsp1 is a promising target for drugs, as inhibition of nsp1 would make SARS-CoV more susceptible to the host antiviral defenses. To gain a better understanding of nsp1 mode of action, we generated and analyzed 38 mutants of the SARS-CoV nsp1, targeting 62 solvent exposed residues out of the 180 amino acid protein. From this work, we identified six classes of mutants that abolished, attenuated or increased nsp1 inhibition of host gene expression and/or antiviral signaling. Each class of mutants clustered on SARS-CoV nsp1 surface and suggested nsp1 interacts with distinct host factors to exert its inhibitory activities. Identification of the nsp1 residues critical for its activities and the pathways involved in these activities should help in the design of drugs targeting nsp1. Significantly, several point mutants increased the inhibitory activity of nsp1, suggesting that coronaviruses could evolve a greater ability to evade the host response through mutations of such residues.

Interpretation of diagnostic laboratory tests for severe acute respiratory syndrome: the Toronto experience

PubMed Central

Tang, Patrick; Louie, Marie; Richardson, Susan E.; Smieja, Marek; Simor, Andrew E.; Jamieson, Frances; Fearon, Margaret; Poutanen, Susan M.; Mazzulli, Tony; Tellier, Raymond; Mahony, James; Loeb, Mark; Petrich, Astrid; Chernesky, Max; McGeer, Allison; Low, Donald E.; Phillips, Elizabeth; Jones, Steven; Bastien, Nathalie; Li, Yan; Dick, Daryl; Grolla, Allen; Fernando, Lisa; Booth, Timothy F.; Henry, Bonnie; Rachlis, Anita R.; Matukas, Larissa M.; Rose, David B.; Lovinsky, Reena; Walmsley, Sharon; Gold, Wayne L.; Krajden, Sigmund

2004-01-01

Background An outbreak of severe acute respiratory syndrome (SARS) began in Canada in February 2003. The initial diagnosis of SARS was based on clinical and epidemiological criteria. During the outbreak, molecular and serologic tests for the SARS-associated coronavirus (SARS-CoV) became available. However, without a “gold standard,” it was impossible to determine the usefulness of these tests. We describe how these tests were used during the first phase of the SARS outbreak in Toronto and offer some recommendations that may be useful if SARS returns. Methods We examined the results of all diagnostic laboratory tests used in 117 patients admitted to hospitals in Toronto who met the Health Canada criteria for suspect or probable SARS. Focusing on tests for SARS-CoV, we attempted to determine the optimal specimen types and timing of specimen collection. Results Diagnostic test results for SARS-CoV were available for 110 of the 117 patients. SARS-CoV was detected by means of reverse-transcriptase polymerase chain reaction (RT-PCR) in at least one specimen in 59 (54.1%) of 109 patients. Serologic test results of convalescent samples were positive in 50 (96.2%) of 52 patients for whom paired serum samples were collected during the acute and convalescent phases of the illness. Of the 110 patients, 78 (70.9%) had specimens that tested positive by means of RT-PCR, serologic testing or both methods. The proportion of RT-PCR test results that were positive was similar between patients who met the criteria for suspect SARS (50.8%, 95% confidence interval [CI] 38.4%–63.2%) and those who met the criteria for probable SARS (58.0%, 95% CI 44.2%–70.7%). SARS-CoV was detected in nasopharyngeal swabs in 33 (32.4%) of 102 patients, in stool specimens in 19 (63.3%) of 30 patients, and in specimens from the lower respiratory tract in 10 (58.8%) of 17 patients. Interpretation These findings suggest that the rapid diagnostic tests in use at the time of the initial outbreak lack sufficient sensitivity to be used clinically to rule out SARS. As tests for SARS-CoV continue to be optimized, evaluation of the clinical presentation and elucidation of a contact history must remain the cornerstone of SARS diagnosis. In patients with SARS, specimens taken from the lower respiratory tract and stool samples test positive by means of RT-PCR more often than do samples taken from other areas. PMID:14707219

Severe acute respiratory syndrome (SARS) S protein production in plants: development of recombinant vaccine.

PubMed

Pogrebnyak, Natalia; Golovkin, Maxim; Andrianov, Vyacheslav; Spitsin, Sergei; Smirnov, Yuriy; Egolf, Richard; Koprowski, Hilary

2005-06-21

In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis.

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

PubMed

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

2014-01-01

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

Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains.

PubMed

Yuan, Yuan; Cao, Duanfang; Zhang, Yanfang; Ma, Jun; Qi, Jianxun; Wang, Qihui; Lu, Guangwen; Wu, Ying; Yan, Jinghua; Shi, Yi; Zhang, Xinzheng; Gao, George F

2017-04-10

The envelope spike (S) proteins of MERS-CoV and SARS-CoV determine the virus host tropism and entry into host cells, and constitute a promising target for the development of prophylactics and therapeutics. Here, we present high-resolution structures of the trimeric MERS-CoV and SARS-CoV S proteins in its pre-fusion conformation by single particle cryo-electron microscopy. The overall structures resemble that from other coronaviruses including HKU1, MHV and NL63 reported recently, with the exception of the receptor binding domain (RBD). We captured two states of the RBD with receptor binding region either buried (lying state) or exposed (standing state), demonstrating an inherently flexible RBD readily recognized by the receptor. Further sequence conservation analysis of six human-infecting coronaviruses revealed that the fusion peptide, HR1 region and the central helix are potential targets for eliciting broadly neutralizing antibodies.

X-ray structure and inhibition of 3C-like protease from porcine epidemic diarrhea virus

DOE PAGES

St. John, Sarah E.; Anson, Brandon J.; Mesecar, Andrew D.

2016-05-13

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CL pro) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 angstrom X-ray structure of 3CL pro from PEDV. Analysis of the PEDV 3CL pro structure and comparison to other coronaviral 3CL pro's from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CL pro's are conserved, with the exception of amore » loop that comprises the protease S-2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL pro, (R)-16, to have inhibitor activity against PEDV 3CL pro, despite that SARS-3CL pro and PEDV 3CL pro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CL pro are conserved in PEDV-3CL pro; however, the sequence variation and positional difference in the loop forming the S-2 pocket may account for large observed difference in IC 50 values. In conclusion, this work advances our understanding of the subtle, but important, differences in coronaviral 3CL pro architecture and contributes to the broader structural knowledge of coronaviral 3CL pro's.« less

X-ray structure and inhibition of 3C-like protease from porcine epidemic diarrhea virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

St. John, Sarah E.; Anson, Brandon J.; Mesecar, Andrew D.

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CL pro) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 angstrom X-ray structure of 3CL pro from PEDV. Analysis of the PEDV 3CL pro structure and comparison to other coronaviral 3CL pro's from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CL pro's are conserved, with the exception of amore » loop that comprises the protease S-2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL pro, (R)-16, to have inhibitor activity against PEDV 3CL pro, despite that SARS-3CL pro and PEDV 3CL pro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CL pro are conserved in PEDV-3CL pro; however, the sequence variation and positional difference in the loop forming the S-2 pocket may account for large observed difference in IC 50 values. In conclusion, this work advances our understanding of the subtle, but important, differences in coronaviral 3CL pro architecture and contributes to the broader structural knowledge of coronaviral 3CL pro's.« less

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

PubMed

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

2016-05-13

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

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

PubMed

Venkataraman, Thiagarajan; Frieman, Matthew B

2017-07-01

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

Exogenous ACE2 Expression Allows Refractory Cell Lines To Support Severe Acute Respiratory Syndrome Coronavirus Replication

PubMed Central

Mossel, Eric C.; Huang, Cheng; Narayanan, Krishna; Makino, Shinji; Tesh, Robert B.; Peters, C. J.

2005-01-01

Of 30 cell lines and primary cells examined, productive severe acute respiratory syndrome coronavirus (Urbani strain) (SARS-CoV) infection after low-multiplicity inoculation was detected in only six: three African green monkey kidney epithelial cell lines (Vero, Vero E6, and MA104), a human colon epithelial line (CaCo-2), a porcine kidney epithelial line [PK(15)], and mink lung epithelial cells (Mv 1 Lu). SARS-CoV produced a lytic infection in Vero, Vero E6, and MA104 cells, but there was no visible cytopathic effect in Caco-2, Mv 1 Lu, or PK(15) cells. Multistep growth kinetics were identical in Vero E6 and MA104 cells, with maximum titer reached 24 h postinoculation (hpi). Virus titer was maximal 96 hpi in CaCo-2 cells, and virus was continually produced from infected CaCo-2 cells for at least 6 weeks after infection. CaCo-2 was the only human cell type of 13 tested that supported efficient SARS-CoV replication. Expression of the SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), resulted in SARS-CoV replication in all refractory cell lines examined. Titers achieved were variable and dependent upon the method of ACE2 expression. PMID:15731278

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

PubMed

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

2015-08-01

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

Recombinant Truncated Nucleocapsid Protein as Antigen in a Novel Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Severe Acute Respiratory Syndrome Coronavirus Infection▿

PubMed Central

Yu, Fuxun; Le, Mai Quynh; Inoue, Shingo; Hasebe, Futoshi; Parquet, Maria del Carmen; Morikawa, Shigeru; Morita, Kouichi

2007-01-01

We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection. PMID:17202310

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

PubMed

Roh, Changhyun

2012-01-01

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

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

PubMed Central

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

2017-01-01

Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1–58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2, an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2−/− mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses. PMID:28592648

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

PubMed

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.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu Yanning; Beijing Center for Diseases Prevention and Control, 16 Hepingli Middle Street, Dongcheng District, Beijing 100013; Neo, T.L.

SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalaninemore » substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.« less

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines.

PubMed

See, Raymond H; Petric, Martin; Lawrence, David J; Mok, Catherine P Y; Rowe, Thomas; Zitzow, Lois A; Karunakaran, Karuna P; Voss, Thomas G; Brunham, Robert C; Gauldie, Jack; Finlay, B Brett; Roper, Rachel L

2008-09-01

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDermott, Jason E.; Mitchell, Hugh D.; Gralinski, Lisa E.

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 identifymore » 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).« less

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

PubMed

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

2014-09-15

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

Severe acute respiratory syndrome coronavirus replication inhibitor that interferes with the nucleic acid unwinding of the viral helicase.

PubMed

Adedeji, Adeyemi O; Singh, Kamalendra; Calcaterra, Nicholas E; DeDiego, Marta L; Enjuanes, Luis; Weiss, Susan; Sarafianos, Stefan G

2012-09-01

Severe acute respiratory syndrome (SARS) is a highly contagious disease, caused by SARS coronavirus (SARS-CoV), for which there are no approved treatments. We report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). We used a Förster resonance energy transfer (FRET)-based helicase assay to screen the Maybridge Hitfinder chemical library. We identified and validated a compound (SSYA10-001) that specifically blocks the double-stranded RNA (dsRNA) and dsDNA unwinding activities of nsp13, with 50% inhibitory concentrations (IC(50)s) of 5.70 and 5.30 μM, respectively. This compound also has inhibitory activity (50% effective concentration [EC(50)] = 8.95 μM) in a SARS-CoV replicon assay, with low cytotoxicity (50% cytotoxic concentration [CC(50)] = >250 μM), suggesting that the helicase plays a still unidentified critical role in the SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a noncompetitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. Moreover, SSYA10-001 does not affect ATP hydrolysis or nsp13 binding to the nucleic acid substrate. SSYA10-001 did not inhibit hepatitis C virus (HCV) helicase, other bacterial and viral RNA-dependent RNA polymerases, or reverse transcriptase. These results suggest that SSYA10-001 specifically blocks nsp13 through a novel mechanism and is less likely to interfere with the functions of cellular enzymes that process nucleic acids or ATP. Hence, it is possible that SSYA10-001 inhibits unwinding by nsp13 by affecting conformational changes during the course of the reaction or translocation on the nucleic acid. SSYA10-001 will be a valuable tool for studying the specific role of nsp13 in the SARS-CoV life cycle, which could be a model for other nidoviruses and also a candidate for further development as a SARS antiviral target.

SARS Surveillance during Emergency Public Health Response, United States, March–July 2003

PubMed Central

Brooks, John T.; Van Beneden, Chris; Parashar, Umesh D.; Griffin, Patricia M.; Anderson, Larry J.; Bellini, William J.; Benson, Robert F.; Erdman, Dean D.; Klimov, Alexander; Ksiazek, Thomas G.; Peret, Teresa C.T.; Talkington, Deborah F.; Thacker, W. Lanier; Tondella, Maria L.; Sampson, Jacquelyn S.; Hightower, Allen W.; Nordenberg, Dale F.; Plikaytis, Brian D.; Khan, Ali S.; Rosenstein, Nancy E.; Treadwell, Tracee A.; Whitney, Cynthia G.; Fiore, Anthony E.; Durant, Tonji M.; Perz, Joseph F.; Wasley, Annemarie; Feikin, Daniel; Herndon, Joy L.; Bower, William A.; Kilbourn, Barbara W.; Levy, Deborah A.; Coronado, Victor G.; Buffington, Joanna; Dykewicz, Clare A.; Khabbaz, Rima F.; Chamberland, Mary E.

2004-01-01

In response to the emergence of severe acute respiratory syndrome (SARS), the United States established national surveillance using a sensitive case definition incorporating clinical, epidemiologic, and laboratory criteria. Of 1,460 unexplained respiratory illnesses reported by state and local health departments to the Centers for Disease Control and Prevention from March 17 to July 30, 2003, a total of 398 (27%) met clinical and epidemiologic SARS case criteria. Of these, 72 (18%) were probable cases with radiographic evidence of pneumonia. Eight (2%) were laboratory-confirmed SARS-coronavirus (SARS-CoV) infections, 206 (52%) were SARS-CoV negative, and 184 (46%) had undetermined SARS-CoV status because of missing convalescent-phase serum specimens. Thirty-one percent (124/398) of case-patients were hospitalized; none died. Travel was the most common epidemiologic link (329/398, 83%), and mainland China was the affected area most commonly visited. One case of possible household transmission was reported, and no laboratory-confirmed infections occurred among healthcare workers. Successes and limitations of this emergency surveillance can guide preparations for future outbreaks of SARS or respiratory diseases of unknown etiology. PMID:15030681

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

PubMed

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

2012-08-01

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

Severe acute respiratory syndrome (SARS) S protein production in plants: Development of recombinant vaccine

PubMed Central

Pogrebnyak, Natalia; Golovkin, Maxim; Andrianov, Vyacheslav; Spitsin, Sergei; Smirnov, Yuriy; Egolf, Richard; Koprowski, Hilary

2005-01-01

In view of a recent spread of severe acute respiratory syndrome (SARS), there is a high demand for production of a vaccine to prevent this disease. Recent studies indicate that SARS-coronavirus (CoV) spike protein (S protein) and its truncated fragments are considered the best candidates for generation of the recombinant vaccine. Toward the development of a safe, effective, and inexpensive vaccine candidate, we have expressed the N-terminal fragment of SARS-CoV S protein (S1) in tomato and low-nicotine tobacco plants. Incorporation of the S1 fragment into plant genomes as well as its transcription was confirmed by PCR and RT-PCR analyses. High levels of expression of recombinant S1 protein were observed in several transgenic lines by Western blot analysis using specific antibodies. Plant-derived antigen was evaluated to induce the systemic and mucosal immune responses in mice. Mice showed significantly increased levels of SARS-CoV-specific IgA after oral ingestion of tomato fruits expressing S1 protein. Sera of mice parenterally primed with tobacco-derived S1 protein revealed the presence of SARS-CoV-specific IgG as detected by Western blot and ELISA analysis. PMID:15956182

Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine.

PubMed

He, Yuxian; Li, Jingjing; Du, Lanying; Yan, Xuxia; Hu, Guangan; Zhou, Yusen; Jiang, Shibo

2006-06-29

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is considered as a major antigen for vaccine design. We previously demonstrated that the receptor-binding domain (RBD: residues 318-510) of S protein contains multiple conformation-dependent neutralizing epitopes (Conf I to VI) and serves as a major target of SARS-CoV neutralization. Here, we further characterized the antigenic structure in the RBD by a panel of novel mAbs isolated from the mice immunized with an inactivated SARS-CoV vaccine. Ten of the RBD-specific mAbs were mapped to four distinct groups of conformational epitopes (designated Group A to D), and all of which had potent neutralizing activity against S protein-pseudotyped SARS viruses. Group A, B, C mAbs target the epitopes that may overlap with the previously characterized Conf I, III, and VI respectively, but they display different capacity to block the receptor binding. Group D mAb (S25) was directed against a unique epitope by its competitive binding. Two anti-RBD mAbs recognizing the linear epitopes (Group E) were mapped to the RBD residues 335-352 and 442-458, respectively, and none of them inhibited the receptor binding and virus entry. Surprisingly, most neutralizing epitopes (Groups A to C) could be completely disrupted by single amino acid substitutions (e.g., D429A, R441A or D454A) or by deletions of several amino acids at the N-terminal or C-terminal region of the RBD; however, the Group D epitope was not sensitive to the mutations, highlighting its importance for vaccine development. These data provide important information for understanding the antigenicity and immunogenicity of SARS-CoV, and this panel of novel mAbs can be used as tools for studying the structure of S protein and for guiding SARS vaccine design.

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

NASA Astrophysics Data System (ADS)

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.

Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model.

PubMed

Lu, Jia-hai; Zhang, Ding-mei; Wang, Guo-ling; Guo, Zhong-min; Zhang, Chuan-hai; Tan, Bing-yan; Ouyang, Li-ping; Lin, Li; Liu, Yi-min; Chen, Wei-qing; Ling, Wen-hua; Yu, Xin-bing; Zhong, Nan-shan

2005-05-05

The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein. The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL(pro).

SARS-CoV related Betacoronavirus and diverse Alphacoronavirus members found in western old-world.

PubMed

Ar Gouilh, Meriadeg; Puechmaille, Sébastien J; Diancourt, Laure; Vandenbogaert, Mathias; Serra-Cobo, Jordi; Lopez Roïg, Marc; Brown, Paul; Moutou, François; Caro, Valérie; Vabret, Astrid; Manuguerra, Jean-Claude

2018-04-01

The emergence of SARS-CoV and MERS-CoV, triggered the discovery of a high diversity of coronaviruses in bats. Studies from Europe have shown that coronaviruses circulate in bats in France but this reflects only a fraction of the whole diversity. In the current study the diversity of coronaviruses circulating in western Europe was extensively explored. Ten alphacoronaviruses in eleven bat species belonging to the Miniopteridae, Vespertilionidae and Rhinolophidae families and, a SARS-CoV-related Betacoronavirus in Rhinolophus ferrumequinum were identified. The diversity and prevalence of bat coronaviruses presently reported from western Europe is much higher than previously described and includes a SARS-CoV sister group. This diversity demonstrates the dynamic evolution and circulation of coronaviruses in this species. That said, the identified coronaviruses were consistently associated with a particular bat species or genus, and these relationships were maintained no matter the geographic location. The observed phylogenetic grouping of coronaviruses from the same species in Europe and Asia, emphasizes the role of host/pathogen coevolution in this group. Copyright © 2018 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akerstroem, Sara; Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 17177 Solna; Gunalan, Vithiagaran

Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we havemore » further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Kailang; Li, Weikai; Peng, Guiqing

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 homologymore » 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.« less

Structural model of the SARS coronavirus E channel in LMPG micelles.

PubMed

Surya, Wahyu; Li, Yan; Torres, Jaume

2018-06-01

Coronaviruses (CoV) cause common colds in humans, but are also responsible for the recent Severe Acute, and Middle East, respiratory syndromes (SARS and MERS, respectively). A promising approach for prevention are live attenuated vaccines (LAVs), some of which target the envelope (E) protein, which is a small membrane protein that forms ion channels. Unfortunately, detailed structural information is still limited for SARS-CoV E, and non-existent for other CoV E proteins. Herein, we report a structural model of a SARS-CoV E construct in LMPG micelles with, for the first time, unequivocal intermolecular NOEs. The model corresponding to the detergent-embedded region is consistent with previously obtained orientational restraints obtained in lipid bilayers and in vivo escape mutants. The C-terminal domain is mostly α-helical, and extramembrane intermolecular NOEs suggest interactions that may affect the TM channel conformation. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

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

2017-06-07

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

Vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease

PubMed Central

Enjuanes, Luis; DeDiego, Marta L.; Álvarez, Enrique; Deming, Damon; Sheahan, Tim; Baric, Ralph

2009-01-01

An important effort has been performed after the emergence of severe acute respiratory syndrome (SARS) epidemic in 2003 to diagnose and prevent virus spreading. Several types of vaccines have been developed including inactivated viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous expression systems, and vaccines derived from SARS-CoV genome by reverse genetics. This review describes several aspects essential to develop SARS-CoV vaccines, such as the correlates of protection, virus serotypes, vaccination side effects, and bio-safeguards that can be engineered into recombinant vaccine approaches based on the SARS-CoV genome. The production of effective and safe vaccines to prevent SARS has led to the development of promising vaccine candidates, in contrast to the design of vaccines for other coronaviruses, that in general has been less successful. After preclinical trials in animal models, efficacy and safety evaluation of the most promising vaccine candidates described has to be performed in humans. PMID:17416434

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

PubMed

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

2014-10-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Barrila, J.; Gabelli, S; Bacha, U

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 ofmore » 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}.« less

Primary Severe Acute Respiratory Syndrome Coronavirus Infection Limits Replication but Not Lung Inflammation upon Homologous Rechallenge

PubMed Central

Clay, Candice; Donart, Nathan; Fomukong, Ndingsa; Knight, Jennifer B.; Lei, Wanli; Price, Lance; Hahn, Fletcher; Van Westrienen, Jesse

2012-01-01

Our knowledge regarding immune-protective and immunopathogenic events in severe acute respiratory syndrome coronavirus (SARS-CoV) infection is limited, and little is known about the dynamics of the immune response at the primary site of disease. Here, an African green monkey (AGM) model was used to elucidate immune mechanisms that facilitate viral clearance but may also contribute to persistent lung inflammation following SARS-CoV infection. During primary infection, SARS-CoV replicated in the AGM lung for up to 10 days. Interestingly, lung inflammation was more prevalent following viral clearance, as leukocyte numbers peaked at 14 days postinfection (dpi) and remained elevated at 28 dpi compared to those of mock-infected controls. Lung macrophages but not dendritic cells were rapidly activated, and both cell types had high activation marker expression at late infection time points. Lung proinflammatory cytokines were induced at 1 to 14 dpi, but most returned to baseline by 28 dpi except interleukin 12 (IL-12) and gamma interferon. In SARS-CoV homologous rechallenge studies, 11 of the 12 animals were free of replicating virus at day 5 after rechallenge. However, incidence and severity of lung inflammation was not reduced despite the limited viral replication upon rechallenge. Evaluating the role of antibodies in immune protection or potentiation revealed a progressive increase in anti-SARS-CoV antibodies in lung and serum that did not correlate temporally or spatially with enhanced viral replication. This study represents one of the first comprehensive analyses of lung immunity, including changes in leukocyte populations, lung-specific cytokines, and antibody responses following SARS-CoV rechallenge in AGMs. PMID:22345460

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

PubMed

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

2015-10-01

Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission. Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats (Rhinolophus sinicus) exhibited <40% amino acid identities to human/civet SARS-CoV in the ORF8 protein. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, China, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed ORF8 proteins with exceptionally high amino acid identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 protein, which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

PubMed Central

Tan, Jinzhi; Vonrhein, Clemens; Smart, Oliver S.; Bricogne, Gerard; Bollati, Michela; Kusov, Yuri; Hansen, Guido; Mesters, Jeroen R.; Schmidt, Christian L.; Hilgenfeld, Rolf

2009-01-01

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 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore 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 SUDcore 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 involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed. PMID:19436709

Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate.

PubMed

Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

2014-01-01

Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318-510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318-536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development.

Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate

PubMed Central

Chen, Wen-Hsiang; Du, Lanying; Chag, Shivali M; Ma, Cuiqing; Tricoche, Nancy; Tao, Xinrong; Seid, Christopher A; Hudspeth, Elissa M; Lustigman, Sara; Tseng, Chien-Te K; Bottazzi, Maria Elena; Hotez, Peter J; Zhan, Bin; Jiang, Shibo

2014-01-01

Development of vaccines for preventing a future pandemic of severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV) and for biodefense preparedness is urgently needed. Our previous studies have shown that a candidate SARS vaccine antigen consisting of the receptor-binding domain (RBD) of SARS-CoV spike protein can induce potent neutralizing antibody responses and protection against SARS-CoV challenge in vaccinated animals. To optimize expression conditions for scale-up production of the RBD vaccine candidate, we hypothesized that this could be potentially achieved by removing glycosylation sites in the RBD protein. In this study, we constructed two RBD protein variants: 1) RBD193-WT (193-aa, residues 318–510) and its deglycosylated forms (RBD193-N1, RBD193-N2, RBD193-N3); 2) RBD219-WT (219-aa, residues 318–536) and its deglycosylated forms (RBD219-N1, RBD219-N2, and RBD219-N3). All constructs were expressed as recombinant proteins in yeast. The purified recombinant proteins of these constructs were compared for their antigenicity, functionality and immunogenicity in mice using alum as the adjuvant. We found that RBD219-N1 exhibited high expression yield, and maintained its antigenicity and functionality. More importantly, RBD219-N1 induced significantly stronger RBD-specific antibody responses and a higher level of neutralizing antibodies in immunized mice than RBD193-WT, RBD193-N1, RBD193-N3, or RBD219-WT. These results suggest that RBD219-N1 could be selected as an optimal SARS vaccine candidate for further development. PMID:24355931

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Kailang; Peng, Guiqing; Wilken, Matthew

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

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus*

PubMed Central

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

2012-01-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals. PMID:22291007

SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity.

PubMed

Muramatsu, Tomonari; Takemoto, Chie; Kim, Yong-Tae; Wang, Hongfei; Nishii, Wataru; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki

2016-11-15

The 3C-like protease (3CL pro ) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4-P1 and P1'. In this study, we determined the crystal structure of 3CL pro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3' position [Phe(P3')] is snugly accommodated in the S3' pocket. Mutations of Phe(P3') impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3' subsite is formed by Phe(P2)-induced conformational changes of 3CL pro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3' subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3'). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3') is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).

DOE Office of Scientific and Technical Information (OSTI.GOV)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R., E-mail: grw7@cornell.ed

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 withmore » 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.« less

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses

PubMed Central

Sheahan, Timothy P.; Sims, Amy C.; Graham, Rachel L.; Menachery, Vineet D.; Gralinski, Lisa E.; Case, James B.; Leist, Sarah R.; Pyrc, Krzysztof; Feng, Joy Y.; Trantcheva, Iva; Bannister, Roy; Park, Yeojin; Babusis, Darius; Clarke, Michael O.; Mackman, Richard L.; Spahn, Jamie E.; Palmiotti, Christopher A.; Siegel, Dustin; Ray, Adrian S.; Cihlar, Tomas; Jordan, Robert; Denison, Mark R.; Baric, Ralph S.

2017-01-01

Emerging viral infections are difficult to control as heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. SARS-CoV and MERS-CoV successively emerged causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. Here we show that a nucleotide prodrug GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat-CoVs, prepandemic bat-CoVs and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory functions. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and possibly most importantly, emerging CoV of the future. PMID:28659436

Mechanisms of Severe Acute Respiratory Syndrome Coronavirus-Induced Acute Lung Injury

PubMed Central

Gralinski, Lisa E.; Bankhead, Armand; Jeng, Sophia; Menachery, Vineet D.; Proll, Sean; Belisle, Sarah E.; Matzke, Melissa; Webb-Robertson, Bobbie-Jo M.; Luna, Maria L.; Shukla, Anil K.; Ferris, Martin T.; Bolles, Meagan; Chang, Jean; Aicher, Lauri; Waters, Katrina M.; Smith, Richard D.; Metz, Thomas O.; Law, G. Lynn; Katze, Michael G.; McWeeney, Shannon; Baric, Ralph S.

2013-01-01

ABSTRACT Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV. PMID:23919993

Factors associated with nosocomial SARS-CoV transmission among healthcare workers in Hanoi, Vietnam, 2003

PubMed Central

Reynolds, Mary G; Anh, Bach Huy; Thu, Vu Hoang; Montgomery, Joel M; Bausch, Daniel G; Shah, J Jina; Maloney, Susan; Leitmeyer, Katrin C; Huy, Vu Quang; Horby, Peter; Plant, Aileen Y; Uyeki, Timothy M

2006-01-01

Background In March of 2003, an outbreak of Severe Acute Respiratory Syndrome (SARS) occurred in Northern Vietnam. This outbreak began when a traveler arriving from Hong Kong sought medical care at a small hospital (Hospital A) in Hanoi, initiating a serious and substantial transmission event within the hospital, and subsequent limited spread within the community. Methods We surveyed Hospital A personnel for exposure to the index patient and for symptoms of disease during the outbreak. Additionally, serum specimens were collected and assayed for antibody to SARS-associated coronavirus (SARS-CoV) antibody and job-specific attack rates were calculated. A nested case-control analysis was performed to assess risk factors for acquiring SARS-CoV infection. Results One hundred and fifty-three of 193 (79.3%) clinical and non-clinical staff consented to participate. Excluding job categories with <3 workers, the highest SARS attack rates occurred among nurses who worked in the outpatient and inpatient general wards (57.1, 47.4%, respectively). Nurses assigned to the operating room/intensive care unit, experienced the lowest attack rates (7.1%) among all clinical staff. Serologic evidence of SARS-CoV infection was detected in 4 individuals, including 2 non-clinical workers, who had not previously been identified as SARS cases; none reported having had fever or cough. Entering the index patient's room and having seen (viewed) the patient were the behaviors associated with highest risk for infection by univariate analysis (odds ratios 20.0, 14.0; 95% confidence intervals 4.1–97.1, 3.6–55.3, respectively). Conclusion This study highlights job categories and activities associated with increased risk for SARS-CoV infection and demonstrates that a broad diversity of hospital workers may be vulnerable during an outbreak. These findings may help guide recommendations for the protection of vulnerable occupational groups and may have implications for other respiratory infections such as influenza. PMID:16907978

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

NASA Astrophysics Data System (ADS)

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

2007-12-01

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

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

PubMed Central

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

2015-01-01

ABSTRACT Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission. IMPORTANCE Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats (Rhinolophus sinicus) exhibited <40% amino acid identities to human/civet SARS-CoV in the ORF8 protein. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, China, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed ORF8 proteins with exceptionally high amino acid identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 protein, which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination. PMID:26269185

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

PubMed

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

2016-09-23

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

Lack of SARS transmission and U.S. SARS case-patient.

PubMed

Peck, Angela J; Newbern, E Claire; Feikin, Daniel R; Issakbaeva, Elmira T; Park, Benjamin J; Fehr, Jason; LaMonte, Ashley C; Le, Thong P; Burger, Terry L; Rhodes, Luther V; Weltman, Andre; Erdman, Dean; Ksiazek, Thomas G; Lingappa, Jairam R

2004-02-01

In early April 2003, severe acute respiratory syndrome (SARS) was diagnosed in a Pennsylvania resident after his exposure to persons with SARS in Toronto, Canada. To identify contacts of the case-patient and evaluate the risk for SARS transmission, a detailed epidemiologic investigation was performed. On the basis of this investigation, 26 persons (17 healthcare workers, 4 household contacts, and 5 others) were identified as having had close contact with this case-patient before infection-control practices were implemented. Laboratory evaluation of clinical specimens showed no evidence of transmission of SARS-associated coronavirus (SARS-CoV) infection to any close contact of this patient. This investigation documents that, under certain circumstances, SARS-CoV is not readily transmitted to close contacts, despite ample unprotected exposures. Improving the understanding of risk factors for transmission will help focus public health control measures.

MERS, SARS and other coronaviruses as causes of pneumonia.

PubMed

Yin, Yudong; Wunderink, Richard G

2018-02-01

Human coronaviruses (HCoVs) have been considered to be relatively harmless respiratory pathogens in the past. However, after the outbreak of the severe acute respiratory syndrome (SARS) and emergence of the Middle East respiratory syndrome (MERS), HCoVs have received worldwide attention as important pathogens in respiratory tract infection. This review focuses on the epidemiology, pathogenesis and clinical characteristics among SARS-coronaviruses (CoV), MERS-CoV and other HCoV infections. © 2017 Asian Pacific Society of Respirology.

Airway Memory CD4(+) T Cells Mediate Protective Immunity against Emerging Respiratory Coronaviruses.

PubMed

Zhao, Jincun; Zhao, Jingxian; Mangalam, Ashutosh K; Channappanavar, Rudragouda; Fett, Craig; Meyerholz, David K; Agnihothram, Sudhakar; Baric, Ralph S; David, Chella S; Perlman, Stanley

2016-06-21

Two zoonotic coronaviruses (CoVs)-SARS-CoV and MERS-CoV-have crossed species to cause severe human respiratory disease. Here, we showed that induction of airway memory CD4(+) T cells specific for a conserved epitope shared by SARS-CoV and MERS-CoV is a potential strategy for developing pan-coronavirus vaccines. Airway memory CD4(+) T cells differed phenotypically and functionally from lung-derived cells and were crucial for protection against both CoVs in mice. Protection was dependent on interferon-γ and required early induction of robust innate and virus-specific CD8(+) T cell responses. The conserved epitope was also recognized in SARS-CoV- and MERS-CoV-infected human leukocyte antigen DR2 and DR3 transgenic mice, indicating potential relevance in human populations. Additionally, this epitope was cross-protective between human and bat CoVs, the progenitors for many human CoVs. Vaccine strategies that induce airway memory CD4(+) T cells targeting conserved epitopes might have broad applicability in the context of new CoVs and other respiratory virus outbreaks. Copyright © 2016 Elsevier Inc. All rights reserved.

SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism.

PubMed

Frieman, Matthew B; Chen, Jun; Morrison, Thomas E; Whitmore, Alan; Funkhouser, William; Ward, Jerrold M; Lamirande, Elaine W; Roberts, Anjeanette; Heise, Mark; Subbarao, Kanta; Baric, Ralph S

2010-04-08

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection often caused severe end stage lung disease and organizing phase diffuse alveolar damage, especially in the elderly. The virus-host interactions that governed development of these acute end stage lung diseases and death are unknown. To address this question, we evaluated the role of innate immune signaling in protection from human (Urbani) and a recombinant mouse adapted SARS-CoV, designated rMA15. In contrast to most models of viral pathogenesis, infection of type I, type II or type III interferon knockout mice (129 background) with either Urbani or MA15 viruses resulted in clinical disease outcomes, including transient weight loss, denuding bronchiolitis and alveolar inflammation and recovery, identical to that seen in infection of wildtype mice. This suggests that type I, II and III interferon signaling play minor roles in regulating SARS pathogenesis in mouse models. In contrast, infection of STAT1-/- mice resulted in severe disease, high virus titer, extensive pulmonary lesions and 100% mortality by day 9 and 30 post-infection with rMA15 or Urbani viruses, respectively. Non-lethal in BALB/c mice, Urbani SARS-CoV infection in STAT1-/- mice caused disseminated infection involving the liver, spleen and other tissues after day 9. These findings demonstrated that SARS-CoV pathogenesis is regulated by a STAT1 dependent but type I, II and III interferon receptor independent, mechanism. In contrast to a well documented role in innate immunity, we propose that STAT1 also protects mice via its role as an antagonist of unrestrained cell proliferation.

Accessory proteins of SARS-CoV and other coronaviruses.

PubMed

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

2014-09-01

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

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

PubMed

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

2015-04-03

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

Cynomolgus Macaque as an Animal Model for Severe Acute Respiratory Syndrome

DTIC Science & Technology

2006-05-01

two viruses . Both icSARS-CoV-challenged animals devel- oped radiographic disease. Of the two animals infected with wild-type virus by a mucosal route...J Virol 78: 11416–11421. 12. Martina BE, Haagmans BL, Kuiken T, Fouchier RA, Rimmelzwaan GF, et al. (2003) Virology: SARS virus infection of cats and...was discovered quite quickly—a new virus known as SARS-associated coronavirus (SARS-CoV) ( corona being a description of the virus’s spiky, crown-like

SARS Basics

MedlinePlus

... Laboratory Testing & Specimens SARS-Associated Coronavirus (SARS-CoV) Sequencing Specimen Collection and Management Laboratory Testing and Diagnosis ... person touches a surface or object contaminated with infectious droplets and then touches his or her mouth, ...

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Hua; Jiang Lifang; Fang Danyun

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 chosenmore » 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.« less

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

PubMed Central

McBride, Ruth; Fielding, Burtram C.

2012-01-01

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

Infectivity of severe acute respiratory syndrome during its incubation period.

PubMed

Zeng, Guang; Xie, Shu-Yun; Li, Qin; Ou, Jian-Ming

2009-12-01

To evaluate the infectivity of severe acute respiratory syndrome (SARS) during its incubation period by investigating chains of transmission and individuals isolated for medical observation with a view to providing scientific evidence for updating protocols of medical isolation. Individuals related with the two SARS chains of transmission in Beijing in 2003 and a group of individuals isolated for medical observation in Haidian district of Beijing during the SARS outbreak were selected as subjects of study. Contactors with SARS patients and those with symptom development following the contacts were investigated via questionnaire. Serum samples were collected from super transmitters and tested for SARS-CoV antibody by neutralization test and enzyme linked immunosorbent assay (ELISA). A total of 1112 contactors were investigated in three surveys. Of them, 669 had a history of close contact with symptomatic SARS patients, 101 developed symptoms with a rate of 15.1%, 363 had a history of close contact with patients in their incubation period, none of whom developed symptoms (0%). Serum samples were collected from 32 highly-exposed individuals, of whom 13 developing SARS symptoms after contact had serum samples positive for SARS-CoV antibody. Samples collected from the asymptomatic contactors were all negative for SARS-CoV antibody. SARS cases are infectious only during their symptomatic period and are non-infectious during the incubation period. Isolation for medical observation should be placed for individuals who are in close contact with symptomatic SARS patients. The results of our study are of decisive significance for the Ministry of Health to the definition of SARS close contactor.

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

PubMed

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

2016-03-01

Viruses with pandemic potential including H1N1, H5N1, and H5N7 influenza viruses, and severe acute respiratory syndrome (SARS)/Middle East respiratory syndrome (MERS) coronaviruses (CoV) have emerged in recent years. SARS-CoV, MERS-CoV, and influenza virus can survive on surfaces for extended periods, sometimes up to months. Factors influencing the survival of these viruses on surfaces include: strain variation, titre, surface type, suspending medium, mode of deposition, temperature and relative humidity, and the method used to determine the viability of the virus. Environmental sampling has identified contamination in field-settings with SARS-CoV and influenza virus, although the frequent use of molecular detection methods may not necessarily represent the presence of viable virus. The importance of indirect contact transmission (involving contamination of inanimate surfaces) is uncertain compared with other transmission routes, principally direct contact transmission (independent of surface contamination), droplet, and airborne routes. However, influenza virus and SARS-CoV may be shed into the environment and be transferred from environmental surfaces to hands of patients and healthcare providers. Emerging data suggest that MERS-CoV also shares these properties. Once contaminated from the environment, hands can then initiate self-inoculation of mucous membranes of the nose, eyes or mouth. Mathematical and animal models, and intervention studies suggest that contact transmission is the most important route in some scenarios. Infection prevention and control implications include the need for hand hygiene and personal protective equipment to minimize self-contamination and to protect against inoculation of mucosal surfaces and the respiratory tract, and enhanced surface cleaning and disinfection in healthcare settings. Copyright © 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine

PubMed Central

Nieto-Torres, Jose L.; DeDiego, Marta L.; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Perlman, Stanley; Enjuanes, Luis

2015-01-01

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV. PMID:26513244

Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species.

PubMed

Yu, Meng; Stevens, Vicky; Berry, Jody D; Crameri, Gary; McEachern, Jennifer; Tu, Changchun; Shi, Zhengli; Liang, Guodong; Weingartl, Hana; Cardosa, Jane; Eaton, Bryan T; Wang, Lin-Fa

2008-02-29

Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402-622) and the N4 fragment (aa 220-336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS-CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS-CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS-CoV, these assays will be a useful tool to trace the origin and transmission of SARS-CoV and to minimise the risk of animal-to-human transmission.

The antiviral action of common household disinfectants and antiseptics against murine hepatitis virus, a potential surrogate for SARS coronavirus.

PubMed

Dellanno, Christine; Vega, Quinn; Boesenberg, Diane

2009-10-01

The 2003 outbreak of severe acute respiratory syndrome (SARS) infected over 8000 people and killed 774. Transmission of SARS occurred through direct and indirect contact and large droplet nuclei. The World Health Organization recommended the use of household disinfectants, which have not been previously tested against SARS coronavirus (SARS-CoV), to disinfect potentially contaminated environmental surfaces. There is a need for a surrogate test system given the limited availability of the SARS-CoV for testing and biosafety requirements necessary to safely handle it. In this study, the antiviral activity of standard household products was assayed against murine hepatitis virus (MHV), as a potential surrogate for SARS-CoV. A surface test method, which involves drying an amount of virus on a surface and then applying the product for a specific contact time, was used to determine the virucidal activity. The virus titers and log reductions were determined by the Reed and Muench tissue culture infective dose (TCID)50 end point method. When tested as directed, common household disinfectants or antiseptics, containing either 0.050% of triclosan, 0.12% of PCMX, 0.21% of sodium hypochlorite, 0.23% of pine oil, or 0.10% of a quaternary compound with 79% of ethanol, demonstrated a 3-log reduction or better against MHV without any virus recovered in a 30-second contact time. Common household disinfectants and antiseptics were effective at inactivating MHV, a possible surrogate for SARS-CoV, from surfaces when used as directed. In an outbreak caused by novel agents, it is important to know the effectiveness of disinfectants and antiseptics to prevent or reduce the possibility of human-to-human transmission via surfaces.

Anti–SARS-CoV Immunoglobulin G in Healthcare Workers, Guangzhou, China

PubMed Central

Lu, Ci-Yong; Wong, Tze-Wai; Ling, Wen-Hua; Lin, Zhong-Ning; Hao, Yuan-Tao; Liu, Qing; Fang, Ji-Qian; He, Yun; Luo, Fu-Tian; Jing, Jin; Ling, Li; Ma, Xiang; Liu, Yi-Min; Chen, Gui-Hua; Huang, Jian; Jiang, Yuan-Sen; Jiang, Wen-Qi; Zou, He-Qun; Yan, Guang-Mei

2005-01-01

To determine the prevalence of inapparent infection with severe acute respiratory syndrome (SARS) among healthcare workers, we performed a serosurvey to test for immunoglobulin (Ig) G antibodies to the SARS coronavirus (SARS-CoV) among 1,147 healthcare workers in 3 hospitals that admitted SARS patients in mid-May 2003. Among them were 90 healthcare workers with SARS. As a reference group, 709 healthcare workers who worked in 2 hospitals that never admitted any SARS patients were similarly tested. The seroprevalence rate was 88.9% (80/90) for healthcare workers with SARS and 1.4% (15/1,057) for healthcare workers who were apparently healthy. The seroprevalence in the reference group was 0.4% (3/709). These findings suggest that inapparent infection is uncommon. Low level of immunity among unaffected healthcare workers reinforces the need for adequate personal protection and other infection control measures in hospitals to prevent future epidemics. PMID:15705328

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

PubMed

Kindler, E; Thiel, V; Weber, F

2016-01-01

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

Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

PubMed

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

2015-10-01

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.

Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-kappaB activation.

PubMed

Kanzawa, Noriyuki; Nishigaki, Kazuo; Hayashi, Takaya; Ishii, Yuichi; Furukawa, Souichi; Niiro, Ayako; Yasui, Fumihiko; Kohara, Michinori; Morita, Kouichi; Matsushima, Kouji; Le, Mai Quynh; Masuda, Takao; Kannagi, Mari

2006-12-22

Severe acute respiratory syndrome (SARS) is characterized by rapidly progressing respiratory failure resembling acute/adult respiratory distress syndrome (ARDS) associated with uncontrolled inflammatory responses. Here, we demonstrated that, among five accessory proteins of SARS coronavirus (SARS-CoV) tested, 3a/X1 and 7a/X4 were capable of activating nuclear factor kappa B (NF-kappaB) and c-Jun N-terminal kinase (JNK), and significantly enhanced interleukin 8 (IL-8) promoter activity. Furthermore, 3a/X1 and 7a/X4 expression in A549 cells enhanced production of inflammatory chemokines that were known to be up-regulated in SARS-CoV infection. Our results suggest potential involvement of 3a/X1 and 7a/X4 proteins in the pathological inflammatory responses in SARS.

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

PubMed Central

Huang, I-Chueh; Bailey, Charles C.; Weyer, Jessica L.; Radoshitzky, Sheli R.; Becker, Michelle M.; Chiang, Jessica J.; Brass, Abraham L.; Ahmed, Asim A.; Chi, Xiaoli; Dong, Lian; Longobardi, Lindsay E.; Boltz, Dutch; Kuhn, Jens H.; Elledge, Stephen J.; Bavari, Sina; Denison, Mark R.; Choe, Hyeryun; Farzan, Michael

2011-01-01

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 (GP1,2) 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. PMID:21253575

High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

DTIC Science & Technology

2010-06-01

viruses . N-glycosylation of viral envelopes is an important such target shared between in- fluenza, HIV, HCV, West Nile virus , SARS-CoV, Hendra virus ...host cells. Therefore, MBL preferentially recognizes glycosylated viruses including influenza virus , human immunodeficiency virus , severe acute...respiratory syndrome coronovirus (SARS-CoV), Ebola virus , and Marburg virus . It also recognizes many glycosylated gram- positive and gram-negative bacteria [1

About Coronavirus

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Severe Acute Respiratory Syndrome Coronaviruses with Mutations in the E Protein Are Attenuated and Promising Vaccine Candidates

PubMed Central

Regla-Nava, Jose A.; Nieto-Torres, Jose L.; Jimenez-Guardeño, Jose M.; Fernandez-Delgado, Raul; Fett, Craig; Castaño-Rodríguez, Carlos; Perlman, Stanley; DeDiego, Marta L.

2015-01-01

ABSTRACT Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouse-adapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoV-MA15-E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4+ and CD8+ T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates. IMPORTANCE Human coronaviruses are important zoonotic pathogens. SARS-CoV caused a worldwide epidemic infecting more than 8,000 people with a mortality of around 10%. Therefore, understanding the virulence mechanisms of this pathogen and developing efficacious vaccines are of high importance to prevent epidemics from this and other human coronaviruses. Previously, we demonstrated that a SARS-CoV lacking the E protein was attenuated in vivo. Here, we show that small deletions and modifications within the E protein led to virus attenuation, manifested by minimal lung injury, limited neutrophil influx to the lungs, reduced expression of proinflammatory cytokines, increased anti-inflammatory cytokine levels, and enhanced CD4+ and CD8+ T cell counts in vivo, suggesting that these phenomena contribute to virus attenuation. The attenuated mutants fully protected mice from challenge with virulent virus. These studies show that mutations in the E protein are not well tolerated and indicate that this protein is an excellent target for vaccine development. PMID:25609816

Quarantine for SARS, Taiwan

PubMed Central

King, Chwan-Chuan; Chen, Cathy W. S.; Ho, Mei-Shang; Lee, Jen-Yu; Liu, Feng-Chi; Wu, Yi-Chun

2005-01-01

During the 2003 outbreak of severe acute respiratory syndrome (SARS) in Taiwan, >150,000 persons were quarantined, 24 of whom were later found to have laboratory-confirmed SARS-coronavirus (SARS-CoV) infection. Since no evidence exists that SARS-CoV is infective before the onset of symptoms and the quarantined persons were exposed but not symptomatic, we thought the quarantine's effectiveness should be investigated. Using the Taiwan quarantine data, we found that the onset-to-diagnosis time of previously quarantined confirmed case-patients was significantly shortened compared to that for those who had not been quarantined. Thus, quarantine for SARS in Taiwan screened potentially infective persons for swift diagnosis and hospitalization after onset, thereby indirectly reducing infections. Full-scale quarantine measures implemented on April 28 led to a significant improvement in onset-to-diagnosis time of all SARS patients, regardless of previous quarantine status. We discuss the temporal effects of quarantine measures and other interventions on detection and isolation as well as the potential usefulness of quarantine in faster identification of persons with SARS and in improving isolation measures. PMID:15752447

Middle East respiratory syndrome coronavirus: current situation and travel-associated concerns.

PubMed

Al-Tawfiq, Jaffar A; Omrani, Ali S; Memish, Ziad A

2016-06-01

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 brought back memories of the occurrence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. More than 1500 MERS-CoV cases were recorded in 42 months with a case fatality rate (CFR) of 40%. Meanwhile, 8000 cases of SARS-CoV were confirmed in six months with a CFR of 10%. The clinical presentation of MERS-CoV ranges from mild and non-specific presentation to progressive and severe pneumonia. No predictive signs or symptoms exist to differentiate MERS-CoV from community-acquired pneumonia in hospitalized patients. An apparent heterogeneity was observed in transmission. Most MERS-CoV cases were secondary to large outbreaks in healthcare settings. These cases were secondary to community-acquired cases, which may also cause family outbreaks. Travel-associated MERS infection remains low. However, the virus exhibited a clear tendency to cause large outbreaks outside the Arabian Peninsula as exemplified by the outbreak in the Republic of Korea. In this review, we summarize the current knowledge about MERS-CoV and highlight travel-related issues.

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

PubMed

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

2013-08-01

A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses.

PubMed

Li, Qiaoli; Zhao, Zhenhuan; Zhou, Dihan; Chen, Yaoqing; Hong, Wei; Cao, Luyang; Yang, Jingyi; Zhang, Yan; Shi, Wei; Cao, Zhijian; Wu, Yingliang; Yan, Huimin; Li, Wenxin

2011-07-01

Outbreaks of SARS-CoV, influenza A (H5N1, H1N1) and measles viruses in recent years have raised serious concerns about the measures available to control emerging and re-emerging infectious viral diseases. Effective antiviral agents are lacking that specifically target RNA viruses such as measles, SARS-CoV and influenza H5N1 viruses, and available vaccinations have demonstrated variable efficacy. Therefore, the development of novel antiviral agents is needed to close the vaccination gap and silence outbreaks. We previously identified mucroporin, a cationic host defense peptide from scorpion venom, which can effectively inhibit standard bacteria. The optimized mucroporin-M1 can inhibit gram-positive bacteria at low concentrations and antibiotic-resistant pathogens. In this investigation, we further tested mucroporin and the optimized mucroporin-M1 for their antiviral activity. Surprisingly, we found that the antiviral activities of mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses were notably increased with an EC₅₀ of 7.15 μg/ml (3.52 μM) and a CC₅₀ of 70.46 μg/ml (34.70 μM) against measles virus, an EC₅₀ of 14.46 μg/ml (7.12 μM) against SARS-CoV and an EC₅₀ of 2.10 μg/ml (1.03 μM) against H5N1, while the original peptide mucroporin showed no antiviral activity against any of these three viruses. The inhibition model could be via a direct interaction with the virus envelope, thereby decreasing the infectivity of virus. This report provides evidence that host defense peptides from scorpion venom can be modified for antiviral activity by rational design and represents a practical approach for developing broad-spectrum antiviral agents, especially against RNA viruses. Copyright © 2011 Elsevier Inc. All rights reserved.

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

PubMed

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

2014-03-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao Jincun; Wang Wei; Yuan Zhihong

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 {beta}-sheets, {beta}5 and {beta}6. 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 {beta}6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the {beta}6 fragment, while the mouse antisera, induced by immunization of BALB/cmore » mice with recombinant S450-650, mainly recognized the {beta}6 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 {beta}6 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 {beta}6 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.« less

Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003

PubMed Central

Vega, Vinsensius B; Ruan, Yijun; Liu, Jianjun; Lee, Wah Heng; Wei, Chia Lin; Se-Thoe, Su Yun; Tang, Kin Fai; Zhang, Tao; Kolatkar, Prasanna R; Ooi, Eng Eong; Ling, Ai Ee; Stanton, Lawrence W; Long, Philip M; Liu, Edison T

2004-01-01

Background The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. Methods We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. Results Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes. Conclusions Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3' – most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs. PMID:15347429

NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion.

PubMed

Mahajan, Mukesh; Chatterjee, Deepak; Bhuvaneswari, Kannaian; Pillay, Shubhadra; Bhattacharjya, Surajit

2018-02-01

The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15 N{ 1 H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV. Copyright © 2017 Elsevier B.V. All rights reserved.

SARS-like WIV1-CoV poised for human emergence

PubMed Central

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

2016-01-01

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Arun K.; Takayama, Jun; Rao, Kalapala Venkateswar

The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead optimization and structure-activity studies to provide a series of improved inhibitors that show potent PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the (S)-Me inhibitor 15h (enzyme IC{sub 50} = 0.56 {mu}M; antiviral EC{sub 50} = 9.1 {mu}M) and the corresponding (R)-Me 15g (IC{sub 50} = 0.32 {mu}M; antiviral EC{sub 50} = 9.1more » {mu}M) are the most potent compounds in this series, with nearly equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15g-bound SARS-CoV PLpro and a corresponding model of 15h docked to PLpro provide intriguing molecular insight into the ligand-binding site interactions.« less

Seroprevalence of SARS coronavirus antibody in household contacts.

PubMed Central

Lee, C-C; Chen, S-Y; Chang, I-J; Tsai, P-C; Lu, T-C; Wu, P-L; Chen, W-J; Huang, L-M; Chang, S-C

2005-01-01

Between March and July 2003, 671 cases of severe acute respiratory syndrome (SARS) were diagnosed in Taiwan with a total of 84 fatalities. After the epidemic, a serological survey was conducted involving the asymptomatic household contacts. Household contacts of 13 index patients were enrolled in the study. Contact history and clinical symptoms of the household contacts were recorded by standardized questionnaires. Blood samples of patients and household contacts were collected at least 28 days after symptom onset in the index patients or household exposure in the contacts for SARS-associated coronavirus (SARS-CoV) IgG testing. On the basis of this investigation, 29 persons (25 adults and 4 children) were identified as having had unprotected exposure to the index cases before infection-control practices were implemented. Laboratory evaluation of clinical specimens showed no evidence of transmission of SARS-CoV infection to any contacts. This investigation demonstrated that subclinical transmission among household contacts was low in the described setting. PMID:16274510

Detection of the Severe Acute Respiratory Syndrome-Related Coronavirus and Alphacoronavirus in the Bat Population of Taiwan.

PubMed

Chen, Y-N; Phuong, V N; Chen, H C; Chou, C-H; Cheng, H-C; Wu, C-H

2016-12-01

Bats have been demonstrated to be natural reservoirs of severe acute respiratory syndrome coronavirus (SARS CoV) and Middle East respiratory syndrome (MERS) CoV. Faecal samples from 248 individuals of 20 bat species were tested for partial RNA-dependent RNA polymerase gene of CoV and 57 faecal samples from eight bat species were tested positive. The highest detection rate of 44% for Scotophilus kuhlii, followed by 30% for Rhinolophus monoceros. Significantly higher detection rates of coronaviral RNA were found in female bats and Scotophilus kuhlii roosting in palm trees. Phylogenetic analysis classified the positive samples into SARS-related (SARSr) CoV, Scotophilus bat CoV 512 close to those from China and Philippines, and Miniopterus bat CoV 1A-related lineages. Coronaviral RNA was also detected in bat guano from Scotophilus kuhlii and Myotis formosus flavus on the ground and had potential risk for human exposure. Diverse bat CoV with zoonotic potential could be introduced by migratory bats and maintained in the endemic bat population in Taiwan. © 2016 Blackwell Verlag GmbH.

Severe Acute Respiratory Syndrome Coronavirus as an Agent of Emerging and Reemerging Infection

PubMed Central

Cheng, Vincent C. C.; Lau, Susanna K. P.; Woo, Patrick C. Y.; Yuen, Kwok Yung

2007-01-01

Before the emergence of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in 2003, only 12 other animal or human coronaviruses were known. The discovery of this virus was soon followed by the discovery of the civet and bat SARS-CoV and the human coronaviruses NL63 and HKU1. Surveillance of coronaviruses in many animal species has increased the number on the list of coronaviruses to at least 36. The explosive nature of the first SARS epidemic, the high mortality, its transient reemergence a year later, and economic disruptions led to a rush on research of the epidemiological, clinical, pathological, immunological, virological, and other basic scientific aspects of the virus and the disease. This research resulted in over 4,000 publications, only some of the most representative works of which could be reviewed in this article. The marked increase in the understanding of the virus and the disease within such a short time has allowed the development of diagnostic tests, animal models, antivirals, vaccines, and epidemiological and infection control measures, which could prove to be useful in randomized control trials if SARS should return. The findings that horseshoe bats are the natural reservoir for SARS-CoV-like virus and that civets are the amplification host highlight the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections. PMID:17934078

Immunogenicity and Protective Efficacy in Mice and Hamsters of a β-Propiolactone Inactivated Whole Virus SARS-CoV Vaccine

PubMed Central

Roberts, Anjeanette; Lamirande, Elaine W.; Vogel, Leatrice; Baras, Benoît; Goossens, Geneviève; Knott, Isabelle; Chen, Jun; Ward, Jerrold M.; Vassilev, Ventzislav

2010-01-01

Abstract The immunogenicity and efficacy of β-propiolactone (BPL) inactivated whole virion SARS-CoV (WI-SARS) vaccine was evaluated in BALB/c mice and golden Syrian hamsters. The vaccine preparation was tested with or without adjuvants. Adjuvant Systems AS01B and AS03A were selected and tested for their capacity to elicit high humoral and cellular immune responses to WI-SARS vaccine. We evaluated the effect of vaccine dose and each adjuvant on immunogenicity and efficacy in mice, and the effect of vaccine dose with or without the AS01B adjuvant on the immunogenicity and efficacy in hamsters. Efficacy was evaluated by challenge with wild-type virus at early and late time points (4 and 18 wk post-vaccination). A single dose of vaccine with or without adjuvant was poorly immunogenic in mice; a second dose resulted in a significant boost in antibody levels, even in the absence of adjuvant. The use of adjuvants resulted in higher antibody titers, with the AS01B-adjuvanted vaccine being slightly more immunogenic than the AS03A-adjuvanted vaccine. Two doses of WI-SARS with and without Adjuvant Systems were highly efficacious in mice. In hamsters, two doses of WI-SARS with and without AS01B were immunogenic, and two doses of 2 μg of WI-SARS with and without the adjuvant provided complete protection from early challenge. Although antibody titers had declined in all groups of vaccinated hamsters 18 wk after the second dose, the vaccinated hamsters were still partially protected from wild-type virus challenge. Vaccine with adjuvant provided better protection than non-adjuvanted WI-SARS vaccine at this later time point. Enhanced disease was not observed in the lungs or liver of hamsters following SARS-CoV challenge, regardless of the level of serum neutralizing antibodies. PMID:20883165

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

PubMed

Hilgenfeld, Rolf; Peiris, Malik

2013-10-01

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

Purified coronavirus Spike protein nanoparticles induce coronavirus neutralizing antibodies in mice

PubMed Central

Mu, Haiyan; Taylor, Justin K; Massare, Michael; Flyer, David C

2014-01-01

Development of vaccination strategies for emerging pathogens are particularly challenging because of the sudden nature of the emergence of these viruses and the long process needed for traditional vaccine development. Therefore, there is a need for development of a rapid method of vaccine development that can respond to emerging pathogens in a short time frame. The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 and Middle East respiratory syndrome (MERS-CoV) in late 2012 demonstrate the importance of coronaviruses as emerging pathogens. The spike glycoproteins of coronaviruses reside on the surface of the virion and are responsible for virus entry. The spike glycoprotein is the major immunodominant antigen of coronaviruses and has proven to be an excellent target for vaccine designs that seek to block coronavirus entry and promote antibody targeting of infected cells. Vaccination strategies for coronaviruses have involved live attenuated virus, recombinant viruses, non-replicative virus-like particles expressing coronavirus proteins or DNA plasmids expressing coronavirus genes. None of these strategies has progressed to an approved human coronavirus vaccine in the ten years since SARS-CoV emerged. Here we describe a novel method for generating MERS-CoV and SARS-CoV full-length spike nanoparticles, which in combination with adjuvants are able to produce high titer antibodies in mice. PMID:24736006

Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection.

PubMed

Cheng, Vincent C C; Lau, Susanna K P; Woo, Patrick C Y; Yuen, Kwok Yung

2007-10-01

Before the emergence of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) in 2003, only 12 other animal or human coronaviruses were known. The discovery of this virus was soon followed by the discovery of the civet and bat SARS-CoV and the human coronaviruses NL63 and HKU1. Surveillance of coronaviruses in many animal species has increased the number on the list of coronaviruses to at least 36. The explosive nature of the first SARS epidemic, the high mortality, its transient reemergence a year later, and economic disruptions led to a rush on research of the epidemiological, clinical, pathological, immunological, virological, and other basic scientific aspects of the virus and the disease. This research resulted in over 4,000 publications, only some of the most representative works of which could be reviewed in this article. The marked increase in the understanding of the virus and the disease within such a short time has allowed the development of diagnostic tests, animal models, antivirals, vaccines, and epidemiological and infection control measures, which could prove to be useful in randomized control trials if SARS should return. The findings that horseshoe bats are the natural reservoir for SARS-CoV-like virus and that civets are the amplification host highlight the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections.

From SARS to MERS: evidence and speculation.

PubMed

Gao, Hainv; Yao, Hangping; Yang, Shigui; Li, Lanjuan

2016-12-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic pathogen. In 2012, the infectious outbreak caused by MERS-CoV in Saudi Arabia has spread to more than 1600 patients in 26 countries, resulting in over 600 deaths.Without a travel history, few clinical and radiological features can reliably differentiate MERS from SARS. But in real world, comparing with SARS, MERS presents more vaguely defined epidemiology, more severe symptoms, and higher case fatality rate. In this review, we summarize the recent findings in the field of MERS-CoV, especially its molecular virology, interspecies mechanisms, clinical features, antiviral therapies, and the further investigation into this disease. As a newly emerging virus, many questions are not fully answered, including the exact mode of transmission chain, geographical distribution, and animal origins. Furthermore, a new protocol needs to be launched to rapidly evaluate the effects of unproven antiviral drugs and vaccine to fasten the clinical application of new drugs.

Fused-ring structure of decahydroisoquinolin as a novel scaffold for SARS 3CL protease inhibitors.

PubMed

Shimamoto, Yasuhiro; Hattori, Yasunao; Kobayashi, Kazuya; Teruya, Kenta; Sanjoh, Akira; Nakagawa, Atsushi; Yamashita, Eiki; Akaji, Kenichi

2015-02-15

The design and evaluation of a novel decahydroisoquinolin scaffold as an inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro)) are described. Focusing on hydrophobic interactions at the S2 site, the decahydroisoquinolin scaffold was designed by connecting the P2 site cyclohexyl group of the substrate-based inhibitor to the main-chain at the α-nitrogen atom of the P2 position via a methylene linker. Starting from a cyclohexene enantiomer obtained by salt resolution, trans-decahydroisoquinolin derivatives were synthesized. All decahydroisoquinolin inhibitors synthesized showed moderate but clear inhibitory activities for SARS 3CL(pro), which confirmed the fused ring structure of the decahydroisoquinolin functions as a novel scaffold for SARS 3CL(pro) inhibitor. X-ray crystallographic analyses of the SARS 3CL(pro) in a complex with the decahydroisoquinolin inhibitor revealed the expected interactions at the S1 and S2 sites, as well as additional interactions at the N-substituent of the inhibitor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers†

PubMed Central

Neuman, Benjamin W.; Stein, David A.; Kroeker, Andrew D.; Churchill, Michael J.; Kim, Alice M.; Kuhn, Peter; Dawson, Philip; Moulton, Hong M.; Bestwick, Richard K.; Iversen, Patrick L.; Buchmeier, Michael J.

2005-01-01

The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targeted P-PMO and a random-sequence control P-PMO showed low inhibitory activity against SARS coronavirus. Certain other virus-targeted P-PMO reduced virus-induced cytopathology and cell-to-cell spread as a consequence of decreasing viral amplification. Active P-PMO were effective when administered at any time prior to peak viral synthesis and exerted sustained antiviral effects while present in culture medium. P-PMO showed low nonspecific inhibitory activity against translation of nontargeted RNA or growth of the arenavirus lymphocytic choriomeningitis virus. Two P-PMO targeting the viral transcription-regulatory sequence (TRS) region in the 5′ untranslated region were the most effective inhibitors tested. After several viral passages in the presence of a TRS-targeted P-PMO, partially drug-resistant SARS-CoV mutants arose which contained three contiguous base point mutations at the binding site of a TRS-targeted P-PMO. Those partially resistant viruses grew more slowly and formed smaller plaques than wild-type SARS-CoV. These results suggest PMO compounds have powerful therapeutic and investigative potential toward coronavirus infection. PMID:16014928

DOE Office of Scientific and Technical Information (OSTI.GOV)

He Yuxian; Li Jingjing; Jiang Shibo

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbsmore » as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.« less

SARS-CoV fusion peptides induce membrane surface ordering and curvature.

PubMed

Basso, Luis G M; Vicente, Eduardo F; Crusca, Edson; Cilli, Eduardo M; Costa-Filho, Antonio J

2016-11-28

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease.

PubMed

Konno, Hiroyuki; Wakabayashi, Masaki; Takanuma, Daiki; Saito, Yota; Akaji, Kenichi

2016-03-15

Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

SARS-CoV fusion peptides induce membrane surface ordering and curvature

PubMed Central

Basso, Luis G. M.; Vicente, Eduardo F.; Crusca Jr., Edson; Cilli, Eduardo M.; Costa-Filho, Antonio J.

2016-01-01

Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed. PMID:27892522

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malet, Hélène; Dalle, Karen; Brémond, Nicolas

The SARS-CoV macro domain was expressed, purified and crystallized. Selenomethionine-labelled crystals diffracted to 1.8 Å resolution. Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1′′-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macromore » domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182–355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 37.5, b = 55.6, c = 108.9 Å, β = 91.4°, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8 Å.« less

Genome Wide Identification of SARS-CoV Susceptibility Loci Using the Collaborative Cross

PubMed Central

Gralinski, Lisa E.; Ferris, Martin T.; Aylor, David L.; Whitmore, Alan C.; Green, Richard; Frieman, Matthew B.; Deming, Damon; Menachery, Vineet D.; Miller, Darla R.; Buus, Ryan J.; Bell, Timothy A.; Churchill, Gary A.; Threadgill, David W.; Katze, Michael G.; McMillan, Leonard; Valdar, William; Heise, Mark T.; Pardo-Manuel de Villena, Fernando; Baric, Ralph S.

2015-01-01

New systems genetics approaches are needed to rapidly identify host genes and genetic networks that regulate complex disease outcomes. Using genetically diverse animals from incipient lines of the Collaborative Cross mouse panel, we demonstrate a greatly expanded range of phenotypes relative to classical mouse models of SARS-CoV infection including lung pathology, weight loss and viral titer. Genetic mapping revealed several loci contributing to differential disease responses, including an 8.5Mb locus associated with vascular cuffing on chromosome 3 that contained 23 genes and 13 noncoding RNAs. Integrating phenotypic and genetic data narrowed this region to a single gene, Trim55, an E3 ubiquitin ligase with a role in muscle fiber maintenance. Lung pathology and transcriptomic data from mice genetically deficient in Trim55 were used to validate its role in SARS-CoV-induced vascular cuffing and inflammation. These data establish the Collaborative Cross platform as a powerful genetic resource for uncovering genetic contributions of complex traits in microbial disease severity, inflammation and virus replication in models of outbred populations. PMID:26452100

Homology models of main proteinase from coronavirus associated with SARS

NASA Astrophysics Data System (ADS)

Liu, Hsuan-Liang; Lin, Jin-Chung; Ho, Yih; Chen, Chin-Wen

2005-01-01

In this study, two homology models of the main proteinase (M pro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed. These models reveal three distinct functional domains, in which an intervening loop connecting domains II and III as well as a catalytic cleft containing the substrate binding subsites S1 and S2 between domains I and II are observed. S2 exhibits structural variations more significantly than S1 during the 200 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft and the amino acid residues lining up this subsite are least conserved. In addition, the higher structural variation of S2 makes it flexible enough to accommodate a bulky hydrophobic residue from the substrate.

A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

PubMed Central

Westerbeck, Jason W.

2015-01-01

ABSTRACT Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. IMPORTANCE CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel antiviral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for antiviral therapeutics. PMID:26136577

Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M.

PubMed

Oostra, M; de Haan, C A M; de Groot, R J; Rottier, P J M

2006-03-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses. It does, however, have several similarities to the coronavirus M proteins; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins, and (iv) they appear to interact with the E and S proteins, as well as with each other. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling. Pulse-chase analysis showed that both proteins were modified, although in different ways. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally, and they were not sensitive to the N-glycosidase enzyme. The SARS-CoV 3a protein, however, was demonstrated to contain sialic acids, indicating the presence of oligosaccharides. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein, like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein, which is N glycosylated.

Prophylactic and therapeutic intranasal administration with an immunomodulator, Hiltonol® (Poly IC:LC), in a lethal SARS-CoV-infected BALB/c mouse model.

PubMed

Kumaki, Yohichi; Salazar, Andres M; Wandersee, Miles K; Barnard, Dale L

2017-03-01

Hiltonol ® , (Poly IC:LC), a potent immunomodulator, is a synthetic, double-stranded polyriboinosinic-polyribocytidylic acid (poly IC) stabilized with Poly-L-lysine and carboxymethyl cellulose (LC). Hiltonol ® was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. Hiltonol ® at 5, 1, 0.5 or 0.25 mg/kg/day by intranasal (i.n.) route resulted in significant survival benefit when administered at selected times 24 h prior to challenge with a lethal dose of mouse-adapted severe acute respiratory syndrome coronavirus (SARS-CoV). The infected BALB/c mice receiving the Hiltonol ® treatments were also significantly effective in protecting mice against weight loss due to infection (p < 0.001). Groups of 20 mice were dosed with Hiltonol ® at 2.5 or 0.75 mg/kg by intranasal instillation 7, 14, and 21 days before virus exposure and a second dose was given 24 h later, prophylactic Hiltonol ® treatments (2.5 mg/kg/day) were completely protective in preventing death, and in causing significant reduction in lung hemorrhage scores, lung weights and lung virus titers. Hiltonol ® was also effective as a therapeutic when give up to 8 h post virus exposure; 100% of the-infected mice were protected against death when Hiltonol ® was administered at 5 mg/kg/day 8 h after infection. Our data suggest that Hiltonol ® treatment of SARS-CoV infection in mice leads to substantial prophylactic and therapeutic effects and could be used for treatment of other virus disease such as those caused by MERS-CoV a related coronavirus. These properties might be therapeutically advantageous if Hiltonol ® is considered for possible clinical use. Published by Elsevier B.V.

Interferon-Beta 1a and SARS Coronavirus Replication

DTIC Science & Technology

2004-02-01

A global outbreak of severe acute respiratory syn- drome ( SARS ) caused by a novel coronavirus began in March 2003. The rapid emergence of SARS and...emerging infectious disease. The etiologic agent was identified as a coronavirus ( SARS -CoV) that is not closely related to any of the previously...some coronaviruses , including avian infectious bronchitis virus, murine hepati- tis virus, and human coronavirus 229E, are susceptible to type I

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

PubMed Central

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

Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

PubMed Central

Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

2013-01-01

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. PMID:24023659

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

PubMed

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

2014-12-19

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

Detection of Severe Acute Respiratory Syndrome-Like, Middle East Respiratory Syndrome-Like Bat Coronaviruses and Group H Rotavirus in Faeces of Korean Bats.

PubMed

Kim, H K; Yoon, S-W; Kim, D-J; Koo, B-S; Noh, J Y; Kim, J H; Choi, Y G; Na, W; Chang, K-T; Song, D; Jeong, D G

2016-08-01

Bat species around the world have recently been recognized as major reservoirs of several zoonotic viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Nipah virus and Hendra virus. In this study, consensus primer-based reverse transcriptase polymerase chain reactions (RT-PCRs) and high-throughput sequencing were performed to investigate viruses in bat faecal samples collected at 11 natural bat habitat sites from July to December 2015 in Korea. Diverse coronaviruses were first detected in Korean bat faeces, including alphacoronaviruses, SARS-CoV-like and MERS-CoV-like betacoronaviruses. In addition, we identified a novel bat rotavirus belonging to group H rotavirus which has only been described in human and pigs until now. Therefore, our results suggest the need for continuing surveillance and additional virological studies in domestic bat. © 2016 Blackwell Verlag GmbH.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeung, Y.-S.; Yip, C.-W.; Hon, C.-C.

We have previously demonstrated that over-expression of spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) or its C-terminal subunit (S2) is sufficient to induce apoptosis in vitro. To further investigate the possible roles of S2 in SARS-CoV-induced apoptosis and pathogenesis of SARS, we characterized the host expression profiles induced upon S2 over-expression in Vero E6 cells by oligonucleotide microarray analysis. Possible activation of mitochondrial apoptotic pathway in S2 expressing cells was suggested, as evidenced by the up-regulation of cytochrome c and down-regulation of the Bcl-2 family anti-apoptotic members. Inhibition of Bcl-2-related anti-apoptotic pathway was further supported by themore » diminution of S2-induced apoptosis in Vero E6 cells over-expressing Bcl-xL. In addition, modulation of CCN E2 and CDKN 1A implied the possible control of cell cycle arrest at G1/S phase. This study is expected to extend our understanding on the pathogenesis of SARS at a molecular level.« less

Accessory proteins 8b and 8ab of severe acute respiratory syndrome coronavirus suppress the interferon signaling pathway by mediating ubiquitin-dependent rapid degradation of interferon regulatory factor 3.

PubMed

Wong, Hui Hui; Fung, To Sing; Fang, Shouguo; Huang, Mei; Le, My Tra; Liu, Ding Xiang

2018-02-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an inefficient inducer of interferon (IFN) response. It expresses various proteins that effectively circumvent IFN production at different levels via distinct mechanisms. Through the construction of recombinant IBV expressing proteins 8a, 8b and 8ab encoded by SARS-CoV ORF8, we demonstrate that expression of 8b and 8ab enables the corresponding recombinant viruses to partially overcome the inhibitory actions of IFN activation to achieve higher replication efficiencies in cells. We also found that proteins 8b and 8ab could physically interact with IRF3. Overexpression of 8b and 8ab resulted in the reduction of poly (I:C)-induced IRF3 dimerization and inhibition of the IFN-β signaling pathway. This counteracting effect was partially mediated by protein 8b/8ab-induced degradation of IRF3 in a ubiquitin-proteasome-dependent manner. Taken together, we propose that SARS-CoV may exploit the unique functions of proteins 8b and 8ab as novel mechanisms to overcome the effect of IFN response during virus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2015-10-01

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

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.

PubMed

Barnard, Dale L; Day, Craig W; Bailey, Kevin; Heiner, Matthew; Montgomery, Robert; Lauridsen, Larry; Winslow, Scott; Hoopes, Justin; Li, Joseph K-K; Lee, Jongdae; Carson, Dennis A; Cottam, Howard B; Sidwell, Robert W

2006-08-01

Because of the conflicting data concerning the SARS-CoV inhibitory efficacy of ribavirin, an inosine monophosphate (IMP) dehydrogenase inhibitor, studies were done to evaluate the efficacy of ribavirin and other IMP dehydrogenase inhibitors (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR), mizoribine, and mycophenolic acid) in preventing viral replication in the lungs of BALB/c mice, a replication model for severe acute respiratory syndrome (SARS) infections (Subbarao, K., McAuliffe, J., Vogel, L., Fahle, G., Fischer, S., Tatti, K., Packard, M., Shieh, W.J., Zaki, S., Murphy, B., 2004. Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus (SARS-CoV) in the respiratory tract of mice. J. Virol. 78, 3572-3577). Ribavirin given at 75 mg/kg 4 h prior to virus exposure and then given twice daily for 3 days beginning at day 0 was found to increase virus lung titers and extend the length of time that virus could be detected in the lungs of mice. Other IMP dehydrogenase inhibitors administered near maximum tolerated doses using the same dosing regimen as for ribavirin were found to slightly enhance virus replication in the lungs. In addition, ribavirin treatment seemed also to promote the production of pro-inflammatory cytokines 4 days after cessation of treatment, although after 3 days of treatment ribavirin inhibited pro-inflammatory cytokine production in infected mice, significantly reducing the levels of the cytokines IL-1alpha, interleukin-5 (IL-5), monocyte chemotactic protein-1 (MCP-1), and granulocyte-macrophage colony stimulating factor (GM-CSF). These findings suggest that ribavirin may actually contribute to the pathogenesis of SARS-CoV by prolonging and/or enhancing viral replication in the lungs. By not inhibiting viral replication in the lungs of infected mice, ribavirin treatment may have provided a continual source of stimulation for the inflammatory response thought to contribute to the pathogenesis of the infection. Our data do not support the use of ribavirin or other IMP dehydrogenase inhibitors for treating SARS infections in humans.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saikatendu, K.S.; Joseph, J.S.; Subramanian, V.

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 1.17Amore » (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.« less

[Surveillance on severe acute respiratory syndrome associated coronavirus in animals at a live animal market of Guangzhou in 2004].

PubMed

Wang, Ming; Jing, Huai-qi; Xu, Hui-fang; Jiang, Xiu-gao; Kan, Biao; Liu, Qi-yong; Wan, Kang-lin; Cui, Bu-yun; Zheng, Han; Cui, Zhi-gang; Yan, Mei-ying; Liang, Wei-li; Wang, Hong-xia; Qi, Xiao-bao; Li, Zhen-jun; Li, Ma-chao; Chen, Kai; Zhang, En-min; Zhang, Shou-yin; Hai, Rong; Yu, Dong-zheng; Xu, Jian-guo

2005-02-01

To study the prevalence of severe acute respiratory syndrome coronavirus (SARS-CoV) like virus in animals at a live animal market of Guanzhou in 2004 before and after culling of wild animal action taken by the local authority, in order to predict the re-emerging of SARS from animal originals in this region. Animals at live animal market were sampled for rectal and throat swabs in triplicate. A single step realtime reverse transcription-polymerase chain reaction (RT-PCR) diagnostic kit was performed for screening SARS-CoV like virus, the manual nested RT- PCR and DNA sequencing were performed for confirmation. Only specimens which tested positive for both of the N and P genes by nested RT-PCR were scored as positive. In 31 animals sampled in January 5 2004 before culling of wild animals at Guangdong Province, including 20 cats (Felis catus), 5 red fox (Vulpes vulpes) and 6 Lesser rice field rats (Rattus losea), 8 (25.8%) animals were tested positive for SARS-CoV like virus by RT-PCR methods, of which 4 cats, 3 red fox and one Lesser rice field rats were included. However, two weeks after culling of animals and disinfection of the market were implemented, in 119 animals sampled in January 20 2004, including 6 rabbits (Oryctolagus cuniculus), 13 cats, 46 red jungle fowl (Gallus gallus), 13 spotbill duck (Anas platyrhynchos), 10 greylag goose (Anser anser), 31 Chinese francolin (Franclinus pintadeanus), only rectal swab from one greylag goose was tested positive for SARS-CoV like virus. Furthermore, in 102 animals that including 14 greylag gooses, 3 cats, 5 rabbits, 9 spotbill duck (Anaspoecilorhyncha), 2 Chinese francolin (Franclinus pintadeanus), 8 common pheasant (Phasianus colchicus), 6 pigeons, 9 Chinese muntjac (Muntiacus reevesi), 19 wild boar (Sus scrofa), 16 Lesser rice field rats, 5 dogs, 1 mink (Mustela vison), 3 goats, 2 green peafowl (Pavo muticus) sampled in April, May, June, July, August and November, only rectal swab from one pig was tested positive. However, of 12 and 10 palm civets sampled in November and December including five of which had been at the live animals market for 2 days, none of them was tested positive. This findings revealed that animals being sampled in April, May, June, July, August and November of 2004, only one rectal swab from a pig was tested positive as SARS-CoV like virus, much lower than the results from the previous year, suggesting that the possibility of re-emerging of human infection from animal origins is low for the winter of 2004-2005.

Molecular interaction study of commercial cyclic peptides and MERS-COV papain-like protease as novel drug candidate for MERS-COV

NASA Astrophysics Data System (ADS)

Nasution, M. A. F.; Azzuhdi, M. G.; Tambunan, U. S. F.

2017-07-01

Middle-east respiratory syndrome coronavirus (MERS-CoV) has become the current outbreak, MERS-CoV infection results in illness at the respiratory system, digestive, and even lead to death with an average mortality caused by MERS-CoV infection reaches 50 %. Until now, there is not any effective vaccine or drug to ward off MERS-CoV infection. Papain-like protease (PLpro) is responsible for cleavage of a nonstructural protein that is essential for viral maturation. Inhibition of PLpro with a ligand will block the cleavage process of nonstructural protein, thus reduce the infection of MERS-CoV. Through of bioinformatics study with molecular docking and binding interaction analysis of commercial cyclic peptides, aldosterone secretion inhibiting factor (1-35) (bovine) was obtained as an inhibitor for PLpro. Thus, aldosterone secretion inhibiting factor (1-35) (bovine) has a potential as a novel candidate drug for treating MERS-CoV.

Preparation of His-tagged armored RNA phage particles as a control for real-time reverse transcription-PCR detection of severe acute respiratory syndrome coronavirus.

PubMed

Cheng, Yangjian; Niu, Jianjun; Zhang, Yongyou; Huang, Jianwei; Li, Qingge

2006-10-01

Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples.

SARS coronavirus protein 7a interacts with human Ap4A-hydrolase.

PubMed

Vasilenko, Natalia; Moshynskyy, Igor; Zakhartchouk, Alexander

2010-02-09

The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm.

Comparative Epidemiology of Human Infections with Middle East Respiratory Syndrome and Severe Acute Respiratory Syndrome Coronaviruses among Healthcare Personnel

PubMed Central

Chu, Yu-Tseng; Wu, Joseph Tsung-Shu; Geng, Xingyi; Zhao, Na; Cheng, Wei; Chen, Enfu; King, Chwan-Chuen

2016-01-01

The largest nosocomial outbreak of Middle East respiratory syndrome (MERS) occurred in South Korea in 2015. Health Care Personnel (HCP) are at high risk of acquiring MERS-Coronavirus (MERS-CoV) infections, similar to the severe acute respiratory syndrome (SARS)-Coronavirus (SARS-CoV) infections first identified in 2003. This study described the similarities and differences in epidemiological and clinical characteristics of 183 confirmed global MERS cases and 98 SARS cases in Taiwan associated with HCP. The epidemiological findings showed that the mean age of MERS-HCP and total MERS cases were 40 (24~74) and 49 (2~90) years, respectively, much older than those in SARS [SARS-HCP: 35 (21~68) years, p = 0.006; total SARS: 42 (0~94) years, p = 0.0002]. The case fatality rates (CFR) was much lower in MERS-HCP [7.03% (9/128)] or SARS-HCP [12.24% (12/98)] than the MERS-non-HCP [36.96% (34/92), p<0.001] or SARS-non-HCP [24.50% (61/249), p<0.001], however, no difference was found between MERS-HCP and SARS-HCP [p = 0.181]. In terms of clinical period, the days from onset to death [13 (4~17) vs 14.5 (0~52), p = 0.045] and to discharge [11 (5~24) vs 24 (0~74), p = 0.010] and be hospitalized days [9.5 (3~22) vs 22 (0~69), p = 0.040] were much shorter in MERS-HCP than SARS-HCP. Similarly, days from onset to confirmation were shorter in MERS-HCP than MERS-non-HCP [6 (1~14) vs 10 (1~21), p = 0.044]. In conclusion, the severity of MERS-HCP and SARS-HCP was lower than that of MERS-non-HCP and SARS-non-HCP due to younger age and early confirmation in HCP groups. However, no statistical difference was found in MERS-HCP and SARS-HCP. Thus, prevention of nosocomial infections involving both novel Coronavirus is crucially important to protect HCP. PMID:26930074

Papain-like protease (PLpro) inhibitory effects of cinnamic amides from Tribulus terrestris fruits.

PubMed

Song, Yeong Hun; Kim, Dae Wook; Curtis-Long, Marcus John; Yuk, Heung Joo; Wang, Yan; Zhuang, Ningning; Lee, Kon Ho; Jeon, Kwon Seok; Park, Ki Hun

2014-01-01

Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria. Subsequent bioactivity-guided fractionation of this extract led to six cinnamic amides (1-6) and ferulic acid (7). Compound 6 emerged as new compound possessing the very rare carbinolamide motif. These compounds (1-7) were evaluated for severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro inhibitory activity to identify their potencies and kinetic behavior. Compounds (1-6) displayed significant inhibitory activity with IC50 values in the range 15.8-70.1 µM. The new cinnamic amide 6 was found to be most potent inhibitor with an IC50 of 15.8 µM. In kinetic studies, all inhibitors exhibited mixed type inhibition. Furthermore, the most active PLpro inhibitors (1-6) were proven to be present in the native fruits in high quantities by HPLC chromatogram and liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS).

Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice.

PubMed

Zheng, Nuoyan; Xia, Ran; Yang, Cuiping; Yin, Bojiao; Li, Yin; Duan, Chengguo; Liang, Liming; Guo, Huishan; Xie, Qi

2009-08-06

Vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. Here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in Nicotiana benthamiana. The rN protein in the agrobacteria-infiltrated plant leaf accumulated up to a concentration of 79 microg per g fresh leaf weight at 3 days post infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund's adjuvant. The rN protein-specific IgG in the mouse sera attained a titer about 1:1,800 following three doses of immunization, which suggested effective B-cell maturation and differentiation in mice. Antibodies of the subclasses IgG1 and IgG2a were abundantly present in the mouse sera. During vaccination of rN protein, the expression of IFN-gamma and IL-10 was evidently up-regulated in splenocytes at different time points, while the expression of IL-2 and IL-4 was not. Up to now, this is the first study that plant-expressed recombinant SARS-CoV N protein can induce strong humoral and cellular responses in mice.

The Middle East Respiratory Syndrome Coronavirus - A Continuing Risk to Global Health Security.

PubMed

Azhar, Esam I; Lanini, Simone; Ippolito, Giuseppe; Zumla, Alimuddin

2017-01-01

Two new zoonotic coronaviruses causing disease in humans (Zumla et al. 2015a; Hui and Zumla 2015; Peiris et al. 2003; Yu et al. 2014) have been the focus of international attention for the past 14 years due to their epidemic potential; (1) The Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) (Peiris et al. 2003) first discovered in China in 2001 caused a major global epidemic of the Severe Acute Respiratory Syndrome (SARS). (2) The Middle East respiratory syndrome coronavirus (MERS-CoV) is a new corona virus isolated for the first time in a patients who died of severe lower respiratory tract infection in Jeddah (Saudi Arabia) in June 2012 (Zaki et al. 2012). The disease has been named Middle East Respiratory Syndrome (MERS) and it has remained on the radar of global public health authorities because of recurrent nosocomial and community outbreaks, and its association with severe disease and high mortality rates (Assiri et al. 2013a; Al-Abdallat et al. 2014; Memish et al. 2013a; Oboho et al. 2015; The WHO MERS-CoV Research Group 2013; Cotten et al. 2013a; Assiri et al. 2013b; Memish et al. 2013b; Azhar et al. 2014; Kim et al. 2015; Wang et al. 2015; Hui et al. 2015a). Cases of MERS have been reported from all continents and have been linked with travel to the Middle East (Hui et al. 2015a; WHO 2015c). The World Health Organization (WHO) have held nine meetings of the Emergency Committee (EC) convened by the Director-General under the International Health Regulations (IHR 2005) regarding MERS-CoV (WHO 2015c). There is wishful anticipation in the political and scientific communities that MERS-CoV like SARS-CoV will disappear with time. However it's been nearly 4 years since the first discovery of MERS-CoV, and MERS cases continue to be reported throughout the year from the Middle East (WHO 2015c). There is a large MERS-CoV camel reservoir, and there is no specific treatment or vaccine (Zumla et al. 2015a). With 10 million people visiting Saudi Arabia every year for Umrah and/or Hajj, the potential risk of global spread is ever present (Memish et al. 2014a; McCloskey et al. 2014; Al-Tawfiq et al. 2014a).

Identifying Monoclonal Antibodies that Potently Inhibit MERS-CoV | Center for Cancer Research

Cancer.gov

The Middle East respiratory syndrome coronavirus (MERS-CoV), first isolated in September 2012, infects cells lining the human airway, causing severe flu-like symptoms that, in some cases, lead to death. As of July 2, 2014, 824 confirmed cases of MERS-CoV infection, including at least 286 related deaths, have been reported to the World Health Organization. While there are currently no effective therapies against the virus, monoclonal antibodies (MAbs) may be a promising candidate. Having previously developed MAbs against other viruses, including the related severe acute respiratory syndrome coronavirus or SARS-CoV, Dimiter Dimitrov, Ph.D., of CCR’s Laboratory of Experimental Immunology (LEI), and his colleagues decided to pan a library of antigen binding fragments (Fab) for activity against MERS-CoV.

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

PubMed Central

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

2017-01-01

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. PMID:28591694

[Expression, purification and antibody preparation of recombinat SARS-CoV X5 protein].

PubMed

Wang, Li-Na; Kong, Jian-Qiang; Zhu, Ping; Du, Guan-Hua; Wang, Wei; Cheng, Ke-Di

2008-11-01

X5 protein is one of the putative unknown proteins of SARS-CoV. The recombinant protein has been successfully expressed in E. coli in the form of insoluble inclusion body. The inclusion body was dissolved in high concentration of urea. Affinity Chromatography was preformed to purify the denatured protein, and then the product was refolded in a series of gradient solutions of urea. The purified protein was obtained with the purity of > 95% and the yield of 93.3 mg x L(-1). Polyclonal antibody of this protein was obtained, and Western blotting assay indicated that the X5 protein has the strong property of antigen. Sixty-eight percent of the recombinant protein sequence was confirmed by LC-ESI-MS/MS analysis.

Two-amino acids change in the nsp4 of SARS coronavirus abolishes viral replication.

PubMed

Sakai, Yusuke; Kawachi, Kengo; Terada, Yutaka; Omori, Hiroko; Matsuura, Yoshiharu; Kamitani, Wataru

2017-10-01

Infection with coronavirus rearranges the host cell membrane to assemble a replication/transcription complex in which replication of the viral genome and transcription of viral mRNA occur. Although coexistence of nsp3 and nsp4 is known to cause membrane rearrangement, the mechanisms underlying the interaction of these two proteins remain unclear. We demonstrated that binding of nsp4 with nsp3 is essential for membrane rearrangement and identified amino acid residues in nsp4 responsible for the interaction with nsp3. In addition, we revealed that the nsp3-nsp4 interaction is not sufficient to induce membrane rearrangement, suggesting the participation of other factors such as host proteins. Finally, we showed that loss of the nsp3-nsp4 interaction eliminated viral replication by using an infectious cDNA clone and replicon system of SARS-CoV. These findings provide clues to the mechanism of the replication/transcription complex assembly of SARS-CoV and could reveal an antiviral target for the treatment of betacoronavirus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

The SARS coronavirus nucleocapsid protein--forms and functions.

PubMed

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

2014-03-01

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

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Rani, Mridula; Bolles, Meagan; Donaldson, Eric F.; Van Blarcom, Thomas; Baric, Ralph; Iverson, Brent

2012-01-01

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection. PMID:22696652

SARS-CoV replicates in primary human alveolar type II cell cultures but not in type I-like cells

PubMed Central

Mossel, Eric C.; Wang, Jieru; Jeffers, Scott; Edeen, Karen E.; Wang, Shuanglin; Cosgrove, Gregory P.; Funk, C. Joel; Manzer, Rizwan; Miura, Tanya A.; Pearson, Leonard D.; Holmes, Kathryn V.; Mason, Robert J.

2008-01-01

Severe acute respiratory syndrome (SARS) is a disease characterized by diffuse alveolar damage. We isolated alveolar type II cells and maintained them in a highly differentiated state. Type II cell cultures supported SARS-CoV replication as evidenced by RT-PCR detection of viral subgenomic RNA and an increase in virus titer. Virus titers were maximal by 24 hours and peaked at approximately 105 pfu/mL. Two cell types within the cultures were infected. One cell type was type II cells, which were positive for SP-A, SP-C, cytokeratin, a type II cell-specific monoclonal antibody, and Ep-CAM. The other cell type was composed of spindle-shaped cells that were positive for vimentin and collagen III and likely fibroblasts. Viral replication was not detected in type I-like cells or macrophages. Hence, differentiated adult human alveolar type II cells were infectible but alveolar type I-like cells and alveolar macrophages did not support productive infection. PMID:18022664

A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant

PubMed Central

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

2014-01-01

ABSTRACT Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. PMID:24667706

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme.

PubMed

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P C; Ovaa, Huib; Drag, Marcin; Lima, Christopher D; Huang, Tony T

2015-06-01

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

From genome to drug lead: identification of a small-molecule inhibitor of the SARS virus.

PubMed

Dooley, Andrea J; Shindo, Nice; Taggart, Barbara; Park, Jewn-Giew; Pang, Yuan-Ping

2006-02-15

Virtual screening, a fast, computational approach to identify drug leads [Perola, E.; Xu, K.; Kollmeyer, T. M.; Kaufmann, S. H.; Prendergast, F. G. J. Med. Chem.2000, 43, 401; Miller, M. A. Nat. Rev. Drug Disc.2002, 1 220], is limited by a known challenge in crystallographically determining flexible regions of proteins. This approach has not been able to identify active inhibitors of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using solely the crystal structures of a SARS-CoV cysteine proteinase with a flexible loop in the active site [Yang, H. T.; Yang, M. J.; Ding, Y.; Liu, Y. W.; Lou, Z. Y. Proc. Natl. Acad. Sci. U.S.A.2003, 100, 13190; Jenwitheesuk, E.; Samudrala, R. Bioorg. Med. Chem. Lett.2003, 13, 3989; Rajnarayanan, R. V.; Dakshanamurthy, S.; Pattabiraman, N. Biochem. Biophys. Res. Commun.2004, 321, 370; Du, Q.; Wang, S.; Wei, D.; Sirois, S.; Chou, K. Anal. Biochem.2005, 337, 262; Du, Q.; Wang, S.; Zhu, Y.; Wei, D.; Guo, H. Peptides2004, 25, 1857; Lee, V.; Wittayanarakul, K.; Remsungenen, T.; Parasuk, V.; Sompornpisut, P. Science (Asia)2003, 29, 181; Toney, J.; Navas-Martin, S.; Weiss, S.; Koeller, A. J. Med. Chem.2004, 47, 1079; Zhang, X. W.; Yap, Y. L. Bioorg. Med. Chem.2004, 12, 2517]. This article demonstrates a genome-to-drug-lead approach that uses terascale computing to model flexible regions of proteins, thus permitting the utilization of genetic information to identify drug leads expeditiously. A small-molecule inhibitor of SARS-CoV, exhibiting an effective concentration (EC50) of 23 microM in cell-based assays, was identified through virtual screening against a computer-predicted model of the cysteine proteinase. Screening against two crystal structures of the same proteinase failed to identify the 23-microM inhibitor. This study suggests that terascale computing can complement crystallography, broaden the scope of virtual screening, and accelerate the development of therapeutics to treat emerging infectious diseases such as SARS and Bird Flu.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joseph, J.S.; Saikatendu, K.S.; Subramanian, V.

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)more » 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.« less

Efficacy of severe acute respiratory syndrome vaccine based on a nonhuman primate adenovirus in the presence of immunity against human adenovirus.

PubMed

Zhi, Yan; Figueredo, Joanita; Kobinger, Gary P; Hagan, Heather; Calcedo, Roberto; Miller, James R; Gao, Guangping; Wilson, James M

2006-05-01

Replication-deficient human adenovirus type 5 (AdH5) vectors can induce strong transgene product-specific cellular and humoral responses. However, many adult humans have neutralizing antibodies (NAbs) against AdH5 as a result of natural infection with this virus. Therefore, a chimpanzee adenovirus C7 (AdC7) vector was developed to circumvent interference by preexisting immunity to AdH5. This study evaluated the impact of preexisting immunity to human adenovirus on the efficacy of adenovirus-based vaccines against the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). Efficacy was assessed after intramuscular injection of the vector into mice and was measured as the frequency of SARS-CoV-specific T cells and NAbs against SARS-CoV. Immunogenicity of the AdH5-based vaccine was significantly attenuated or completely abolished when the preexisting anti-AdH5 NAb titer was higher than 40. Because 27% of human serum samples from the United States tested so far have an anti-AdH5 NAb titer higher than 40, our results suggested that a significant percentage of humans with preexisting anti-AdH5 immunity would not be candidates for vaccination with an AdH5-based genetic vaccine. In contrast, preexisting anti-AdH5 NAbs have a minimal effect on the potency of the AdC7-based genetic vaccine. Taken together, our studies warrant the further development of AdC7 as a vaccine carrier for human trials.

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

PubMed Central

Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

2016-01-01

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 frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus. PMID:26919232

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

PubMed

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.

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

PubMed

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.

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

PubMed

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

2017-01-15

Currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like SARS- and MERS-coronavirus. We here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin A-analog, inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. Nevertheless, our data provide a basis to further explore the potential of Cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2017-08-22

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Follis, Kathryn E.; York, Joanne; Nunberg, Jack H.

The fusogenic potential of Class I viral envelope glycoproteins is activated by proteloytic cleavage of the precursor glycoprotein to generate the mature receptor-binding and transmembrane fusion subunits. Although the coronavirus (CoV) S glycoproteins share membership in this class of envelope glycoproteins, cleavage to generate the respective S1 and S2 subunits appears absent in a subset of CoV species, including that responsible for the severe acute respiratory syndrome (SARS). To determine whether proteolytic cleavage of the S glycoprotein might be important for the newly emerged SARS-CoV, we introduced a furin recognition site at single basic residues within the putative S1-S2 junctionalmore » region. We show that furin cleavage at the modified R667 position generates discrete S1 and S2 subunits and potentiates membrane fusion activity. This effect on the cell-cell fusion activity by the S glycoprotein is not, however, reflected in the infectivity of pseudotyped lentiviruses bearing the cleaved glycoprotein. The lack of effect of furin cleavage on virion infectivity mirrors that observed in the normally cleaved S glycoprotein of the murine coronavirus and highlights an additional level of complexity in coronavirus entry.« less

Routes of transmission of influenza A H1N1, SARS CoV, and norovirus in air cabin: Comparative analyses.

PubMed

Lei, H; Li, Y; Xiao, S; Lin, C-H; Norris, S L; Wei, D; Hu, Z; Ji, S

2018-05-01

Identifying the exact transmission route(s) of infectious diseases in indoor environments is a crucial step in developing effective intervention strategies. In this study, we proposed a comparative analysis approach and built a model to simulate outbreaks of 3 different in-flight infections in a similar cabin environment, that is, influenza A H1N1, severe acute respiratory syndrome (SARS) coronavirus (CoV), and norovirus. The simulation results seemed to suggest that the close contact route was probably the most significant route (contributes 70%, 95% confidence interval [CI]: 67%-72%) in the in-flight transmission of influenza A H1N1 transmission; as a result, passengers within 2 rows of the index case had a significantly higher infection risk than others in the outbreak (relative risk [RR]: 13.4, 95% CI: 1.5-121.2, P = .019). For SARS CoV, the airborne, close contact, and fomite routes contributed 21% (95% CI: 19%-23%), 29% (95% CI: 27%-31%), and 50% (95% CI: 48%-53%), respectively. For norovirus, the simulation results suggested that the fomite route played the dominant role (contributes 85%, 95% CI: 83%-87%) in most cases; as a result, passengers in aisle seats had a significantly higher infection risk than others (RR: 9.5, 95% CI: 1.2-77.4, P = .022). This work highlighted a method for using observed outbreak data to analyze the roles of different infection transmission routes. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology

PubMed Central

Honda-Okubo, Yoshikazu; Barnard, Dale; Ong, Chun Hao; Peng, Bi-Hung; Tseng, Chien-Te Kent

2014-01-01

ABSTRACT Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses. IMPORTANCE Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines. PMID:25520500

Biosafety level 3 laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects.

PubMed

Li, Ling; Gu, Jiang; Shi, Xicheng; Gong, Encong; Li, Xingwang; Shao, Hongquan; Shi, Xueying; Jiang, Huijun; Gao, Xiaoqiang; Cheng, Daiyun; Guo, Lizhu; Wang, Hao; Shi, Xiaohong; Wang, Peizhi; Zhang, Qianying; Shen, Bing

2005-09-15

During the outbreak of the emergent severe acute respiratory syndrome (SARS) infection, >30% of the approximately 8000 infected persons were health care workers. The highly infectious nature of SARS coronavirus (SARS-CoV) compelled our pathologists to consider biosafety issues in the autopsy room and for tissue processing procedures. A specially designed biosafety level 3 (BSL-3) autopsy laboratory was constructed and divided into a clean area, a semicontaminated area, a contaminated area, and 2 buffer zones. High-efficiency particulate air filters were placed in the air supply and exhaust systems. Laminar air flow was from the clean areas to the less clean areas. The negative pressures of the contaminated, semicontaminated, and clean areas were approximately -50 pa, -25 pa, and -5 pa, respectively. Personal protective equipment, including gas mask, impermeable protective clothing, and 3 layers of gloves worn during autopsies; the equipment was decontaminated before it was allowed to exit the facility. Strict BSL-3 practices were followed. When a given concentration of particulate sarin simulant was introduced into the contaminated area, it could not be detected in either the semicontaminated area or clean area, and particles >0.3 microm in size were not detected in the exhaust air. A total of 16 complete postmortem examinations for probable and suspected SARS were performed during a 2-month period. Of these, 7 reported confirmed cases of SARS. None of the 23 pathologists and technicians who participated in these autopsies was infected with SARS-CoV. Our experience suggests that BSL-3 laboratory operating principles should be among the special requirements for performing autopsies of contaminated bodies and that they can safeguard the clinicians and the environment involved in these procedures.

Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

PubMed Central

Broer, Rene; Boson, Bertrand; Spaan, Willy; Cosset, François-Loïc; Corver, Jeroen

2006-01-01

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity. PMID:16415007

DOE Office of Scientific and Technical Information (OSTI.GOV)

Follis, Kathryn E.; York, Joanne; Nunberg, Jack H.

The fusion subunit of the SARS-CoV S glycoprotein contains two regions of hydrophobic heptad-repeat amino acid sequences that have been shown in biophysical studies to form a six-helix bundle structure typical of the fusion-active core found in Class I viral fusion proteins. Here, we have applied serine-scanning mutagenesis to the C-terminal-most heptad-repeat region in the SARS-CoV S glycoprotein to investigate the functional role of this region in membrane fusion. We show that hydrophobic sidechains at a and d positions only within the short helical segment of the C-terminal heptad-repeat region (I1161, I1165, L1168, A1172, and L1175) are critical for cell-cellmore » fusion. Serine mutations at outlying heptad-repeat residues that form an extended chain in the core structure (V1158, L1179, and L1182) do not affect fusogenicity. Our study provides genetic evidence for the important role of {alpha}-helical packing in promoting S glycoprotein-mediated membrane fusion.« less

SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

PubMed Central

Békés, Miklós; Rut, Wioletta; Kasperkiewicz, Paulina; Mulder, Monique P. C.; Ovaa, Huib; Drag, Marcin; Lima, Christopher D.; Huang, Tony T.

2015-01-01

Ubiquitin (Ub) and the ubiquitin-like modifier interferon stimulated gene 15 (ISG15) participate in the host defense of viral infections. Viruses, including the Severe Acute Respiratory Syndrome human coronavirus (SARS hCoV), have co-opted Ub/ISG15-conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub/ISG15-conjugated host proteins. Here, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle Eastern Respiratory Syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that similar to SARS PLpro, MERS PLpro is both a deubiquitinating and a deISGylating enzyme. Further analysis of the intrinsic deubiquitinating enzyme (DUB) activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, while SARS PLpro prefers to cleave Lys48-linked polyUb chains. Second, MERS PLpro cleaves polyUb chains in a “mono-distributive” manner (one Ub at a time), and SARS PLpro prefers to cleave K48-linked poly-Ub chains by sensing a di-Ub moiety as a minimal recognition element using a “di-distributive” cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses. PMID:25764917

Porcine Epidemic Diarrhea Virus 3C-Like Protease-Mediated Nucleocapsid Processing: Possible Link to Viral Cell Culture Adaptability.

PubMed

Jaru-Ampornpan, Peera; Jengarn, Juggragarn; Wanitchang, Asawin; Jongkaewwattana, Anan

2017-01-15

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high mortality rates in newborn piglets, leading to massive losses to the swine industry worldwide during recent epidemics. Intense research efforts are now focusing on defining viral characteristics that confer a growth advantage, pathogenicity, or cell adaptability in order to better understand the PEDV life cycle and identify suitable targets for antiviral or vaccine development. Here, we report a unique phenomenon of PEDV nucleocapsid (N) cleavage by the PEDV-encoded 3C-like protease (3Cpro) during infection. The identification of the 3Cpro cleavage site at the C terminus of N supported previous observations that PEDV 3Cpro showed a substrate requirement slightly different from that of severe acute respiratory syndrome coronavirus (SARS-CoV) 3Cpro and revealed a greater flexibility in its substrate recognition site. This cleavage motif is present in the majority of cell culture-adapted PEDV strains but is missing in emerging field isolates. Remarkably, reverse-genetics-derived cell culture-adapted PEDV AVCT12 harboring uncleavable N displayed growth retardation in Vero E6-APN cells compared to the wild-type virus. These observations altogether shed new light on the investigation and characterization of the PEDV nucleocapsid protein and its possible link to cell culture adaptation. Recurrent PEDV outbreaks have resulted in enormous economic losses to swine industries worldwide. To gain the upper hand in combating this disease, it is necessary to understand how this virus replicates and evades host immunity. Characterization of viral proteins provides important clues to mechanisms by which viruses survive and spread. Here, we characterized an intriguing phenomenon in which the nucleocapsids of some PEDV strains are proteolytically processed by the virally encoded main protease. Growth retardation in recombinant PEDV carrying uncleavable N suggests a replication advantage provided by the cleavage event, at least in the cell culture system. These findings may direct us to a more complete understanding of PEDV replication and pathogenicity. Copyright © 2017 American Society for Microbiology.

Repurposing of Clinically Developed Drugs for Treatment of Middle East Respiratory Syndrome Coronavirus Infection

PubMed Central

Dyall, Julie; Coleman, Christopher M.; Hart, Brit J.; Venkataraman, Thiagarajan; Holbrook, Michael R.; Kindrachuk, Jason; Johnson, Reed F.; Olinger, Gene G.; Jahrling, Peter B.; Laidlaw, Monique; Johansen, Lisa M.; Lear-Rooney, Calli M.; Glass, Pamela J.; Hensley, Lisa E.

2014-01-01

Outbreaks of emerging infections present health professionals with the unique challenge of trying to select appropriate pharmacologic treatments in the clinic with little time available for drug testing and development. Typically, clinicians are left with general supportive care and often untested convalescent-phase plasma as available treatment options. Repurposing of approved pharmaceutical drugs for new indications presents an attractive alternative to clinicians, researchers, public health agencies, drug developers, and funding agencies. Given the development times and manufacturing requirements for new products, repurposing of existing drugs is likely the only solution for outbreaks due to emerging viruses. In the studies described here, a library of 290 compounds was screened for antiviral activity against Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Selection of compounds for inclusion in the library was dependent on current or previous FDA approval or advanced clinical development. Some drugs that had a well-defined cellular pathway as target were included. In total, 27 compounds with activity against both MERS-CoV and SARS-CoV were identified. The compounds belong to 13 different classes of pharmaceuticals, including inhibitors of estrogen receptors used for cancer treatment and inhibitors of dopamine receptor used as antipsychotics. The drugs identified in these screens provide new targets for in vivo studies as well as incorporation into ongoing clinical studies. PMID:24841273

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

PubMed Central

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

Bats as reservoirs of severe emerging infectious diseases.

PubMed

Han, Hui-Ju; Wen, Hong-ling; Zhou, Chuan-Min; Chen, Fang-Fang; Luo, Li-Mei; Liu, Jian-wei; Yu, Xue-Jie

2015-07-02

In recent years severe infectious diseases have been constantly emerging, causing panic in the world. Now we know that many of these terrible diseases are caused by viruses originated from bats (Table 1), such as Ebola virus, Marburg, SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), Nipah virus (NiV) and Hendra virus (HeV). These viruses have co-evolved with bats due to bats' special social, biological and immunological features. Although bats are not in close contact with humans, spillover of viruses from bats to intermediate animal hosts, such as horses, pigs, civets, or non-human primates, is thought to be the most likely mode to cause human infection. Humans may also become infected with viruses through aerosol by intruding into bat roosting caves or via direct contact with bats, such as catching bats or been bitten by bats. Copyright © 2015 Elsevier B.V. All rights reserved.

Crystal structures reveal an induced-fit binding of a substrate-like Aza-peptide epoxide to SARS coronavirus main peptidase.

PubMed

Lee, Ting-Wai; Cherney, Maia M; Liu, Jie; James, Karen Ellis; Powers, James C; Eltis, Lindsay D; James, Michael N G

2007-02-23

The SARS coronavirus main peptidase (SARS-CoV M(pro)) plays an essential role in the life-cycle of the virus and is a primary target for the development of anti-SARS agents. Here, we report the crystal structure of M(pro) at a resolution of 1.82 Angstroms, in space group P2(1) at pH 6.0. In contrast to the previously reported structure of M(pro) in the same space group at the same pH, the active sites and the S1 specificity pockets of both protomers in the structure of M(pro) reported here are in the catalytically competent conformation, suggesting their conformational flexibility. We report two crystal structures of M(pro) having an additional Ala at the N terminus of each protomer (M(+A(-1))(pro)), both at a resolution of 2.00 Angstroms, in space group P4(3)2(1)2: one unbound and one bound by a substrate-like aza-peptide epoxide (APE). In the unbound form, the active sites and the S1 specificity pockets of both protomers of M(+A(-1))(pro) are observed in a collapsed (catalytically incompetent) conformation; whereas they are in an open (catalytically competent) conformation in the APE-bound form. The observed conformational flexibility of the active sites and the S1 specificity pockets suggests that these parts of M(pro) exist in dynamic equilibrium. The structural data further suggest that the binding of APE to M(pro) follows an induced-fit model. The substrate likely also binds in an induced-fit manner in a process that may help drive the catalytic cycle.

Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease

PubMed Central

Chan, Jasper F. W.; Lau, Susanna K. P.; To, Kelvin K. W.; Cheng, Vincent C. C.; Woo, Patrick C. Y.

2015-01-01

SUMMARY The source of the severe acute respiratory syndrome (SARS) epidemic was traced to wildlife market civets and ultimately to bats. Subsequent hunting for novel coronaviruses (CoVs) led to the discovery of two additional human and over 40 animal CoVs, including the prototype lineage C betacoronaviruses, Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5; these are phylogenetically closely related to the Middle East respiratory syndrome (MERS) CoV, which has affected more than 1,000 patients with over 35% fatality since its emergence in 2012. All primary cases of MERS are epidemiologically linked to the Middle East. Some of these patients had contacted camels which shed virus and/or had positive serology. Most secondary cases are related to health care-associated clusters. The disease is especially severe in elderly men with comorbidities. Clinical severity may be related to MERS-CoV's ability to infect a broad range of cells with DPP4 expression, evade the host innate immune response, and induce cytokine dysregulation. Reverse transcription-PCR on respiratory and/or extrapulmonary specimens rapidly establishes diagnosis. Supportive treatment with extracorporeal membrane oxygenation and dialysis is often required in patients with organ failure. Antivirals with potent in vitro activities include neutralizing monoclonal antibodies, antiviral peptides, interferons, mycophenolic acid, and lopinavir. They should be evaluated in suitable animal models before clinical trials. Developing an effective camel MERS-CoV vaccine and implementing appropriate infection control measures may control the continuing epidemic. PMID:25810418

MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

DOE Office of Scientific and Technical Information (OSTI.GOV)

Menachery, Vineet D.; Schäfer, Alexandra; Burnum-Johnson, Kristin E.

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways in order to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems based approach, we examined differential regulation of IFNγ dependent genes following infection with highly pathogenic viruses including influenza (H5N1-VN1203, H1N1-CA04) and coronaviruses (SARS-CoV, MERS-CoV). Categorizing by function, we observed down regulation of genes associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down regulation of antigenmore » presentation genes and was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation rather than histone modification plays a crucial role in MERS-CoV mediated antagonism of antigen presentation genes; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Together, the results indicate a common approach utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.« less

MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

DOE PAGES

Menachery, Vineet D.; Schafer, Alexandra; Burnum-Johnson, Kristin E.; ...

2018-01-16

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways in order to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems based approach, we examined differential regulation of IFNγ dependent genes following infection with highly pathogenic viruses including influenza (H5N1-VN1203, H1N1-CA04) and coronaviruses (SARS-CoV, MERS-CoV). Categorizing by function, we observed down regulation of genes associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down regulation of antigenmore » presentation genes and was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation rather than histone modification plays a crucial role in MERS-CoV mediated antagonism of antigen presentation genes; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Altogether, the results indicate a common approach utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.« less

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

PubMed

Corman, V M; Eckerle, I; Bleicker, T; Zaki, A; Landt, O; Eschbach-Bludau, M; van Boheemen, S; Gopal, R; Ballhause, M; Bestebroer, T M; Muth, D; Müller, M A; Drexler, J F; Zambon, M; Osterhaus, A D; Fouchier, R M; Drosten, C

2012-09-27

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5–6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.

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

PubMed Central

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

2016-01-01

Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm. Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection. PMID:26953343

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsao, Y.-P.; Lin, J.-Y.; Jan, J.-T.

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{sub b}ind/), ten each potential N- and S-specific HLA-A2.1-binding peptides were synthesized. The high affinity HLA-A2.1 peptides were validated bymore » 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-{gamma} stimulation of blood CD8{sup +} 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.« less

SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice.

PubMed

Shen, Zu T; Sigalov, Alexander B

2016-06-28

During the co-evolution of viruses and their hosts, the viruses have evolved numerous strategies to counter and evade host antiviral immune responses in order to establish a successful infection, replicate and persist in the host. Recently, based on our model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, we suggested specific molecular mechanisms used by different viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV) to modulate the host immune response mediated by members of the family of multichain immune recognition receptors (MIRRs). This family includes T cell receptor (TCR) that is critically involved in immune diseases such as autoimmune arthritis. In the present study, we provide compelling experimental in vivo evidence in support of our hypothesis. Using the SCHOOL approach and the SARS-CoV fusion peptide sequence, we rationally designed a novel immunomodulatory peptide that targets TCR. We showed that this peptide ameliorates collagen-induced arthritis in DBA/1J mice and protects against bone and cartilage damage. Incorporation of the peptide into self-assembling lipopeptide nanoparticles that mimic native human high density lipoproteins significantly increases peptide dosage efficacy. Together, our data further confirm that viral immune evasion strategies that target MIRRs can be transferred to therapeutic strategies that require similar functionalities.

Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jun; Shen, Wei; Liao, Ming, E-mail: mliao@scau.edu.cn

The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M{sup pro} was overexpressed inmore » Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6{sub 1}22. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.« less

Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA.

PubMed

Ferron, François; Subissi, Lorenzo; Silveira De Morais, Ana Theresa; Le, Nhung Thi Tuyet; Sevajol, Marion; Gluais, Laure; Decroly, Etienne; Vonrhein, Clemens; Bricogne, Gérard; Canard, Bruno; Imbert, Isabelle

2018-01-09

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3'-5' exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3'-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5'-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

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

PubMed

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.

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

PubMed Central

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.

2014-01-01

Background. 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. Methods. 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. Results. 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. Conclusions. 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. PMID:24253287

Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential.

PubMed

Kindler, Eveline; Jónsdóttir, Hulda R; Muth, Doreen; Hamming, Ole J; Hartmann, Rune; Rodriguez, Regulo; Geffers, Robert; Fouchier, Ron A M; Drosten, Christian; Müller, Marcel A; Dijkman, Ronald; Thiel, Volker

2013-02-19

The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

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

PubMed

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

2016-04-22

Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus

PubMed Central

Thai, Hong Thi Cam; Le, Mai Quynh; Vuong, Cuong Duc; Parida, Manmohan; Minekawa, Harumi; Notomi, Tsugunori; Hasebe, Futoshi; Morita, Kouichi

2004-01-01

The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 102 PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries. PMID:15131154

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petit, Chad M.; Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803; Chouljenko, Vladimir N.

The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion wasmore » reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.« less

Detection of coronavirus genomes in Moluccan naked-backed fruit bats in Indonesia.

PubMed

Anindita, Paulina Duhita; Sasaki, Michihito; Setiyono, Agus; Handharyani, Ekowati; Orba, Yasuko; Kobayashi, Shintaro; Rahmadani, Ibnu; Taha, Siswatiana; Adiani, Sri; Subangkit, Mawar; Nakamura, Ichiro; Sawa, Hirofumi; Kimura, Takashi

2015-04-01

Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus.

Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

PubMed Central

Mielech, Anna M.; Deng, Xufang; Chen, Yafang; Kindler, Eveline; Wheeler, Dorthea L.; Mesecar, Andrew D.; Thiel, Volker; Perlman, Stanley

2015-01-01

ABSTRACT Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis. PMID:25694594

MERS-CoV Accessory ORFs Play Key Role for Infection and Pathogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Menachery, Vineet D.; Mitchell, Hugh D.; Cockrell, Adam S.

ABSTRACT While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation.In vitroreplication attenuation also extends toin vivomodels, allowing use of dORF3-5 as a live attenuated vaccine platform.more » Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward. IMPORTANCEThe initial emergence and periodic outbreaks of MERS-CoV highlight a continuing threat posed by zoonotic pathogens to global public health. In these studies, mutant virus generation demonstrates the necessity of accessory ORFs in regard to MERS-CoV infection and pathogenesis. With this in mind, accessory ORF functions can be targeted for both therapeutic and vaccine treatments in response to MERS-CoV and related group 2C coronaviruses. In addition, disruption of accessory ORFs in parallel may offer a rapid response platform to attenuation of future emergent strains based on both SARS- and MERS-CoV accessory ORF mutants.« less

Efficient Replication of the Novel Human Betacoronavirus EMC on Primary Human Epithelium Highlights Its Zoonotic Potential

PubMed Central

Kindler, Eveline; Jónsdóttir, Hulda R.; Muth, Doreen; Hamming, Ole J.; Hartmann, Rune; Rodriguez, Regulo; Geffers, Robert; Fouchier, Ron A. M.; Drosten, Christian; Müller, Marcel A.; Dijkman, Ronald; Thiel, Volker

2013-01-01

ABSTRACT The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. PMID:23422412

Global patterns in coronavirus diversity

PubMed Central

Johnson, Christine K.; Greig, Denise J.; Kramer, Sarah; Che, Xiaoyu; Wells, Heather; Hicks, Allison L.; Joly, Damien O.; Wolfe, Nathan D.; Daszak, Peter; Karesh, William; Lipkin, W. I.; Morse, Stephen S.; Mazet, Jonna A. K.

2017-01-01

Abstract Since the emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrom Coronavirus (MERS-CoV) it has become increasingly clear that bats are important reservoirs of CoVs. Despite this, only 6% of all CoV sequences in GenBank are from bats. The remaining 94% largely consist of known pathogens of public health or agricultural significance, indicating that current research effort is heavily biased towards describing known diseases rather than the ‘pre-emergent’ diversity in bats. Our study addresses this critical gap, and focuses on resource poor countries where the risk of zoonotic emergence is believed to be highest. We surveyed the diversity of CoVs in multiple host taxa from twenty countries to explore the factors driving viral diversity at a global scale. We identified sequences representing 100 discrete phylogenetic clusters, ninety-one of which were found in bats, and used ecological and epidemiologic analyses to show that patterns of CoV diversity correlate with those of bat diversity. This cements bats as the major evolutionary reservoirs and ecological drivers of CoV diversity. Co-phylogenetic reconciliation analysis was also used to show that host switching has contributed to CoV evolution, and a preliminary analysis suggests that regional variation exists in the dynamics of this process. Overall our study represents a model for exploring global viral diversity and advances our fundamental understanding of CoV biodiversity and the potential risk factors associated with zoonotic emergence. PMID:28630747

Network-based analysis of comorbidities risk during an infection: SARS and HIV case studies.

PubMed

Moni, Mohammad Ali; Liò, Pietro

2014-10-24

Infections are often associated to comorbidity that increases the risk of medical conditions which can lead to further morbidity and mortality. SARS is a threat which is similar to MERS virus, but the comorbidity is the key aspect to underline their different impacts. One UK doctor says "I'd rather have HIV than diabetes" as life expectancy among diabetes patients is lower than that of HIV. However, HIV has a comorbidity impact on the diabetes. We present a quantitative framework to compare and explore comorbidity between diseases. By using neighbourhood based benchmark and topological methods, we have built comorbidity relationships network based on the OMIM and our identified significant genes. Then based on the gene expression, PPI and signalling pathways data, we investigate the comorbidity association of these 2 infective pathologies with other 7 diseases (heart failure, kidney disorder, breast cancer, neurodegenerative disorders, bone diseases, Type 1 and Type 2 diabetes). Phenotypic association is measured by calculating both the Relative Risk as the quantified measures of comorbidity tendency of two disease pairs and the ϕ-correlation to measure the robustness of the comorbidity associations. The differential gene expression profiling strongly suggests that the response of SARS affected patients seems to be mainly an innate inflammatory response and statistically dysregulates a large number of genes, pathways and PPIs subnetworks in different pathologies such as chronic heart failure (21 genes), breast cancer (16 genes) and bone diseases (11 genes). HIV-1 induces comorbidities relationship with many other diseases, particularly strong correlation with the neurological, cancer, metabolic and immunological diseases. Similar comorbidities risk is observed from the clinical information. Moreover, SARS and HIV infections dysregulate 4 genes (ANXA3, GNS, HIST1H1C, RASA3) and 3 genes (HBA1, TFRC, GHITM) respectively that affect the ageing process. It is notable that HIV and SARS similarly dysregulated 11 genes and 3 pathways. Only 4 significantly dysregulated genes are common between SARS-CoV and MERS-CoV, including NFKBIA that is a key regulator of immune responsiveness implicated in susceptibility to infectious and inflammatory diseases. Our method presents a ripe opportunity to use data-driven approaches for advancing our current knowledge on disease mechanism and predicting disease comorbidities in a quantitative way.

A Rapid and Specific Assay for the Detection of MERS-CoV

PubMed Central

Huang, Pei; Wang, Hualei; Cao, Zengguo; Jin, Hongli; Chi, Hang; Zhao, Jincun; Yu, Beibei; Yan, Feihu; Hu, Xingxing; Wu, Fangfang; Jiao, Cuicui; Hou, Pengfei; Xu, Shengnan; Zhao, Yongkun; Feng, Na; Wang, Jianzhong; Sun, Weiyang; Wang, Tiecheng; Gao, Yuwei; Yang, Songtao; Xia, Xianzhu

2018-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that can cause human respiratory disease. The development of a detection method for this virus that can lead to rapid and accurate diagnosis would be significant. In this study, we established a nucleic acid visualization technique that combines the reverse transcription loop-mediated isothermal amplification technique and a vertical flow visualization strip (RT-LAMP-VF) to detect the N gene of MERS-CoV. The RT-LAMP-VF assay was performed in a constant temperature water bath for 30 min, and the result was visible by the naked eye within 5 min. The RT-LAMP-VF assay was capable of detecting 2 × 101 copies/μl of synthesized RNA transcript and 1 × 101 copies/μl of MERS-CoV RNA. The method exhibits no cross-reactivities with multiple CoVs including SARS-related (SARSr)-CoV, HKU4, HKU1, OC43 and 229E, and thus exhibits high specificity. Compared to the real-time RT-PCR (rRT-PCR) method recommended by the World Health Organization (WHO), the RT-LAMP-VF assay is easy to handle, does not require expensive equipment and can rapidly complete detection within 35 min. PMID:29896174

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

PubMed

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.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Hugh D.; Eisfeld, Amie J.; Sims, Amy

Respiratory infections stemming from influenza viruses and the Severe Acute Respiratory Syndrome corona virus (SARS-CoV) represent a serious public health threat as emerging pandemics. Despite efforts to identify the critical interactions of these viruses with host machinery, the key regulatory events that lead to disease pathology remain poorly targeted with therapeutics. Here we implement an integrated network interrogation approach, in which proteome and transcriptome datasets from infection of both viruses in human lung epithelial cells are utilized to predict regulatory genes involved in the host response. We take advantage of a novel “crowd-based” approach to identify and combine ranking metricsmore » that isolate genes/proteins likely related to the pathogenicity of SARS-CoV and influenza virus. Subsequently, a multivariate regression model is used to compare predicted lung epithelial regulatory influences with data derived from other respiratory virus infection models. We predicted a small set of regulatory factors with conserved behavior for consideration as important components of viral pathogenesis that might also serve as therapeutic targets for intervention. Our results demonstrate the utility of integrating diverse ‘omic datasets to predict and prioritize regulatory features conserved across multiple pathogen infection models.« less

Structural basis for the development of SARS 3CL protease inhibitors from a peptide mimic to an aza-decaline scaffold.

PubMed

Teruya, Kenta; Hattori, Yasunao; Shimamoto, Yasuhiro; Kobayashi, Kazuya; Sanjoh, Akira; Nakagawa, Atsushi; Yamashita, Eiki; Akaji, Kenichi

2016-11-04

Design of inhibitors against severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL(pro) ) is a potentially important approach to fight against SARS. We have developed several synthetic inhibitors by structure-based drug design. In this report, we reveal two crystal structures of SARS 3CL(pro) complexed with two new inhibitors based on our previous work. These structures combined with six crystal structures complexed with a series of related ligands reported by us are collectively analyzed. To these eight complexes, the structural basis for inhibitor binding was analyzed by the COMBINE method, which is a chemometrical analysis optimized for the protein-ligand complex. The analysis revealed that the first two latent variables gave a cumulative contribution ratio of r(2)  = 0.971. Interestingly, scores using the second latent variables for each complex were strongly correlated with root mean square deviations (RMSDs) of side-chain heavy atoms of Met(49) from those of the intact crystal structure of SARS-3CL(pro) (r = 0.77) enlarging the S2 pocket. The substantial contribution of this side chain (∼10%) for the explanation of pIC50 s was dependent on stereochemistry and the chemical structure of the ligand adapted to the S2 pocket of the protease. Thus, starting from a substrate mimic inhibitor, a design for a central scaffold for a low molecular weight inhibitor was evaluated to develop a further potent inhibitor. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 391-403, 2016. © 2015 Wiley Periodicals, Inc.

Lack of association between HLA-A, -B and -DRB1 alleles and the development of SARS: a cohort of 95 SARS-recovered individuals in a population of Guangdong, southern China.

PubMed

Xiong, P; Zeng, X; Song, M S; Jia, S W; Zhong, M H; Xiao, L L; Lan, W; Cai, C; Wu, X W; Gong, F L; Wang, W

2008-02-01

Severe acute respiratory syndrome (SARS), caused by infection with a novel coronavirus (SARS-CoV), was the first major novel infectious disease at the beginning of the 21st century, with China especially affected. SARS was characterized by high infectivity, morbidity and mortality, and the confined pattern of the disease spreading among the countries of South-East and East Asia suggested the existence of susceptible factor(s) in these populations. Studies in the populations of Hong Kong and Taiwan showed an association of human leucocyte antigen (HLA) polymorphisms with the development and/or severity of SARS, respectively. The aim of the present study was to define the genotypic patterns of HLA-A, -B and -DRB1 loci in SARS patients and a co-resident population of Guangdong province, southern China, where the first SARS case was reported. The samples comprised 95 cases of recovered SARS patients and 403 unrelated healthy controls. HLA -A, -B and -DRB1 alleles were genotyped using polymerase chain reaction with sequence-specific primers. The severity of the disease was assessed according to the history of lung infiltration, usage of assisted ventilation and occurrence of lymphocytopenia. Although the allelic frequencies of A23, A34, B60, DRB1*12 in the SARS group were slightly higher, and A33, -B58 and -B61 were lower than in the controls, no statistical significance was found when the Pc value was considered. Similarly, no association of HLA alleles with the severity of the disease was detected. Thus, variations in the major histocompatibility complex are unlikely to have contributed significantly to either the susceptibility or the severity of SARS in the population of Guangdong.

Genetic Characteristics of Coronaviruses from Korean Bats in 2016.

PubMed

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.

Pathogenic Influenza Viruses and Coronaviruses Utilize Similar and Contrasting Approaches To Control Interferon-Stimulated Gene Responses

PubMed Central

Menachery, Vineet D.; Eisfeld, Amie J.; Schäfer, Alexandra; Josset, Laurence; Sims, Amy C.; Proll, Sean; Fan, Shufang; Li, Chengjun; Neumann, Gabriele; Tilton, Susan C.; Chang, Jean; Gralinski, Lisa E.; Long, Casey; Green, Richard; Williams, Christopher M.; Weiss, Jeffrey; Matzke, Melissa M.; Webb-Robertson, Bobbie-Jo; Schepmoes, Athena A.; Shukla, Anil K.; Metz, Thomas O.; Smith, Richard D.; Waters, Katrina M.; Katze, Michael G.; Kawaoka, Yoshihiro

2014-01-01

ABSTRACT The broad range and diversity of interferon-stimulated genes (ISGs) function to induce an antiviral state within the host, impeding viral pathogenesis. While successful respiratory viruses overcome individual ISG effectors, analysis of the global ISG response and subsequent viral antagonism has yet to be examined. Employing models of the human airway, transcriptomics and proteomics datasets were used to compare ISG response patterns following highly pathogenic H5N1 avian influenza (HPAI) A virus, 2009 pandemic H1N1, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) infection. The results illustrated distinct approaches utilized by each virus to antagonize the global ISG response. In addition, the data revealed that highly virulent HPAI virus and MERS-CoV induce repressive histone modifications, which downregulate expression of ISG subsets. Notably, influenza A virus NS1 appears to play a central role in this histone-mediated downregulation in highly pathogenic influenza strains. Together, the work demonstrates the existence of unique and common viral strategies for controlling the global ISG response and provides a novel avenue for viral antagonism via altered histone modifications. PMID:24846384

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

PubMed

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

2014-10-13

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

Detection of group 1 coronaviruses in bats in North America

USGS Publications Warehouse

Dominguez, S.R.; O'Shea, T.J.; Oko, L.M.; Holmes, K.V.

2007-01-01

The epidemic of severe acute respiratory syndrome (SARS) was caused by a newly emerged coronavirus (SARS-CoV). Bats of several species in southern People's Republic of China harbor SARS-like CoVs and may be reservoir hosts for them. To determine whether bats in North America also harbor coronaviruses, we used reverse transcription-PCR to detect coronavirus RNA in bats. We found coronavirus RNA in 6 of 28 fecal specimens from bats of 2 of 7 species tested. The prevalence of viral RNA shedding was high: 17% in Eptesicus fuscus and 50% in Myotis occultus. Sequence analysis of a 440-bp amplicon in gene 1b showed that these Rocky Mountain bat coronaviruses formed 3 clusters in phylogenetic group 1 that were distinct from group 1 coronaviruses of Asian bats. Because of the potential for bat coronaviruses to cause disease in humans and animals, further surveillance and characterization of bat coronaviruses in North America are needed.

Structure based computational assessment of channel properties of assembled ORF-8a from SARS-CoV.

PubMed

Hsu, Hao-Jen; Lin, Meng-Han; Schindler, Christina; Fischer, Wolfgang B

2015-02-01

ORF 8a is a short 39 amino acid bitopic membrane protein encoded by severe acute respiratory syndrome causing corona virus (SARS-CoV). It has been identified to increase permeability of the lipid membrane for cations. Permeability is suggested to occur due to the assembly of helical bundles. Computational models of a pentameric assembly of 8a peptides are generated using the first 22 amino acids, which include the transmembrane domain. Low energy structures reveal a hydrophilic pore mantled by residues Thr-8, and -18, Ser-11, Cys-13, and Arg-22. Potential of mean force (PMF) profiles for mono (Na(+) , K(+) , Cl(-) ) and divalent (Ca(2+) ) ions along the pore are calculated. The data support experimental findings of a weak cation selectivity of the channel. Calculations on 8a are compared to data derived for a pentameric bundle consisting of the M2 helices of the bacterial pentameric ligand gated ion channel GLIC (3EHZ). PMF curves of both, bundles 8a and M2, show sigmoidal shaped profiles. In comparison to the data for the M2-GLIC model, data of the 8a bundle show lower amplitude of the PMF values between maximum and minimum and less discrimination amongst ions. © 2014 Wiley Periodicals, Inc.

EDs on heightened alert for MERS-CoV as first cases reach the US.

PubMed

2014-07-01

The first cases of Middle East Respiratory Syndrome coronavirus (MERS-CoV) have turned up in the United States. First, in late April, a patient tested positive for the virus at a hospital in Munster, IN, and then shortly thereafter, a second patient tested positive at a hospital in Orlando, FL. While both patients have since recovered from the virus and been released, the cases have raised awareness of the infectious threat of MERS-CoV, and they have put EDs and other frontline providers on heightened alert for patients with severe respiratory symptoms and other risk factors. While MERS-CoV is not yet as contagious as seasonal influenza or the severe acute respiratory syndrome (SARS) that started in China and then swept around the globe in 2003, it is more deadly. The World Health Organization reports that roughly one-quarter of 514 people who have tested positive for the virus have died. Experts note that health care workers make up a large percentage of the documented cases of MERS-CoV, and they point out that most human-to-human transmissions of the virus occur in the hospital setting. Public health officials urge emergency personnel to pay strict attention to infection control practices, and to query patients who present with fever and respiratory distress about recent travel to the Arabian Peninsula and/or close contact with a person who has a confirmed or probable case of MERS-CoV.

Emerging infectious diseases: Focus on infection control issues for novel coronaviruses (Severe Acute Respiratory Syndrome-CoV and Middle East Respiratory Syndrome-CoV), hemorrhagic fever viruses (Lassa and Ebola), and highly pathogenic avian influenza viruses, A(H5N1) and A(H7N9).

PubMed

Weber, David J; Rutala, William A; Fischer, William A; Kanamori, Hajime; Sickbert-Bennett, Emily E

2016-05-02

Over the past several decades, we have witnessed the emergence of many new infectious agents, some of which are major public threats. New and emerging infectious diseases which are both transmissible from patient-to-patient and virulent with a high mortality include novel coronaviruses (SARS-CoV, MERS-CV), hemorrhagic fever viruses (Lassa, Ebola), and highly pathogenic avian influenza A viruses, A(H5N1) and A(H7N9). All healthcare facilities need to have policies and plans in place for early identification of patients with a highly communicable diseases which are highly virulent, ability to immediately isolate such patients, and provide proper management (e.g., training and availability of personal protective equipment) to prevent transmission to healthcare personnel, other patients and visitors to the healthcare facility. Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Menachery, Vineet D.; Gralinski, Lisa E.; Mitchell, Hugh D.

ABSTRACT 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 impactmore » 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 effectively targets the highly conserved 2'O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2'O-MTase function across CoVs.« less

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

PubMed Central

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.

2017-01-01

ABSTRACT 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 and in 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. IMPORTANCE Coronavirus (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 effectively targets the highly conserved 2′O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2′O-MTase function across CoVs. PMID:29152578

The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

PubMed

Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

2015-09-01

RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.

Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

PubMed Central

Kuo, Lili; Hurst-Hess, Kelley R.; Koetzner, Cheri A.

2016-01-01

ABSTRACT The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. IMPORTANCE The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins. PMID:26889024

Intelligent Adversary Risk Analysis: A Bioterrorism Risk Management Model (PREPRINT)

DTIC Science & Technology

2009-02-20

Dengue • Listeria monocytogenes • Filoviruses coronavirus ( SARS -CoV) • Campylobacter jejuni • Ebola • Yersinia enterocolitica) • Marburg...OF: 17. LIMITATION OF ABSTRACT Same as Report ( SAR ) 18. NUMBER OF PAGES 29 19a. NAME OF RESPONSIBLE PERSON a. REPORT unclassified b. ABSTRACT...which may be to maximize the consequences they can inflict (Golany et al., Figure 2: Decision Tree Example  7    Submitted to Risk Analysis

Structural insights of a self-assembling 9-residue peptide from the C-terminal tail of the SARS corona virus E-protein in DPC and SDS micelles: A combined high and low resolution spectroscopic study.

PubMed

Ghosh, Anirban; Bhattacharyya, Dipita; Bhunia, Anirban

2018-02-01

In recent years, several studies based on the interaction of self-assembling short peptides derived from viroporins with model membranes, have improved our understanding of the molecular mechanism of corona virus (CoV) infection under physiological conditions. In this study, we have characterized the mechanism of membrane interaction of a short, 9-residue peptide TK9 (T 55 VYVYSRVK 63 ) that had been derived from the carboxyl terminal of the Severe Acute Respiratory Syndrome (SARS) corona virus (SARS CoV) envelope (E) protein. The peptide has been studied for its physical changes in the presence of both zwitterionic DPC and negatively charged SDS model membrane micelles, respectively, with the help of a battery of biophysical techniques including two-dimensional solution state NMR spectroscopy. Interestingly, in both micellar environments, TK9 adopted an alpha helical conformation; however, the helical propensities were much higher in the case of DPC compared to those of SDS micelle, suggesting that TK9 has more specificity towards eukaryotic cell membrane than the bacterial cell membrane. The orientation of the peptide TK9 also varies in the different micellar environments. The peptide's affinity was further manifested by its pronounced membrane disruption ability towards the mammalian compared to the bacterial membrane mimic. Collectively, the in-depth structural information on the interaction of TK9 with different membrane environments explains the host specificity and membrane orientation owing to subsequent membrane disruption implicated in the viral pathogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang,W.; Lin, Y.; Santelli, E.

Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that caused pandemic spread in 2003. The etiological agent of SARS is a novel coronavirus (SARS-CoV). The coronaviral surface spike protein S is a type I transmembrane glycoprotein that mediates initial host binding via the cell surface receptor angiotensin-converting enzyme 2 (ACE2), as well as the subsequent membrane fusion events required for cell entry. Here we report the crystal structure of the S1 receptor binding domain (RBD) in complex with a neutralizing antibody, 80R, at 2.3 {angstrom} resolution, as well as the structure of the uncomplexed S1 RBD atmore » 2.2 {angstrom} resolution. We show that the 80R-binding epitope on the S1 RBD overlaps very closely with the ACE2-binding site, providing a rationale for the strong binding and broad neutralizing ability of the antibody. We provide a structural basis for the differential effects of certain mutations in the spike protein on 80R versus ACE2 binding, including escape mutants, which should facilitate the design of immunotherapeutics to treat a future SARS outbreak. We further show that the RBD of S1 forms dimers via an extensive interface that is disrupted in receptor- and antibody-bound crystal structures, and we propose a role for the dimer in virus stability and infectivity.« less

Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells.

PubMed

Millet, Jean Kaoru; Whittaker, Gary R

2018-04-01

During viral entry, enveloped viruses require the fusion of their lipid envelope with host cell membranes. For coronaviruses, this critical step is governed by the virally-encoded spike (S) protein, a class I viral fusion protein that has several unique features. Coronavirus entry is unusual in that it is often biphasic in nature, and can occur at or near the cell surface or in late endosomes. Recent advances in structural, biochemical and molecular biology of the coronavirus S protein has shed light on the intricacies of coronavirus entry, in particular the molecular triggers of coronavirus S-mediated membrane fusion. Furthermore, characterization of the coronavirus fusion peptide (FP), the segment of the fusion protein that inserts to a target lipid bilayer during membrane fusion, has revealed its particular attributes which imparts some of the unusual properties of the S protein, such as Ca 2+ -dependency. These unusual characteristics can explain at least in part the biphasic nature of coronavirus entry. In this review, using severe acute respiratory syndrome coronavirus (SARS-CoV) as model virus, we give an overview of advances in research on the coronavirus fusion peptide with an emphasis on its role and properties within the biological context of host cell entry. Copyright © 2017 Elsevier Inc. All rights reserved.

Unique signatures of long noncoding RNA expression in response to virus infection and altered innate immune signaling.

PubMed

Peng, Xinxia; Gralinski, Lisa; Armour, Christopher D; Ferris, Martin T; Thomas, Matthew J; Proll, Sean; Bradel-Tretheway, Birgit G; Korth, Marcus J; Castle, John C; Biery, Matthew C; Bouzek, Heather K; Haynor, David R; Frieman, Matthew B; Heise, Mark; Raymond, Christopher K; Baric, Ralph S; Katze, Michael G

2010-10-26

Studies of the host response to virus infection typically focus on protein-coding genes. However, non-protein-coding RNAs (ncRNAs) are transcribed in mammalian cells, and the roles of many of these ncRNAs remain enigmas. Using next-generation sequencing, we performed a whole-transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection across four founder mouse strains of the Collaborative Cross. We observed differential expression of approximately 500 annotated, long ncRNAs and 1,000 nonannotated genomic regions during infection. Moreover, studies of a subset of these ncRNAs and genomic regions showed the following. (i) Most were similarly regulated in response to influenza virus infection. (ii) They had distinctive kinetic expression profiles in type I interferon receptor and STAT1 knockout mice during SARS-CoV infection, including unique signatures of ncRNA expression associated with lethal infection. (iii) Over 40% were similarly regulated in vitro in response to both influenza virus infection and interferon treatment. These findings represent the first discovery of the widespread differential expression of long ncRNAs in response to virus infection and suggest that ncRNAs are involved in regulating the host response, including innate immunity. At the same time, virus infection models provide a unique platform for studying the biology and regulation of ncRNAs.

Unique Signatures of Long Noncoding RNA Expression in Response to Virus Infection and Altered Innate Immune Signaling

PubMed Central

Peng, Xinxia; Gralinski, Lisa; Armour, Christopher D.; Ferris, Martin T.; Thomas, Matthew J.; Proll, Sean; Bradel-Tretheway, Birgit G.; Korth, Marcus J.; Castle, John C.; Biery, Matthew C.; Bouzek, Heather K.; Haynor, David R.; Frieman, Matthew B.; Heise, Mark; Raymond, Christopher K.; Baric, Ralph S.; Katze, Michael G.

2010-01-01

Studies of the host response to virus infection typically focus on protein-coding genes. However, non-protein-coding RNAs (ncRNAs) are transcribed in mammalian cells, and the roles of many of these ncRNAs remain enigmas. Using next-generation sequencing, we performed a whole-transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection across four founder mouse strains of the Collaborative Cross. We observed differential expression of approximately 500 annotated, long ncRNAs and 1,000 nonannotated genomic regions during infection. Moreover, studies of a subset of these ncRNAs and genomic regions showed the following. (i) Most were similarly regulated in response to influenza virus infection. (ii) They had distinctive kinetic expression profiles in type I interferon receptor and STAT1 knockout mice during SARS-CoV infection, including unique signatures of ncRNA expression associated with lethal infection. (iii) Over 40% were similarly regulated in vitro in response to both influenza virus infection and interferon treatment. These findings represent the first discovery of the widespread differential expression of long ncRNAs in response to virus infection and suggest that ncRNAs are involved in regulating the host response, including innate immunity. At the same time, virus infection models provide a unique platform for studying the biology and regulation of ncRNAs. PMID:20978541

Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

PubMed Central

Nieto-Torres, Jose L.; DeDiego, Marta L.; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M.; Enjuanes, Luis

2014-01-01

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

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

PubMed

Vergara-Alert, Júlia; Vidal, Enric; Bensaid, Albert; Segalés, Joaquim

2017-06-01

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.

Recognition of the murine coronavirus genomic RNA packaging signal depends on the second RNA-binding domain of the nucleocapsid protein.

PubMed

Kuo, Lili; Koetzner, Cheri A; Hurst, Kelley R; Masters, Paul S

2014-04-01

The coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was replaced by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging. This work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective packaging of genomic RNA into assembled virions.

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.

PubMed

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 development. Copyright © 2017 John Wiley & Sons, Ltd.

Genomic analysis and geographic visualization of H5N1 and SARS-CoV.

PubMed

Hill, Andrew W; Alexandrov, Boyan; Guralnick, Robert P; Janies, Daniel

2007-10-11

Emerging infectious diseases and organisms present critical issues of national security public health, and economic welfare. We still understand little about the zoonotic potential of many viruses. To this end, we are developing novel database tools to manage comparative genomic datasets. These tools add value because they allow us to summarize the direction, frequency and order of genomic changes. We will perform numerous real world tests with our tools with both Avian Influenza and Coronaviruses.

Optimization of the Production Process and Characterization of the Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1), a SARS Vaccine Candidate.

PubMed

Chen, Wen-Hsiang; Chag, Shivali M; Poongavanam, Mohan V; Biter, Amadeo B; Ewere, Ebe A; Rezende, Wanderson; Seid, Christopher A; Hudspeth, Elissa M; Pollet, Jeroen; McAtee, C Patrick; Strych, Ulrich; Bottazzi, Maria Elena; Hotez, Peter J

2017-08-01

From 2002 to 2003, a global pandemic of severe acute respiratory syndrome (SARS) spread to 5 continents and caused 8000 respiratory infections and 800 deaths. To ameliorate the effects of future outbreaks as well as to prepare for biodefense, a process for the production of a recombinant protein vaccine candidate is under development. Previously, we reported the 5 L scale expression and purification of a promising recombinant SARS vaccine candidate, RBD219-N1, the 218-amino acid residue receptor-binding domain (RBD) of SARS coronavirus expressed in yeast-Pichia pastoris X-33. When adjuvanted with aluminum hydroxide, this protein elicited high neutralizing antibody titers and high RBD-specific antibody titers. However, the yield of RBD219-N1 (60 mg RBD219-N1 per liter of fermentation supernatant; 60 mg/L FS) still required improvement to reach our target of >100 mg/L FS. In this study, we optimized the 10 L scale production process and increased the fermentation yield 6- to 7-fold to 400 mg/L FS with purification recovery >50%. A panel of characterization tests indicated that the process is reproducible and that the purified, tag-free RBD219-N1 protein has high purity and a well-defined structure and is therefore a suitable candidate for production under current Good Manufacturing Practice and future phase-1 clinical trials. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History

PubMed Central

Tao, Ying; Shi, Mang; Chommanard, Christina; Queen, Krista; Zhang, Jing; Markotter, Wanda; Kuzmin, Ivan V.; Holmes, Edward C.

2017-01-01

ABSTRACT Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5′ and 3′ ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination “hot spot” in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances. IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential. PMID:28077633

Risk and Outbreak Communication: Lessons from Taiwan's Experiences in the Post-SARS Era.

PubMed

Hsu, Yu-Chen; Chen, Yu-Ling; Wei, Han-Ning; Yang, Yu-Wen; Chen, Ying-Hwei

In addition to the impact of a disease itself, public reaction could be considered another outbreak to be controlled during an epidemic. Taiwan's experience with SARS in 2003 highlighted the critical role played by the media during crisis communication. After the SARS outbreak, Taiwan's Centers for Disease Control (Taiwan CDC) followed the WHO outbreak communication guidelines on trust, early announcements, transparency, informing the public, and planning, in order to reform its risk communication systems. This article describes the risk communication framework in Taiwan, which has been used to respond to the 2009-2016 influenza epidemics, Ebola in West Africa (2014-16), and MERS-CoV in South Korea (2015) during the post-SARS era. Many communication strategies, ranging from traditional media to social and new media, have been implemented to improve transparency in public communication and promote civic engagement. Taiwan CDC will continue to maintain the strengths of its risk communication systems and resolve challenges as they emerge through active evaluation and monitoring of public opinion to advance Taiwan's capacity in outbreak communication and control. Moreover, Taiwan CDC will continue to implement the IHR (2005) and to promote a global community working together to fight shared risks and to reach the goal of "One World, One Health."

Risk and Outbreak Communication: Lessons from Taiwan's Experiences in the Post-SARS Era

PubMed Central

Chen, Yu-Ling; Wei, Han-Ning; Yang, Yu-Wen; Chen, Ying-Hwei

2017-01-01

In addition to the impact of a disease itself, public reaction could be considered another outbreak to be controlled during an epidemic. Taiwan's experience with SARS in 2003 highlighted the critical role played by the media during crisis communication. After the SARS outbreak, Taiwan's Centers for Disease Control (Taiwan CDC) followed the WHO outbreak communication guidelines on trust, early announcements, transparency, informing the public, and planning, in order to reform its risk communication systems. This article describes the risk communication framework in Taiwan, which has been used to respond to the 2009-2016 influenza epidemics, Ebola in West Africa (2014-16), and MERS-CoV in South Korea (2015) during the post-SARS era. Many communication strategies, ranging from traditional media to social and new media, have been implemented to improve transparency in public communication and promote civic engagement. Taiwan CDC will continue to maintain the strengths of its risk communication systems and resolve challenges as they emerge through active evaluation and monitoring of public opinion to advance Taiwan's capacity in outbreak communication and control. Moreover, Taiwan CDC will continue to implement the IHR (2005) and to promote a global community working together to fight shared risks and to reach the goal of “One World, One Health.” PMID:28418746

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lien, Shu-Pei; Shih, Yi-Ping; Chen, Hsin-Wei

Three peptides, D1 (amino acid residues 175-201), D2 (a.a. 434-467), and TM (a.a. 1128-1159), corresponding to the spike protein (S) of severe acute respiratory syndrome corona virus (SARS CoV) were synthesized and their immunological functions were investigated in three different animals models (mice, guinea pigs, and rabbits). The peptides mixture formulated either with Freund's adjuvant or synthetic adjuvant Montanide ISA-51/oligodeoxy nucleotide CpG (ISA/CpG) could elicit antisera in immunized animals which were capable of inhibiting SARS/HIV pseudovirus entry into HepG2 cells. The neutralizing epitopes were identified using peptides to block the neutralizing effect of guinea pig antisera. The major neutralizing epitopemore » was located on the D2 peptide, and the amino acid residue was fine mapped to 434-453. In BALB/c mice T-cell proliferation assay revealed that only D2 peptide contained T-cell epitope, the sequence of which corresponded to amino acid residue 434-448. The ISA/CpG formulation generated anti-D2 IgG titer comparable to those obtained from Freund's adjuvant formulation, but generated fewer antibodies against D1 or TM peptides. The highly immunogenic D2 peptide contains both neutralizing and Th cell epitopes. These results suggest that synthetic peptide D2 would be useful as a component of SARS vaccine candidates.« less

A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

PubMed Central

Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

2016-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

Middle East Respiratory Syndrome Coronavirus Intra-Host Populations Are Characterized by Numerous High Frequency Variants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borucki, Monica K.; Lao, Victoria; Hwang, Mona

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater thanmore » 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Lastly, many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.« less

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

PubMed

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.

Small molecules targeting viral RNA.

PubMed

Hermann, Thomas

2016-11-01

Highly conserved noncoding RNA (ncRNA) elements in viral genomes and transcripts offer new opportunities to expand the repertoire of drug targets for the development of antiinfective therapy. Ligands binding to ncRNA architectures are able to affect interactions, structural stability or conformational changes and thereby block processes essential for viral replication. Proof of concept for targeting functional RNA by small molecule inhibitors has been demonstrated for multiple viruses with RNA genomes. Strategies to identify antiviral compounds as inhibitors of ncRNA are increasingly emphasizing consideration of drug-like properties of candidate molecules emerging from screening and ligand design. Recent efforts of antiviral lead discovery for RNA targets have provided drug-like small molecules that inhibit viral replication and include inhibitors of human immunodeficiency virus (HIV), hepatitis C virus (HCV), severe respiratory syndrome coronavirus (SARS CoV), and influenza A virus. While target selectivity remains a challenge for the discovery of useful RNA-binding compounds, a better understanding is emerging of properties that define RNA targets amenable for inhibition by small molecule ligands. Insight from successful approaches of targeting viral ncRNA in HIV, HCV, SARS CoV, and influenza A will provide a basis for the future exploration of RNA targets for therapeutic intervention in other viral pathogens which create urgent, unmet medical needs. Viruses for which targeting ncRNA components in the genome or transcripts may be promising include insect-borne flaviviruses (Dengue, Zika, and West Nile) and filoviruses (Ebola and Marburg). WIREs RNA 2016, 7:726-743. doi: 10.1002/wrna.1373 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

University Marine Biological Station Millport Annual Report

DTIC Science & Technology

1987-07-31

courses cover a variety of topics including marine biology, ecology, behaviour, marine microbiology, advanced studies on selected marine invertebrates ... marine invertebrates and on a more detailed kinetic study of phenoloxidase, prophenoloxidase and protease activities in tunicate coelomic fluid (C...DOTIC FILE COV () NUNIVERSITY OF LONDON and UNIVERSITY OF GLASGOW VO"- DTIC SELECTE University Marine Biological Station Millport DIThZUYTNSTAEEiT

New Metrics for Evaluating Viral Respiratory Pathogenesis

PubMed Central

Menachery, Vineet D.; Gralinski, Lisa E.; Baric, Ralph S.; Ferris, Martin T.

2015-01-01

Viral pathogenesis studies in mice have relied on markers of severe systemic disease, rather than clinically relevant measures, to evaluate respiratory virus infection; thus confounding connections to human disease. Here, whole-body plethysmography was used to directly measure changes in pulmonary function during two respiratory viral infections. This methodology closely tracked with traditional pathogenesis metrics, distinguished both virus- and dose-specific responses, and identified long-term respiratory changes following both SARS-CoV and Influenza A Virus infection. Together, the work highlights the utility of examining respiratory function following infection in order to fully understand viral pathogenesis. PMID:26115403

3D tissue-like assemblies: A novel approach to investigate virus-cell interactions.

PubMed

Goodwin, Thomas J; McCarthy, Maureen; Cohrs, Randall J; Kaufer, Benedikt B

2015-11-15

Virus-host cell interactions are most commonly analyzed in cells maintained in vitro as two-dimensional tissue cultures. However, these in vitro conditions vary quite drastically from the tissues that are commonly infected in vivo. Over the years, a number of systems have been developed that allow the establishment of three-dimensional (3D) tissue structures that have properties similar to their in vivo 3D counterparts. These 3D systems have numerous applications including drug testing, maintenance of large tissue explants, monitoring migration of human lymphocytes in tissues, analysis of human organ tissue development and investigation of virus-host interactions including viral latency. Here, we describe the establishment of tissue-like assemblies for human lung and neuronal tissue that we infected with a variety of viruses including the respiratory pathogens human parainfluenza virus type 3 (PIV3), respiratory syncytial virus (RSV) and SARS corona virus (SARS-CoV) as well as the human neurotropic herpesvirus, varicella-zoster virus (VZV). Copyright © 2015 Elsevier Inc. All rights reserved.

[Emerging infectious agents].

PubMed

Chidiac, C; Ferry, T

2016-11-01

Emergence of many emerging or re-emerging infectious diseases have occurred over the past decade, some of which are major public threat. SARS, MERS-CoV, highly pathogenic avian influenza A(H5N1), Ebola virus disease have raised concerns because of their virulence, their mortality, and/or their modality of transmission, or their impact on maternofoetal transmission (Zika virus). The witness of these emergences have conducted health authorities to have policies and plans and to imagine new organizations for health systems in order to identify any case of highly communicable virulent disease for immediate isolation, and adequate management. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Middle East Respiratory Syndrome Coronavirus Intra-Host Populations Are Characterized by Numerous High Frequency Variants

DOE PAGES

Borucki, Monica K.; Lao, Victoria; Hwang, Mona; ...

2016-01-20

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater thanmore » 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Lastly, many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.« less

A novel peptide with potent and broad-spectrum antiviral activities against multiple respiratory viruses

PubMed Central

Zhao, Hanjun; Zhou, Jie; Zhang, Ke; Chu, Hin; Liu, Dabin; Poon, Vincent Kwok-Man; Chan, Chris Chung-Sing; Leung, Ho-Chuen; Fai, Ng; Lin, Yong-Ping; Zhang, Anna Jin-Xia; Jin, Dong-Yan; Yuen, Kwok-Yung; Zheng, Bo-Jian

2016-01-01

A safe, potent and broad-spectrum antiviral is urgently needed to combat emerging respiratory viruses. In light of the broad antiviral activity of β-defensins, we tested the antiviral activity of 11 peptides derived from mouse β-defensin-4 and found that a short peptide, P9, exhibited potent and broad-spectrum antiviral effects against multiple respiratory viruses in vitro and in vivo, including influenza A virus H1N1, H3N2, H5N1, H7N7, H7N9, SARS-CoV and MERS-CoV. The antiviral activity of P9 was attributed to its high-affinity binding to viral glycoproteins, as well as the abundance of basic amino acids in its composition. After binding viral particles through viral surface glycoproteins, P9 entered into cells together with the viruses via endocytosis and prevented endosomal acidification, which blocked membrane fusion and subsequent viral RNA release. This study has paved the avenue for developing new prophylactic and therapeutic agents with broad-spectrum antiviral activities. PMID:26911565

Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease.

PubMed

Békés, Miklós; van der Heden van Noort, Gerbrand J; Ekkebus, Reggy; Ovaa, Huib; Huang, Tony T; Lima, Christopher D

2016-05-19

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections. Copyright © 2016 Elsevier Inc. All rights reserved.

3D Tissue-Like Assemblies: A Novel Approach to Investigate Virus-Cell Interactions

PubMed Central

Goodwin, Thomas J.; McCarthy, Maureen; Cohrs, Randall J.; Kaufer, Benedikt B.

2017-01-01

Virus-host cell interactions are most commonly analyzed in cells maintained in vitro as two-dimensional tissue cultures. However, these in vitro conditions vary quite drastically from the tissues that are commonly infected in vivo. Over the years, a number of systems have been developed that allow the establishment of three-dimensional (3D) tissue structures that have properties similar to their in vivo 3D counterparts. These 3D systems have numerous applications including drug testing, maintenance of large tissue explants, monitoring migration of human lymphocytes in tissues, analysis of human organ tissue development and investigation of virus-host interactions including viral latency. Here, we describe the establishment of tissue-like assemblies for human lung and neuronal tissue that we infected with a variety of viruses including the respiratory pathogens human parainfluenza virus type 3 (PIV3), respiratory syncytial virus (RSV) and SARS corona virus (SARS-CoV) as well as the human neurotropic herpesvirus, varicella-zoster virus (VZV) PMID:25986169

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein.

PubMed

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

2012-04-02

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 noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.

Relations of SARS-Related Stressors and Coping to Chinese College Students' Psychological Adjustment during the 2003 Beijing SARS Epidemic

ERIC Educational Resources Information Center

Main, Alexandra; Zhou, Qing; Ma, Yue; Luecken, Linda J.; Liu, Xin

2011-01-01

This study examined the main and interactive relations of stressors and coping related to severe acute respiratory syndrome (SARS) with Chinese college students' psychological adjustment (psychological symptoms, perceived general health, and life satisfaction) during the 2003 Beijing SARS epidemic. All the constructs were assessed by self-report…

Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays.

PubMed

Jadhao, Samadhan J; Anderson, Larry J

2016-01-01

Enzyme immunoassays (EIAs) to detect and quantify antibodies against respiratory syncytial virus (RSV) and RSV proteins in human plasma or sera are described. The first EIA uses RSV lysate antigens produced in HEp-2 cell line. The second EIA uses RSV F or G gene-expressed antigen in HEp-2 cells. The third EIA uses 30-amino acid synthetic peptides from central conserved region of G protein of RSV A2 or RSV B1 virus and a peptide from the SARS CoV nucleoprotein as a negative control peptide. All three EIAs have been evaluated for detecting and quantifying the respective antibodies in human sera or plasma.

Insight into the evolution of nidovirus endoribonuclease based on the finding that Nsp15 from porcine deltacoronavirus functions as a dimer.

PubMed

Zheng, Anjun; Shi, Yuejun; Shen, Zhou; Wang, Gang; Shi, Jiale; Xiong, Qiqi; Fang, Liurong; Xiao, Shaobo; Fu, Zhen F; Peng, Guiqing

2018-06-10

Nidovirus endoribonucleases (NendoUs) include Nsp15 from coronaviruses and Nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E and MHV Nsp15 indicate that it mainly forms a functional hexamer, whereas Nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine deltacoronavirus (PDCoV) Nsp15 primarily exists as dimers and monomers in vitro. Biological experiments reveal that a PDCoV Nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (NTD, Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV Nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV Nsp15. This result may explain why PDCoV Nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells and that NendoU from arterivirus gained ability to form dimers and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV Nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Conductance and amantadine binding of a pore formed by a lysine-flanked transmembrane domain of SARS coronavirus envelope protein

PubMed Central

Torres, Jaume; Maheswari, Uma; Parthasarathy, Krupakar; Ng, Lifang; Liu, Ding Xiang; Gong, Xiandi

2007-01-01

The coronavirus responsible for the severe acute respiratory syndrome (SARS-CoV) contains a small envelope protein, E, with putative involvement in host cell apoptosis and virus morphogenesis. It has been suggested that E protein can form a membrane destabilizing transmembrane (TM) hairpin, or homooligomerize to form a regular TM α-helical bundle. We have shown previously that the topology of the α-helical putative TM domain of E protein (ETM), flanked by two lysine residues at C and N termini to improve solubility, is consistent with a regular TM α-helix, with orientational parameters in lipid bilayers that are consistent with a homopentameric model. Herein, we show that this peptide, reconstituted in lipid bilayers, shows sodium conductance. Channel activity is inhibited by the anti-influenza drug amantadine, which was found to bind our preparation with moderate affinity. Results obtained from single or double mutants indicate that the organization of the transmembrane pore is consistent with our previously reported pentameric α-helical bundle model. PMID:17766393

BCX4430 - A broad-spectrum antiviral adenosine nucleoside analog under development for the treatment of Ebola virus disease.

PubMed

Taylor, Raymond; Kotian, Pravin; Warren, Travis; Panchal, Rekha; Bavari, Sina; Julander, Justin; Dobo, Sylvia; Rose, Angela; El-Kattan, Yahya; Taubenheim, Brian; Babu, Yarlagadda; Sheridan, William P

2016-01-01

The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. Cellular kinases phosphorylate BCX4430 to a triphosphate that mimics ATP; viral RNA polymerases incorporate the drug's monophosphate nucleotide into the growing RNA chain, causing premature chain termination. BCX4430 is active in vitro against many RNA viral pathogens, including the filoviruses and emerging infectious agents such as MERS-CoV and SARS-CoV. In vivo, BCX4430 is active after intramuscular, intraperitoneal, and oral administration in a variety of experimental infections. In nonclinical studies involving lethal infections with Ebola virus, Marburg virus, Rift Valley fever virus, and Yellow Fever virus, BCX4430 has demonstrated pronounced efficacy. In experiments conducted in several models, both a reduction in the viral load and an improvement in survival were found to be related to the dose of BCX4430. A Phase 1 clinical trial of intramuscular administration of BCX4430 in healthy subjects is currently ongoing. Copyright © 2016 King Saud Bin Abdulaziz University for Health Sciences. All rights reserved.

Structural and biochemical basis for the difference in the helicase activity of two different constructs of SARS-CoV helicase.

PubMed

Adedeji, A O; Singh, K; Sarafianos, S G

2012-12-22

The non—structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS—CoV) is a helicase that separates double—stranded RNA or DNA with a 5'—3' polarity, using the energy of nucleotide hydrolysis. We have previously determined the minimal mechanism of helicase function by nsp13 where we demonstrated that the enzyme unwinds nucleic acid in discrete steps of 9.3 base—pairs each with a catalytic rate of 30 steps per second. In that study we used different constructs of nsp13 (GST and H6 constructs). GST—nsp13 showed much more efficient nucleic acid unwinding than the H6—tagged counterpart. At 0.1 second, more than 50% of the ATP is hydrolyzed by GST—nsp13 compared to less than 5% ATP hydrolysis by H6—nsp13. Interestingly, the two constructs have the same binding affinity for nucleic acids. We, therefore propose that the difference in the catalytic efficiency of these two constructs is due to the interference of ATP binding by the histidine tag at the amino—terminus of nsp13.

Nucleocapsid protein-dependent assembly of the RNA packaging signal of Middle East respiratory syndrome coronavirus.

PubMed

Hsin, Wei-Chen; Chang, Chan-Hua; Chang, Chi-You; Peng, Wei-Hao; Chien, Chung-Liang; Chang, Ming-Fu; Chang, Shin C

2018-05-24

Middle East respiratory syndrome coronavirus (MERS-CoV) consists of a positive-sense, single-stranded RNA genome and four structural proteins: the spike, envelope, membrane, and nucleocapsid protein. The assembly of the viral genome into virus particles involves viral structural proteins and is believed to be mediated through recognition of specific sequences and RNA structures of the viral genome. A culture system for the production of MERS coronavirus-like particles (MERS VLPs) was determined and established by electron microscopy and the detection of coexpressed viral structural proteins. Using the VLP system, a 258-nucleotide RNA fragment, which spans nucleotides 19,712 to 19,969 of the MERS-CoV genome (designated PS258(19712-19969) ME ), was identified to function as a packaging signal. Assembly of the RNA packaging signal into MERS VLPs is dependent on the viral nucleocapsid protein. In addition, a 45-nucleotide stable stem-loop substructure of the PS258(19712-19969) ME interacted with both the N-terminal domain and the C-terminal domain of the viral nucleocapsid protein. Furthermore, a functional SARS-CoV RNA packaging signal failed to assemble into the MERS VLPs, which indicated virus-specific assembly of the RNA genome. A MERS-oV RNA packaging signal was identified by the detection of GFP expression following an incubation of MERS VLPs carrying the heterologous mRNA GFP-PS258(19712-19969) ME with virus permissive Huh7 cells. The MERS VLP system could help us in understanding virus infection and morphogenesis.

On the characteristics of a residual external signal seen in coefficients of main geomagnetic field models

NASA Astrophysics Data System (ADS)

Stefan, Cristiana; Demetrescu, Crisan; Dobrica, Venera

2014-05-01

Several recently developed main geomagnetic field models, based on both observatory and satellite data (e.g. IGRF, CHAOS, GRIMM, COV-OBS), as well as the historical model gufm1, have been designed to describe only the internal part of the field, except for the COV-OBS that also accounts for the external dipole. In this paper we analyze data and coefficients from two main field models, namely gufm1 (Jackson et al., 2000) and COV-OBS (Gillet et al., 2013), by means of low pass filters with a cutoff period of 11-year, to evidence a residual signal with seemingly external sources, superimposed on the internal part of the field. The characteristics of the residual signal in the dipole and non-dipole coefficients are discussed.

Ebola Virus and Severe Acute Respiratory Syndrome Coronavirus Display Late Cell Entry Kinetics: Evidence that Transport to NPC1+ Endolysosomes Is a Rate-Defining Step

PubMed Central

Mingo, Rebecca M.; Simmons, James A.; Shoemaker, Charles J.; Nelson, Elizabeth A.; Schornberg, Kathryn L.; D'Souza, Ryan S.; Casanova, James E.

2014-01-01

ABSTRACT Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1+ LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. IMPORTANCE Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1+ LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1+ LE/Lys, as a therapeutic target for SARS and EBOV. PMID:25552710

Ebola virus and severe acute respiratory syndrome coronavirus display late cell entry kinetics: evidence that transport to NPC1+ endolysosomes is a rate-defining step.

PubMed

Mingo, Rebecca M; Simmons, James A; Shoemaker, Charles J; Nelson, Elizabeth A; Schornberg, Kathryn L; D'Souza, Ryan S; Casanova, James E; White, Judith M

2015-03-01

Ebola virus (EBOV) causes hemorrhagic fevers with high mortality rates. During cellular entry, the virus is internalized by macropinocytosis and trafficked through endosomes until fusion between the viral and an endosomal membrane is triggered, releasing the RNA genome into the cytoplasm. We found that while macropinocytotic uptake of filamentous EBOV viruslike particles (VLPs) expressing the EBOV glycoprotein (GP) occurs relatively quickly, VLPs only begin to enter the cytoplasm after a 30-min lag, considerably later than particles bearing the influenza hemagglutinin or GP from lymphocytic choriomeningitis virus, which enter through late endosomes (LE). For EBOV, the long lag is not due to the large size or unusual shape of EBOV filaments, the need to prime EBOV GP to the 19-kDa receptor-binding species, or a need for unusually low endosomal pH. In contrast, since we observed that EBOV entry occurs upon arrival in Niemann-Pick C1 (NPC1)-positive endolysosomes (LE/Lys), we propose that trafficking to LE/Lys is a key rate-defining step. Additional experiments revealed, unexpectedly, that severe acute respiratory syndrome (SARS) S-mediated entry also begins only after a 30-min lag. Furthermore, although SARS does not require NPC1 for entry, SARS entry also begins after colocalization with NPC1. Since the only endosomal requirement for SARS entry is cathepsin L activity, we tested and provide evidence that NPC1(+) LE/Lys have higher cathepsin L activity than LE, with no detectable activity in earlier endosomes. Our findings suggest that both EBOV and SARS traffic deep into the endocytic pathway for entry and that they do so to access higher cathepsin activity. Ebola virus is a hemorrhagic fever virus that causes high fatality rates when it spreads from zoonotic vectors into the human population. Infection by severe acute respiratory syndrome coronavirus (SARS-CoV) causes severe respiratory distress in infected patients. A devastating outbreak of EBOV occurred in West Africa in 2014, and there was a significant outbreak of SARS in 2003. No effective vaccine or treatment has yet been approved for either virus. We present evidence that both viruses traffic late into the endocytic pathway, to NPC1(+) LE/Lys, in order to enter host cells, and that they do so to access high levels of cathepsin activity, which both viruses use in their fusion-triggering mechanisms. This unexpected similarity suggests an unexplored vulnerability, trafficking to NPC1(+) LE/Lys, as a therapeutic target for SARS and EBOV. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

NCI Scientists Solve Structure of Protein that Enables MERS Virus to Spread | Poster

Cancer.gov

Scientists at the Frederick National Lab have produced three crystal structures that reveal a specific part of a protein that can be targeted to fight the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an emerging viral respiratory illness. Senior Investigator David Waugh, Ph.D., Macromolecular Crystallography Laboratory, has solved the structure of an enzyme known as the 3C-like protease (3CLpro), which, if blocked, can prevent the virus from replicating...

Transmembrane Domains of Highly Pathogenic Viral Fusion Proteins Exhibit Trimeric Association In Vitro

PubMed Central

Webb, Stacy R.; Smith, Stacy E.; Fried, Michael G.

2018-01-01

ABSTRACT Enveloped viruses require viral fusion proteins to promote fusion of the viral envelope with a target cell membrane. To drive fusion, these proteins undergo large conformational changes that must occur at the right place and at the right time. Understanding the elements which control the stability of the prefusion state and the initiation of conformational changes is key to understanding the function of these important proteins. The construction of mutations in the fusion protein transmembrane domains (TMDs) or the replacement of these domains with lipid anchors has implicated the TMD in the fusion process. However, the structural and molecular details of the role of the TMD in these fusion events remain unclear. Previously, we demonstrated that isolated paramyxovirus fusion protein TMDs associate in a monomer-trimer equilibrium, using sedimentation equilibrium analytical ultracentrifugation. Using a similar approach, the work presented here indicates that trimeric interactions also occur between the fusion protein TMDs of Ebola virus, influenza virus, severe acute respiratory syndrome coronavirus (SARS CoV), and rabies virus. Our results suggest that TM-TM interactions are important in the fusion protein function of diverse viral families. IMPORTANCE Many important human pathogens are enveloped viruses that utilize membrane-bound glycoproteins to mediate viral entry. Factors that contribute to the stability of these glycoproteins have been identified in the ectodomain of several viral fusion proteins, including residues within the soluble ectodomain. Although it is often thought to simply act as an anchor, the transmembrane domain of viral fusion proteins has been implicated in protein stability and function as well. Here, using a biophysical approach, we demonstrated that the fusion protein transmembrane domains of several deadly pathogens—Ebola virus, influenza virus, SARS CoV, and rabies virus—self-associate. This observation across various viral families suggests that transmembrane domain interactions may be broadly relevant and serve as a new target for therapeutic development. PMID:29669880

Spatial pattern of severe acute respiratory syndrome in-out flow in 2003 in Mainland China.

PubMed

Xu, Chengdong; Wang, Jinfeng; Wang, Li; Cao, Chunxiang

2014-12-31

Severe acute respiratory syndrome (SARS) spread to 32 countries and regions within a few months in 2003. There were 5327 SARS cases from November 2002 to May 2003 in Mainland China, which involved 29 provinces, resulted in 349 deaths, and directly caused economic losses of $18.3 billion. This study used an in-out flow model and flow mapping to visualize and explore the spatial pattern of SARS transmission in different regions. In-out flow is measured by the in-out degree and clustering coefficient of SARS. Flow mapping is an exploratory method of spatial visualization for interaction data. The findings were as follows. (1) SARS in-out flow had a clear hierarchy. It formed two main centers, Guangdong in South China and Beijing in North China, and two secondary centers, Shanxi and Inner Mongolia, both connected to Beijing. (2) "Spring Festival travel" strengthened external flow, but "SARS panic effect" played a more significant role and pushed the external flow to the peak. (3) External flow and its three typical kinds showed obvious spatial heterogeneity, such as self-spreading flow (spatial displacement of SARS cases only within the province or municipality of onset and medical locations); hospitalized flow (spatial displacement of SARS cases that had been seen by a hospital doctor); and migrant flow (spatial displacement of SARS cases among migrant workers). (4) Internal and external flow tended to occur in younger groups, and occupational differentiation was particularly evident. Low-income groups of male migrants aged 19-35 years were the main routes of external flow. During 2002-2003, SARS in-out flow played an important role in countrywide transmission of the disease in Mainland China. The flow had obvious spatial heterogeneity, which was influenced by migrants' behavior characteristics. In addition, the Chinese holiday effect led to irregular spread of SARS, but the panic effect was more apparent in the middle and late stages of the epidemic. These findings constitute valuable input to prevent and control future serious infectious diseases like SARS.

Human monoclonal antibodies as candidate therapeutics against emerging viruses and HIV-1.

PubMed

Zhu, Zhongyu; Prabakaran, Ponraj; Chen, Weizao; Broder, Christopher C; Gong, Rui; Dimitrov, Dimiter S

2013-04-01

More than 40 monoclonal antibodies (mAbs) have been approved for a number of disease indications with only one of these (Synagis) - for a viral disease, and not for therapy but for prevention. However, in the last decade novel potent mAbs have been discovered and characterized with potential as therapeutics against viruses of major importance for public health and biosecurity including Hendra virus (HeV), Nipah virus (NiV), severe acute respiratory syndrome coronavirus (SARS-CoV), Ebola virus (EBOV), West Nile virus (WNV), influenza virus (IFV) and human immunodeficiency virus type 1 (HIV-1). Here, we review such mAbs with an emphasis on antibodies of human origin, and highlight recent results as well as technologies and mechanisms related to their potential as therapeutics.

Public response to MERS-CoV in the Middle East: iPhone survey in six countries.

PubMed

Alqahtani, Amani S; Rashid, Harunor; Basyouni, Mada H; Alhawassi, Tariq M; BinDhim, Nasser F

Gulf Cooperation Council (GCC) countries bear the heaviest brunt of MERS-CoV. This study aims to compare public awareness and practice around MERS-CoV across GCC countries. A cross-sectional survey was conducted using the Gulf Indicators (GI) smartphone app among people in the six GCC countries, namely Saudi Arabia, Kuwait, the United Arab Emirates, Qatar, Bahrain, and Oman. A total of 1812 participants recruited. All were aware of MERS-CoV, yet the perception and practice around MERS-CoV varied widely between countries. Over two thirds were either "not concerned" or "slightly concerned" about contracting MERS-CoV; believing that they were under Allah's (God's) protection (40%) was the most cited reason. While 79% were aware that the disease can transmit through droplet from infected person, only 12% stated that MERS-CoV transmits via camels; people in Saudi Arabia were better aware of the transmission. Nevertheless, only 22% of respondents believed that camels are the zoonotic reservoir of MERS-CoV. Those who were concerned about contracting MERS-CoV (aOR: 1.6, 95% CI: 1.2-2.1, p<0.01) and those who thought MERS-CoV to be a severe disease only for those with high-risk conditions (aOR: 1.5, 95% CI: 1.1-2.1, p<0.01) were more likely to believe that camels are the zoonotic source. However, residents of KSA (aOR: 0.03, 95% CI: 0.01-0.07, p<0.01), UAE (aOR: 0.01, 95% CI: 0.004-0.02, p<0.01) and Kuwait (aOR: 0.03, 95% CI: 0.01-0.07, p<0.01) were less likely to believe that camels are the main zoonotic source compared to respondents from the other countries. Hygienic measures were more commonly adopted than avoidance of camels or their raw products, yet there was a discrepancy between the countries. This study highlights that despite being aware of the ongoing MERS-CoV epidemic; many people lack accurate understanding about MERS-CoV transmission, prevention, and are not fully compliant with preventive measures. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism

PubMed Central

Phakthanakanok, Krongsakda; Ratanakhanokchai, Khanok; Kyu, Khin Lay; Sompornpisut, Pornthep; Watts, Aaron; Pinitglang, Surapong

2009-01-01

Background SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach. Results The one turn α-helix chain (residues 47–54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide. Conclusion MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide. PMID:19208150

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

PubMed Central

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

2017-01-01

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

[SARS (Severe Acute Respiratory Syndrome). Emergent transmissible disease].

PubMed

Ivan, A; Azoicăi, Doina

2003-01-01

Of the reemergent transmissible diseases of the past decades, SARS is probably not the last to express the alterations occurring in the relationships of the human being with its global ecosystem. The life of contemporary man is characterized, among others, by a huge thirst for traveling, for varied reasons, consequence of the globalization process. SARS virus, mutant belonging to Coronaviridae, occurred in one of the most densely populated areas of the world. There are two main moments marking the reemergence and evolution of SARS: firstly, the onset of the epidemic in China in November 2002 followed by the worldwide spread of the epidemiological process, and secondly the discovery of SARS virus as a mutant of coronaviruses in March-April 2003 in USA, Canada, and Hong Kong. The possibilities of general and special prevention, and particularly vaccine prevention are likely to bring this disease under control.

Detection of feline coronavirus using microcantilever sensors

NASA Astrophysics Data System (ADS)

Velanki, Sreepriya; Ji, Hai-Feng

2006-11-01

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

Surveillance of Severe Acute Respiratory Infection (SARI) for Hospitalized Patients in Northern Vietnam, 2011-2014.

PubMed

Nguyen, Hang Khanh Le; Nguyen, Son Vu; Nguyen, Anh Phuong; Hoang, Phuong Mai Vu; Le, Thanh Thi; Nguyen, Thach Co; Hoang, Huong Thu; Vuong, Cuong Duc; Tran, Loan Thi Thanh; Le, Mai Quynh

2017-09-25

Severe acute respiratory infections (SARI) are leading causes of hospitalization, morbidity, and mortality in children worldwide. The aim of this study was to identify viral pathogens responsible for SARI in northern Vietnam in the period from 2011 to 2014. Throat swabs and tracheal aspirates were collected from SARI patients according to WHO guidelines. The presence of 13 different viral pathogens (influenza A[H1N1]pdm09; A/H3N2; A/H5; A/H7 and B; para influenza 1,2,3; RSV; HMPV; adeno; severe acute respiratory syndrome-CoV and rhino) was tested by conventional/real-time reverse transcription-polymerase chain reaction. During the study period, 975 samples were collected and tested. More than 30% (32.1%, 313 samples) of the samples showed evidence of infection with influenza viruses, including A/H3N2 (48 samples), A (H1N1) pdm09 (221 samples), influenza B (42 samples), and co-infection of A (H1N1) pdm09 or A/H3N2 and influenza B (2 samples). Other respiratory pathogens were detected in 101 samples, including rhinovirus (73 samples), adenovirus (10 samples), hMPV (9 samples), parainfluenza 3 (5 samples), parainfluenza 2 (3 samples), and RSV (1 sample). Influenza A/H5, A/H7, or SARS-CoV were not detected. Respiratory viral infection, particularly infection of influenza and rhinoviruses, were associated with high rates of SARI hospitalization, and future studies correlating the clinical aspects are needed to design interventions, including targeted vaccination.

[Preliminary result on the nosocomial infection of severe acute respiratory syndrome in one hospital of Beijing].

PubMed

He, Yao; Jiang, Yong; Xing, Yu-bin; Zhong, Guang-lin; Wang, Lei; Sun, Zheng-ji; Jia, Hong; Chang, Qing; Wang, Yong; Ni, Bin; Chen, Shi-ping

2003-07-01

To study the transmission route of severe acute respiratory syndrome (SARS) nosocomial infection. Ten identified SARS patients were selected from a general hospital in March. Survey was carried out through a standardized questionnaire provided by Chinese Center for Disease Control and Prevention. Contents of the questionnaire would include: history of contact with SARS patient, route of infection, methods used for protection and so on. (1) Distribution os SARS patients were confined to 3 wards: 4, 5, and 6 on the 7, 8, 12, 13 and 14 floors in the west unit of the inpatient building. Most of the inpatients were elderly and having severe original diseases. (2) Index patients were the first generation source of transmission and they infected inpatients and medical staff, making them the second generation. People with latent infection who had close contact with SARS patients might also serve as the possible source of transmission. (3) The major transmission routes were: near distant droplet infection and close contact infection. There was also a clue to the probability of aerosol or droplet nuclei infection through air-conditioning and ventilation system. Nosocomial infection appeared to be the main characteristic of the SARS epidemic in the early stage of this hospital. Other than close contact and near space airborne transmission of SARS virus, the possibility of long-distance aerosol transmission called for further epidemiological and experimental studies in the future.

Healthcare Workers Emotions, Perceived Stressors and Coping Strategies During a MERS-CoV Outbreak.

PubMed

Khalid, Imran; Khalid, Tabindeh J; Qabajah, Mohammed R; Barnard, Aletta G; Qushmaq, Ismael A

2016-03-01

Healthcare workers (HCWs) are at high risk of contracting Middle East respiratory syndrome coronavirus (MERS-CoV) during an epidemic. We explored the emotions, perceived stressors, and coping strategies of healthcare workers who worked during a MERS-CoV outbreak in our hospital. A cross-sectional descriptive survey design. A tertiary care hospital. HCWs (150) who worked in high risk areas during the April-May 2014 MERS-CoV outbreak that occurred in Jeddah, Saudi Arabia. We developed and administered a "MERS-CoV staff questionnaire" to study participants. The questionnaire consisted of 5 sections with 72 questions. The sections evaluated hospital staffs emotions, perceived stressors, factors that reduced their stress, coping strategies, and motivators to work during future outbreaks. Responses were scored on a scale from 0-3. The varying levels of stress or effectiveness of measures were reported as mean and standard deviation, as appropriate. Completed questionnaires were returned by 117 (78%) of the participants. The results had many unique elements. HCWs ethical obligation to their profession pushed them to continue with their jobs. The main sentiments centered upon fear of personal safety and well-being of colleagues and family. Positive attitudes in the workplace, clinical improvement of infected colleagues, and stoppage of disease transmission among HCWs after adopting strict protective measures alleviated their fear and drove them through the epidemic. They appreciated recognition of their efforts by hospital management and expected similar acknowledgment, infection control guidance, and equipment would entice them to work during future epidemics. The MERS-CoV outbreak was a distressing time for our staff. Hospitals can enhance HCWs experiences during any future MERS-CoV outbreak by focusing on the above mentioned aspects. © 2016 Marshfield Clinic.

Nosocomial infection control in healthcare settings: Protection against emerging infectious diseases.

PubMed

Fu, Chuanxi; Wang, Shengyong

2016-04-12

The Middle East respiratory syndrome (MERS) outbreak in Korea in 2015 may be attributable to poor nosocomial infection control procedures implemented. Strict infection control measures were taken in the hospital where an imported case with MERS was treated in southern China and 53 health care workers were confirmed to be MERS-CoV negative. Infection control in healthcare settings, in which patients with emerging infectious diseases such as MERS, Ebola virus disease, and the severe acute respiratory syndrome (SARS) are diagnosed and treated, are often imperfect. When it comes to emerging or unknown infectious diseases, before the imported case was finally identified or community transmission was reported, cases have often occurred in clusters in healthcare settings. Nosocomial infection control measures should be further strengthened among the workers and inpatients in designated healthcare settings that accommodate suspected cases suffering from emerging or unknown infectious diseases.

Thin-Section Computed Tomography Manifestations During Convalescence and Long-Term Follow-Up of Patients with Severe Acute Respiratory Syndrome (SARS).

PubMed

Wu, Xiaohua; Dong, Dawei; Ma, Daqing

2016-08-08

BACKGROUND SARS is not only an acute disease, but also leads to long-term impaired lung diffusing capacity in some survivors. However, there is a paucity of data regarding long-term CT findings in survivors after SARS. The aim of this study was to assess the changes in lung function and lung thin-section computed tomography (CT) features in patients recovering from severe acute respiratory syndrome (SARS), especially the dynamic changes in ground-glass opacity (GGO). MATERIAL AND METHODS Clinical and radiological data from 11 patients with SARS were collected. The serial follow-up thin-section CTs were evaluated at 3, 6, and 84 months after SARS presentation. The distribution and predominant thin-section CT findings of lesions were evaluated. RESULTS The extent of the lesions on the CT scans of the 11 patients decreased at 6 and 84 months compared to 3 months. The number of segments involved on 84-month follow-up CTs was less than those at 6 months (P<0.05). The predominant thin-section CT manifestation at 84 months (intralobular and interlobular septal thickening) was different than that at 6 months, at which GGO was predominant. CONCLUSIONS During convalescence after SARS, GGO and intralobular and interlobular septal thickening were the main thin-section CT manifestation. Intralobular and interlobular septal thickening predominated over GGO at 84 months.

Knowledge of severe acute respiratory syndrome among community physicians, nurses, and emergency medical responders.

PubMed

Tice, Alan Douglas; Kishimoto, Mitsumasa; Dinh, Chuong Hoang; Lam, Geoffrey Tak-Kin; Marineau, Michelle

2006-01-01

The preparedness levels of front-line clinicians including physicians, nurses, emergency medical responders (EMRs), and other medical staff working in clinics, offices and ambulatory care centers must be assessed, so these personnel are able to deal with communicable and potentially lethal diseases, such as severe acute respiratory syndrome (SARS). In order to determine the knowledge of these clinicians, a survey of their understanding of SARS and their use of educational resources was administered. A questionnaire was distributed to physicians, nurses, and EMRs attending conferences on SARS in the summer of 2003. Questions related to information sources, knowledge of SARS, and plans implemented in their workplace to deal with it. Statistical analysis was performed using the Statistical Package for the Social Sciences (10.1 Program, SPSS Inc., Chicago, Illinois). A total of 201 community healthcare providers (HCPs) participated in the study. A total of 51% of the participants correctly identified the incubation period of SARS; 48% correctly identified the symptoms of SARS; and 60% knew the recommended infection control precautions to take for families. There was little difference in knowledge among the physicians, nurses, and EMRs evaluated. Media outlets such as newspapers, journals, television, and radio were reported as the main sources of information on SARS. However, there appears to be a growing use of the Internet, which correlated best with the correct answers on symptoms of SARS. Fewer than one-third of respondents were aware of a protocol for SARS in their workplace. A total of 60% reported that N-95 masks were available in their workplace. These findings suggest the need for more effective means of education and training for front-line clinicians, as well as the institution of policies and procedures in medical offices, clinics, and emergency services in the community.

A computerized scheme of SARS detection in early stage based on chest image of digital radiograph

NASA Astrophysics Data System (ADS)

Zheng, Zhong; Lan, Rihui; Lv, Guozheng

2004-05-01

A computerized scheme for early severe acute respiratory syndrome(SARS) lesion detection in digital chest radiographs is presented in this paper. The total scheme consists of two main parts: the first part is to determine suspect lesions by the theory of locally orderless images(LOI) and their spatial features; the second part is to select real lesions among these suspect ones by their frequent features. The method we used in the second part is firstly developed by Katsuragawa et al with necessary modification. Preliminary results indicate that these features are good criterions to tell early SARS lesions apart from other normal lung structures.

How did general practitioners protect themselves, their family, and staff during the SARS epidemic in Hong Kong?

PubMed Central

Wong, W; Lee, A; Tsang, K; Wong, S

2004-01-01

Context: Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease and how the frontline community doctors respond to it is not known. Objectives: To explore the impact of SARS on general practitioners (GPs) in Hong Kong. Design: A cross sectional survey. Setting: Community based primary care clinics. Participants: 183 family medicine tutors affiliated with a local university. Postal survey sent to all tutors with a 74.8% response rate. Main outcome measures: Change of clinical behaviour and practices during the epidemic; anxiety level of primary care doctors. Results: All agreed SARS had changed their clinical practices. Significant anxiety was found in family doctors. Three quarters of respondents recalled requesting more investigations while a quarter believed they had over-prescribed antibiotics. GPs who were exposed to SARS or who had worked in high infection districts were less likely to quarantine themselves (10.8% versus 33.3%; p<0.01; 6.5% versus 27.5%; p<0.01 respectively). Exposure to SARS, the infection rates in their working district, and anxiety levels had significant impact on the level of protection or prescribing behaviour. Conclusion: The clinical practice of GPs changed significantly as a result of SARS. Yet, those did not quarantine themselves suggesting other factors may have some part to play. As failure to apply isolation precautions to suspected cases of SARS was one major reason for its spread, a contingency plan from the government to support family doctors is of utmost importance. Interface between private and public sectors are needed in Hong Kong to prepare for any future epidemics. PMID:14966227

Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells.

PubMed

Bleau, Christian; Filliol, Aveline; Samson, Michel; Lamontagne, Lucie

2015-10-01

Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain. Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells

PubMed Central

Bleau, Christian; Filliol, Aveline; Samson, Michel

2015-01-01

ABSTRACT Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain. IMPORTANCE Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion. PMID:26202229

Alphacoronaviruses in New World Bats: Prevalence, Persistence, Phylogeny, and Potential for Interaction with Humans

USGS Publications Warehouse

Osborne, Christina; Cryan, Paul M.; O'Shea, Thomas J.; Oko, Lauren M.; Ndaluka, Christina; Calisher, Charles H.; Berglund, Andrew D.; Klavetter, Mead L.; Holmes, Kathryn V.; Dominguez, Samuel R.; Montgomery, Joel Mark

2011-01-01

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.

Alphacoronaviruses in new World bats: Prevalence, persistence, phylogeny, and potential for interaction with humans

USGS Publications Warehouse

Osborne, C.; Cryan, P.M.; O'Shea, T.J.; Oko, L.M.; Ndaluka, C.; Calisher, C.H.; Berglund, A.D.; Klavetter, M.L.; Bowen, R.A.; Holmes, K.V.; Dominguez, S.R.

2011-01-01

Bats are reservoirs for many different coronaviruses (CoVs) as well as many other important zoonotic viruses. We sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within Colorado from 2007 to 2009. We detected alphacoronavirus RNA in bats of 4 species: big brown bats (Eptesicus fuscus), 10% prevalence; long-legged bats (Myotis volans), 8% prevalence; little brown bats (Myotis lucifugus), 3% prevalence; and western long-eared bats (Myotis evotis), 2% prevalence. Overall, juvenile bats were twice as likely to be positive for CoV RNA as adult bats. At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. CoV RNA was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. Individually tagged big brown bats that were positive for CoV RNA and later sampled again all became CoV RNA negative. Nucleotide sequences in the RdRp gene fell into 3 main clusters, all distinct from those of Old World bats. Similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a CoV may be capable of infecting bats of different genera. These data suggest that ongoing evolution of CoVs in bats creates the possibility of a continued threat for emergence into hosts of other species. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. Further CoV surveillance studies in bats throughout the Americas are warranted.

[Development of a SPA-ELISA method for detecting anti-coronavirus IgG antibodies in serum samples from fulvous fruit bats].

PubMed

Zhou, Jie; Liao, Yu-xue; Chen, Zhong; Li, Yu-chun; Gao, Lu-Lu; Chen, Yi-xiong; Cai, Lian-gong; Chen, Qing; Yu, Shou-yi

2008-05-01

To develop an simple and sensitive method for detecting anti-coronavirus IgG antibodies in bat sera based on enzyme-linked immunosorbent assay (ELISA). A commercial ELISA kit for detecting SARS-CoV antibody was modified for detecting coronavirus antibodies in bat serum samples. The second antibody in the kit was replaced with horseradish peroxidase-conjugated protein-A (HRP-SPA) based on the characteristics of binding between Staphylococcus aureus protein A (SPA) and mammal IgG Fc fragment. The sera of 55 fulvous fruit bats (Rousettus dasymallus) were tested using the SPA-ELISA. The test results of the positive and negative controls in the kit and the serum samples from convalescent ;patient were consistent with expectation. Coronavirus antibody was detected in 2 out of the 55 bat serum samples. Serum neutralization test confirmed the validity of the SPA-ELISA method. This SPA-ELISA method is applicable for detecting coronavirus antibody in bat sera.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allen, J; Velsko, S

This report explores the question of whether meaningful conclusions can be drawn regarding the transmission relationship between two microbial samples on the basis of differences observed between the two sample's respective genomes. Unlike similar forensic applications using human DNA, the rapid rate of microbial genome evolution combined with the dynamics of infectious disease require a shift in thinking on what it means for two samples to 'match' in support of a forensic hypothesis. Previous outbreaks for SARS-CoV, FMDV and HIV were examined to investigate the question of how microbial sequence data can be used to draw inferences that link twomore » infected individuals by direct transmission. The results are counter intuitive with respect to human DNA forensic applications in that some genetic change rather than exact matching improve confidence in inferring direct transmission links, however, too much genetic change poses challenges, which can weaken confidence in inferred links. High rates of infection coupled with relatively weak selective pressure observed in the SARS-CoV and FMDV data lead to fairly low confidence for direct transmission links. Confidence values for forensic hypotheses increased when testing for the possibility that samples are separated by at most a few intermediate hosts. Moreover, the observed outbreak conditions support the potential to provide high confidence values for hypothesis that exclude direct transmission links. Transmission inferences are based on the total number of observed or inferred genetic changes separating two sequences rather than uniquely weighing the importance of any one genetic mismatch. Thus, inferences are surprisingly robust in the presence of sequencing errors provided the error rates are randomly distributed across all samples in the reference outbreak database and the novel sequence samples in question. When the number of observed nucleotide mutations are limited due to characteristics of the outbreak or the availability of only partial rather than whole genome sequencing, indel information was shown to have the potential to improve performance but only for select outbreak conditions. In examined HIV transmission cases, extended evolution proved to be the limiting factor in assigning high confidence to transmission links, however, the potential to correct for extended evolution not associated with transmission events is demonstrated. Outbreak specific conditions such as selective pressure (in the form of varying mutation rate), are shown to impact the strength of inference made and a Monte Carlo simulation tool is introduced, which is used to provide upper and lower bounds on the confidence values associated with a forensic hypothesis.« less

Chalcone scaffolds as anti-infective agents: structural and molecular target perspectives.

PubMed

Mahapatra, Debarshi Kar; Bharti, Sanjay Kumar; Asati, Vivek

2015-08-28

In recent years, widespread outbreak of numerous infectious diseases across the globe has created havoc among the population. Particularly, the inhabitants of tropical and sub-tropical regions are mainly affected by these pathogens. Several natural and (semi) synthetic chalcones deserve the credit of being potential anti-infective candidates that inhibit various parasitic, malarial, bacterial, viral, and fungal targets like cruzain-1/2, trypanopain-Tb, trans-sialidase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fumarate reductase, falcipain-1/2, β-hematin, topoisomerase-II, plasmepsin-II, lactate dehydrogenase, protein kinases (Pfmrk and PfPK5), and sorbitol-induced hemolysis, DEN-1 NS3, H1N1, HIV (Integrase/Protease), protein tyrosine phosphatase A/B (Ptp-A/B), FtsZ, FAS-II, lactate/isocitrate dehydrogenase, NorA efflux pump, DNA gyrase, fatty acid synthase, chitin synthase, and β-(1,3)-glucan synthase. In this review, a comprehensive study (from Jan. 1982 to May 2015) of the structural features of anti-infective chalcones, their mechanism of actions (MOAs) and structure activity relationships (SARs) have been highlighted. With the knowledge of molecular targets, structural insights and SARs, this review may be helpful for (medicinal) chemists to design more potent, safe, selective and cost effective anti-infective agents. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Super-spreaders and the rate of transmission of the SARS virus

NASA Astrophysics Data System (ADS)

Small, Michael; Tse, C. K.; Walker, David M.

2006-03-01

We describe a stochastic small-world network model of transmission of the SARS virus. Unlike the standard Susceptible-Infected-Removed models of disease transmission, our model exhibits both geographically localised outbreaks and “super-spreaders”. Moreover, the combination of localised and long range links allows for more accurate modelling of partial isolation and various public health policies. From this model, we derive an expression for the probability of a widespread outbreak and a condition to ensure that the epidemic is controlled. Moreover, multiple simulations are used to make predictions of the likelihood of various eventual scenarios for fixed initial conditions. The main conclusions of this study are: (i) “super-spreaders” may occur even if the infectiousness of all infected individuals is constant; (ii) consistent with previous reports, extended exposure time beyond 3-5 days (i.e. significant nosocomial transmission) was the key factor in the severity of the SARS outbreak in Hong Kong; and, (iii) the spread of SARS can be effectively controlled by either limiting long range links (imposing a partial quarantine) or enforcing rapid hospitalisation and isolation of symptomatic individuals.

Replication and shedding of MERS-CoV in Jamaican fruit bats (Artibeus jamaicensis)

PubMed Central

Munster, Vincent J.; Adney, Danielle R.; van Doremalen, Neeltje; Brown, Vienna R.; Miazgowicz, Kerri L.; Milne-Price, Shauna; Bushmaker, Trenton; Rosenke, Rebecca; Scott, Dana; Hawkinson, Ann; de Wit, Emmie; Schountz, Tony; Bowen, Richard A.

2016-01-01

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) highlights the zoonotic potential of Betacoronaviruses. Investigations into the origin of MERS-CoV have focused on two potential reservoirs: bats and camels. Here, we investigated the role of bats as a potential reservoir for MERS-CoV. In vitro, the MERS-CoV spike glycoprotein interacted with Jamaican fruit bat (Artibeus jamaicensis) dipeptidyl peptidase 4 (DPP4) receptor and MERS-CoV replicated efficiently in Jamaican fruit bat cells, suggesting there is no restriction at the receptor or cellular level for MERS-CoV. To shed light on the intrinsic host-virus relationship, we inoculated 10 Jamaican fruit bats with MERS-CoV. Although all bats showed evidence of infection, none of the bats showed clinical signs of disease. Virus shedding was detected in the respiratory and intestinal tract for up to 9 days. MERS-CoV replicated transiently in the respiratory and, to a lesser extent, the intestinal tracts and internal organs; with limited histopathological changes observed only in the lungs. Analysis of the innate gene expression in the lungs showed a moderate, transient induction of expression. Our results indicate that MERS-CoV maintains the ability to replicate in bats without clinical signs of disease, supporting the general hypothesis of bats as ancestral reservoirs for MERS-CoV. PMID:26899616

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

PubMed

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

2016-01-01

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

Using autoregressive integrated moving average (ARIMA) models to predict and monitor the number of beds occupied during a SARS outbreak in a tertiary hospital in Singapore.

PubMed

Earnest, Arul; Chen, Mark I; Ng, Donald; Sin, Leo Yee

2005-05-11

The main objective of this study is to apply autoregressive integrated moving average (ARIMA) models to make real-time predictions on the number of beds occupied in Tan Tock Seng Hospital, during the recent SARS outbreak. This is a retrospective study design. Hospital admission and occupancy data for isolation beds was collected from Tan Tock Seng hospital for the period 14th March 2003 to 31st May 2003. The main outcome measure was daily number of isolation beds occupied by SARS patients. Among the covariates considered were daily number of people screened, daily number of people admitted (including observation, suspect and probable cases) and days from the most recent significant event discovery. We utilized the following strategy for the analysis. Firstly, we split the outbreak data into two. Data from 14th March to 21st April 2003 was used for model development. We used structural ARIMA models in an attempt to model the number of beds occupied. Estimation is via the maximum likelihood method using the Kalman filter. For the ARIMA model parameters, we considered the simplest parsimonious lowest order model. We found that the ARIMA (1,0,3) model was able to describe and predict the number of beds occupied during the SARS outbreak well. The mean absolute percentage error (MAPE) for the training set and validation set were 5.7% and 8.6% respectively, which we found was reasonable for use in the hospital setting. Furthermore, the model also provided three-day forecasts of the number of beds required. Total number of admissions and probable cases admitted on the previous day were also found to be independent prognostic factors of bed occupancy. ARIMA models provide useful tools for administrators and clinicians in planning for real-time bed capacity during an outbreak of an infectious disease such as SARS. The model could well be used in planning for bed-capacity during outbreaks of other infectious diseases as well.

One-Health: a Safe, Efficient, Dual-Use Vaccine for Humans and Animals against Middle East Respiratory Syndrome Coronavirus and Rabies Virus.

PubMed

Wirblich, Christoph; Coleman, Christopher M; Kurup, Drishya; Abraham, Tara S; Bernbaum, John G; Jahrling, Peter B; Hensley, Lisa E; Johnson, Reed F; Frieman, Matthew B; Schnell, Matthias J

2017-01-15

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 and is a highly pathogenic respiratory virus. There are no treatment options against MERS-CoV for humans or animals, and there are no large-scale clinical trials for therapies against MERS-CoV. To address this need, we developed an inactivated rabies virus (RABV) that contains the MERS-CoV spike (S) protein expressed on its surface. Our initial recombinant vaccine, BNSP333-S, expresses a full-length wild-type MERS-CoV S protein; however, it showed significantly reduced viral titers compared to those of the parental RABV strain and only low-level incorporation of full-length MERS-CoV S into RABV particles. Therefore, we developed a RABV-MERS vector that contained the MERS-CoV S1 domain of the MERS-CoV S protein fused to the RABV G protein C terminus (BNSP333-S1). BNSP333-S1 grew to titers similar to those of the parental vaccine vector BNSP333, and the RABV G-MERS-CoV S1 fusion protein was efficiently expressed and incorporated into RABV particles. When we vaccinated mice, chemically inactivated BNSP333-S1 induced high-titer neutralizing antibodies. Next, we challenged both vaccinated mice and control mice with MERS-CoV after adenovirus transduction of the human dipeptidyl peptidase 4 (hDPP4) receptor and then analyzed the ability of mice to control MERS-CoV infection. Our results demonstrated that vaccinated mice were fully protected from the MERS-CoV challenge, as indicated by the significantly lower MERS-CoV titers and MERS-CoV and mRNA levels in challenged mice than those in unvaccinated controls. These data establish that an inactivated RABV-MERS S-based vaccine may be effective for use in animals and humans in areas where MERS-CoV is endemic. Rabies virus-based vectors have been proven to be efficient dual vaccines against rabies and emergent infectious diseases such as Ebola virus. Here we show that inactivated rabies virus particles containing the MERS-CoV S1 protein induce potent immune responses against MERS-CoV and RABV. This novel vaccine is easy to produce and may be useful to protect target animals, such as camels, as well as humans from deadly MERS-CoV and RABV infections. Our results indicate that this vaccine approach can prevent disease, and the RABV-based vaccine platform may be a valuable tool for timely vaccine development against emerging infectious diseases. Copyright © 2017 American Society for Microbiology.

Big Data and the Global Public Health Intelligence Network (GPHIN)

PubMed Central

Dion, M; AbdelMalik, P; Mawudeku, A

2015-01-01

Background Globalization and the potential for rapid spread of emerging infectious diseases have heightened the need for ongoing surveillance and early detection. The Global Public Health Intelligence Network (GPHIN) was established to increase situational awareness and capacity for the early detection of emerging public health events. Objective To describe how the GPHIN has used Big Data as an effective early detection technique for infectious disease outbreaks worldwide and to identify potential future directions for the GPHIN. Findings Every day the GPHIN analyzes over more than 20,000 online news reports (over 30,000 sources) in nine languages worldwide. A web-based program aggregates data based on an algorithm that provides potential signals of emerging public health events which are then reviewed by a multilingual, multidisciplinary team. An alert is sent out if a potential risk is identified. This process proved useful during the Severe Acute Respiratory Syndrome (SARS) outbreak and was adopted shortly after by a number of countries to meet new International Health Regulations that require each country to have the capacity for early detection and reporting. The GPHIN identified the early SARS outbreak in China, was credited with the first alert on MERS-CoV and has played a significant role in the monitoring of the Ebola outbreak in West Africa. Future developments are being considered to advance the GPHIN’s capacity in light of other Big Data sources such as social media and its analytical capacity in terms of algorithm development. Conclusion The GPHIN’s early adoption of Big Data has increased global capacity to detect international infectious disease outbreaks and other public health events. Integration of additional Big Data sources and advances in analytical capacity could further strengthen the GPHIN’s capability for timely detection and early warning. PMID:29769954

Effective inhibition of MERS-CoV infection by resveratrol.

PubMed

Lin, Shih-Chao; Ho, Chi-Tang; Chuo, Wen-Ho; Li, Shiming; Wang, Tony T; Lin, Chi-Chen

2017-02-13

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is an emerging viral pathogen that causes severe morbidity and mortality. Up to date, there is no approved or licensed vaccine or antiviral medicines can be used to treat MERS-CoV-infected patients. Here, we analyzed the antiviral activities of resveratrol, a natural compound found in grape seeds and skin and in red wine, against MERS-CoV infection. We performed MTT and neutral red uptake assays to assess the survival rates of MERS-infected Vero E6 cells. In addition, quantitative PCR, western blotting, and immunofluorescent assays determined the intracellular viral RNA and protein expression. For viral productivity, we utilized plaque assays to confirm the antiviral properties of resveratrol against MERS-CoV. Resveratrol significantly inhibited MERS-CoV infection and prolonged cellular survival after virus infection. We also found that the expression of nucleocapsid (N) protein essential for MERS-CoV replication was decreased after resveratrol treatment. Furthermore, resveratrol down-regulated the apoptosis induced by MERS-CoV in vitro. By consecutive administration of resveratrol, we were able to reduce the concentration of resveratrol while achieving inhibitory effectiveness against MERS-CoV. In this study, we first demonstrated that resveratrol is a potent anti-MERS agent in vitro. We perceive that resveratrol can be a potential antiviral agent against MERS-CoV infection in the near future.

A comprehensive collection of systems biology data characterizing the host response to viral infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aevermann, Brian D.; Pickett, Brett E.; Kumar, Sanjeev

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archivedmore » at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). As a result, by comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.« less

Middle East respiratory syndrome coronavirus (MERS-CoV): animal to human interaction

PubMed Central

Omrani, Ali S.; Al-Tawfiq, Jaffar A.

2015-01-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel enzootic betacoronavirus that was first described in September 2012. The clinical spectrum of MERS-CoV infection in humans ranges from an asymptomatic or mild respiratory illness to severe pneumonia and multi-organ failure; overall mortality is around 35.7%. Bats harbour several betacoronaviruses that are closely related to MERS-CoV but more research is needed to establish the relationship between bats and MERS-CoV. The seroprevalence of MERS-CoV antibodies is very high in dromedary camels in Eastern Africa and the Arabian Peninsula. MERS-CoV RNA and viable virus have been isolated from dromedary camels, including some with respiratory symptoms. Furthermore, near-identical strains of MERS-CoV have been isolated from epidemiologically linked humans and camels, confirming inter-transmission, most probably from camels to humans. Though inter-human spread within health care settings is responsible for the majority of reported MERS-CoV cases, the virus is incapable at present of causing sustained human-to-human transmission. Clusters can be readily controlled with implementation of appropriate infection control procedures. Phylogenetic and sequencing data strongly suggest that MERS-CoV originated from bat ancestors after undergoing a recombination event in the spike protein, possibly in dromedary camels in Africa, before its exportation to the Arabian Peninsula along the camel trading routes. MERS-CoV serosurveys are needed to investigate possible unrecognized human infections in Africa. Amongst the important measures to control MERS-CoV spread are strict regulation of camel movement, regular herd screening and isolation of infected camels, use of personal protective equipment by camel handlers and enforcing rules banning all consumption of unpasteurized camel milk and urine. PMID:26924345

A comprehensive collection of systems biology data characterizing the host response to viral infection

PubMed Central

Aevermann, Brian D.; Pickett, Brett E.; Kumar, Sanjeev; Klem, Edward B.; Agnihothram, Sudhakar; Askovich, Peter S.; Bankhead, Armand; Bolles, Meagen; Carter, Victoria; Chang, Jean; Clauss, Therese R.W.; Dash, Pradyot; Diercks, Alan H.; Eisfeld, Amie J.; Ellis, Amy; Fan, Shufang; Ferris, Martin T.; Gralinski, Lisa E.; Green, Richard R.; Gritsenko, Marina A.; Hatta, Masato; Heegel, Robert A.; Jacobs, Jon M.; Jeng, Sophia; Josset, Laurence; Kaiser, Shari M.; Kelly, Sara; Law, G. Lynn; Li, Chengjun; Li, Jiangning; Long, Casey; Luna, Maria L.; Matzke, Melissa; McDermott, Jason; Menachery, Vineet; Metz, Thomas O.; Mitchell, Hugh; Monroe, Matthew E.; Navarro, Garnet; Neumann, Gabriele; Podyminogin, Rebecca L.; Purvine, Samuel O.; Rosenberger, Carrie M.; Sanders, Catherine J.; Schepmoes, Athena A.; Shukla, Anil K.; Sims, Amy; Sova, Pavel; Tam, Vincent C.; Tchitchek, Nicolas; Thomas, Paul G.; Tilton, Susan C.; Totura, Allison; Wang, Jing; Webb-Robertson, Bobbie-Jo; Wen, Ji; Weiss, Jeffrey M.; Yang, Feng; Yount, Boyd; Zhang, Qibin; McWeeney, Shannon; Smith, Richard D.; Waters, Katrina M.; Kawaoka, Yoshihiro; Baric, Ralph; Aderem, Alan; Katze, Michael G.; Scheuermann, Richard H.

2014-01-01

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection. PMID:25977790

SARS related preventive and risk behaviours practised by Hong Kong-mainland China cross border travellers during the outbreak of the SARS epidemic in Hong Kong

PubMed Central

Lau, J.; Yang, X.; Tsui, H; Pang, E.

2004-01-01

Objectives: To investigate patterns of behaviours and attitudes related to SARS prevention in the Hong Kong cross border traveller population. Settings: A survey was carried out at the Hong Kong-China cross border checkpoint in the middle of the epidemic. Participants: A total of 839 Hong Kong adult residents returning to Hong Kong from mainland China were surveyed. Main outcome measures: Practice of preventive measures and relevant behaviours and attitudes. Results: Around 40% of the respondents were using masks all or most of the time in public places or washing their hands frequently (>10 times per day) and about one third avoided visiting crowded places in mainland China. Such figures were however lower than those practised by the general public in Hong Kong. SARS related perceptions, such as perceived risk of transmission and efficacy, etc, were associated with mask use and not visiting crowded places, but not with hand washing, which was associated with duration of stay. Gender differences were also observed. Around 70% of the travellers would have delayed medical consultation for influenza-like illness in China; 12.7% would not wear masks during such episodes of illness. Furthermore, about 30% of the respondents used to wear masks in Hong Kong but not in mainland China. Conclusions: The findings have implications on cross border prevention of SARS. It seems that those travelling during the SARS epidemic were a "self selected" group, and they were using less preventive measures. Special attention and intervention need to be provided to travellers to prevent a second wave cross border transmission of the disease. PMID:15547057

A comprehensive collection of systems biology data characterizing the host response to viral infection

DOE PAGES

Aevermann, Brian D.; Pickett, Brett E.; Kumar, Sanjeev; ...

2014-10-14

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archivedmore » at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). As a result, by comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.« less

Disinfection of aircraft : Appropriate disinfectants and standard operating procedures for highly infectious diseases.

PubMed

Klaus, Joachim; Gnirs, Peter; Hölterhoff, Sabine; Wirtz, Angela; Jeglitza, Matthias; Gaber, Walter; Gottschalk, Rene

2016-12-01

For infectious diseases caused by highly pathogenic agents (e. g., Ebola/Lassa fever virus, SARS-/MERS-CoV, pandemic influenza virus) which have the potential to spread over several continents within only a few days, international Health Protection Authorities have taken appropriate measures to limit the consequences of a possible spread. A crucial point in this context is the disinfection of an aircraft that had a passenger on board who is suspected of being infected with one of the mentioned diseases. Although, basic advice on hygiene and sanitation on board an aircraft is given by the World Health Organization, these guidelines lack details on available and effective substances as well as standardized operating procedures (SOP). The purpose of this paper is to give guidance on the choice of substances that were tested by a laboratory of Lufthansa Technik and found compatible with aircraft components, as well as to describe procedures which ensure a safe and efficient disinfection of civil aircrafts. This guidance and the additional SOPs are made public and are available as mentioned in this paper.

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

NASA Astrophysics Data System (ADS)

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

Conformational Flexibility of a Short Loop near the Active Site of the SARS-3CLpro is Essential to Maintain Catalytic Activity

NASA Astrophysics Data System (ADS)

Li, Chunmei; Teng, Xin; Qi, Yifei; Tang, Bo; Shi, Hailing; Ma, Xiaomin; Lai, Luhua

2016-02-01

The SARS 3C-like proteinase (SARS-3CLpro), which is the main proteinase of the SARS coronavirus, is essential to the virus life cycle. This enzyme has been shown to be active as a dimer in which only one protomer is active. However, it remains unknown how the dimer structure maintains an active monomer conformation. It has been observed that the Ser139-Leu141 loop forms a short 310-helix that disrupts the catalytic machinery in the inactive monomer structure. We have tried to disrupt this helical conformation by mutating L141 to T in the stable inactive monomer G11A/R298A/Q299A. The resulting tetra-mutant G11A/L141T/R298A/Q299A is indeed enzymatically active as a monomer. Molecular dynamics simulations revealed that the L141T mutation disrupts the 310-helix and helps to stabilize the active conformation. The coil-310-helix conformational transition of the Ser139-Leu141 loop serves as an enzyme activity switch. Our study therefore indicates that the dimer structure can stabilize the active conformation but is not a required structure in the evolution of the active enzyme, which can also arise through simple mutations.

A comprehensive collection of systems biology data characterizing the host response to viral infection.

PubMed

Aevermann, Brian D; Pickett, Brett E; Kumar, Sanjeev; Klem, Edward B; Agnihothram, Sudhakar; Askovich, Peter S; Bankhead, Armand; Bolles, Meagen; Carter, Victoria; Chang, Jean; Clauss, Therese R W; Dash, Pradyot; Diercks, Alan H; Eisfeld, Amie J; Ellis, Amy; Fan, Shufang; Ferris, Martin T; Gralinski, Lisa E; Green, Richard R; Gritsenko, Marina A; Hatta, Masato; Heegel, Robert A; Jacobs, Jon M; Jeng, Sophia; Josset, Laurence; Kaiser, Shari M; Kelly, Sara; Law, G Lynn; Li, Chengjun; Li, Jiangning; Long, Casey; Luna, Maria L; Matzke, Melissa; McDermott, Jason; Menachery, Vineet; Metz, Thomas O; Mitchell, Hugh; Monroe, Matthew E; Navarro, Garnet; Neumann, Gabriele; Podyminogin, Rebecca L; Purvine, Samuel O; Rosenberger, Carrie M; Sanders, Catherine J; Schepmoes, Athena A; Shukla, Anil K; Sims, Amy; Sova, Pavel; Tam, Vincent C; Tchitchek, Nicolas; Thomas, Paul G; Tilton, Susan C; Totura, Allison; Wang, Jing; Webb-Robertson, Bobbie-Jo; Wen, Ji; Weiss, Jeffrey M; Yang, Feng; Yount, Boyd; Zhang, Qibin; McWeeney, Shannon; Smith, Richard D; Waters, Katrina M; Kawaoka, Yoshihiro; Baric, Ralph; Aderem, Alan; Katze, Michael G; Scheuermann, Richard H

2014-01-01

The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.

Sparse evidence of MERS-CoV infection among animal workers living in Southern Saudi Arabia during 2012

PubMed Central

Memish, Ziad A; Alsahly, Ahmad; Masri, Malak al; Heil, Gary L; Anderson, Benjamin D; Peiris, Malik; Khan, Salah Uddin; Gray, Gregory C

2015-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging viral pathogen that primarily causes respiratory illness. We conducted a seroprevalence study of banked human serum samples collected in 2012 from Southern Saudi Arabia. Sera from 300 animal workers (17% with daily camel exposure) and 50 non-animal-exposed controls were examined for serological evidence of MERS-CoV infection by a pseudoparticle MERS-CoV spike protein neutralization assay. None of the sera reproducibly neutralized the MERS-CoV-pseudotyped lentiviral vector. These data suggest that serological evidence of zoonotic transmission of MERS-CoV was not common among animal workers in Southern Saudi Arabia during July 2012. PMID:25470665

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

PubMed

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.

3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012.

PubMed

Johnson, Reed F; Bagci, Ulas; Keith, Lauren; Tang, Xianchun; Mollura, Daniel J; Zeitlin, Larry; Qin, Jing; Huzella, Louis; Bartos, Christopher J; Bohorova, Natasha; Bohorov, Ognian; Goodman, Charles; Kim, Do H; Paulty, Michael H; Velasco, Jesus; Whaley, Kevin J; Johnson, Joshua C; Pettitt, James; Ork, Britini L; Solomon, Jeffrey; Oberlander, Nicholas; Zhu, Quan; Sun, Jiusong; Holbrook, Michael R; Olinger, Gene G; Baric, Ralph S; Hensley, Lisa E; Jahrling, Peter B; Marasco, Wayne A

2016-03-01

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10(6) PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a human anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment. Published by Elsevier Inc.

Prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels in Abu Dhabi Emirate, United Arab Emirates.

PubMed

Yusof, Mohammed F; Eltahir, Yassir M; Serhan, Wissam S; Hashem, Farouk M; Elsayed, Elsaeid A; Marzoug, Bahaaeldin A; Abdelazim, Assem Si; Bensalah, Oum Keltoum A; Al Muhairi, Salama S

2015-06-01

High seroprevalence of Middle East respiratory syndrome corona virus (MERS-CoV) in dromedary camels has been previously reported in United Arab Emirates (UAE). However, the molecular detection of the virus has never been reported before in UAE. Of the 7,803 nasal swabs tested in the epidemiological survey, MERS-CoV nucleic acid was detected by real-time PCR in a total of 126 (1.6 %) camels. Positive camels were detected at the borders with Saudi Arabia and Oman and in camels' slaughter houses. MERS-CoV partial sequences obtained from UAE camels were clustering with human- and camel-derived MERS-CoV sequences in the same geographic area. Results provide further evidence of MERS-CoV zoonosis.

Statistical properties of superactive regions during solar cycles 19-23

NASA Astrophysics Data System (ADS)

Chen, A. Q.; Wang, J. X.; Li, J. W.; Feynman, J.; Zhang, J.

2011-10-01

Context. Each solar activity cycle is characterized by a small number of superactive regions (SARs) that produce the most violent of space weather events with the greatest disastrous influence on our living environment. Aims: We aim to re-parameterize the SARs and study the latitudinal and longitudinal distributions of SARs. Methods: We select 45 SARs in solar cycles 21-23, according to the following four parameters: 1) the maximum area of sunspot group, 2) the soft X-ray flare index, 3) the 10.7 cm radio peak flux, and 4) the variation in the total solar irradiance. Another 120 SARs given by previous studies of solar cycles 19-23 are also included. The latitudinal and longitudinal distributions of the 165 SARs in both the Carrington frame and the dynamic reference frame during solar cycles 19-23 are studied statistically. Results: Our results indicate that these 45 SARs produced 44% of all the X class X-ray flares during solar cycles 21-23, and that all the SARs are likely to produce a very fast CME. The latitudinal distributions of SARs display the Maunder butterfly diagrams and SARs occur preferentially in the maximum period of each solar cycle. Northern hemisphere SARs dominated in solar cycles 19 and 20 and southern hemisphere SARs dominated in solar cycles 21 and 22. In solar cycle 23, however, SARs occurred about equally in each hemisphere. There are two active longitudes in both the northern and southern hemispheres, about 160°-200° apart. Applying the improved dynamic reference frame to SARs, we find that SARs rotate faster than the Carrington rate and there is no significant difference between the two hemispheres. The synodic periods are 27.19 days and 27.25 days for the northern and southern hemispheres, respectively. The longitudinal distribution of SARs is significantly non-axisymmetric and about 75% SARs occurred near two active longitudes with half widths of 45°. Appendix A is available in electronic form at http://www.aanda.org

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

PubMed

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.

Evaluation of candidate vaccine approaches for MERS-CoV

DOE PAGES

Wang, Lingshu; Shi, Wei; Joyce, M. Gordon; ...

2015-07-28

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanismsmore » were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.« less

SARS Grid--an AG-based disease management and collaborative platform.

PubMed

Hung, Shu-Hui; Hung, Tsung-Chieh; Juang, Jer-Nan

2006-01-01

This paper describes the development of the NCHC's Severe Acute Respiratory Syndrome (SARS) Grid project-An Access Grid (AG)-based disease management and collaborative platform that allowed for SARS patient's medical data to be dynamically shared and discussed between hospitals and doctors using AG's video teleconferencing (VTC) capabilities. During the height of the SARS epidemic in Asia, SARS Grid and the SARShope website significantly curved the spread of SARS by helping doctors manage the in-hospital and in-home care of quarantined SARS patients through medical data exchange and the monitoring of the patient's symptoms. Now that the SARS epidemic has ended, the primary function of the SARS Grid project is that of a web-based informatics tool to increase pubic awareness of SARS and other epidemic diseases. Additionally, the SARS Grid project can be viewed and further studied as an outstanding model of epidemic disease prevention and/or containment.

Active screening and surveillance in the United Kingdom for Middle East respiratory syndrome coronavirus in returning travellers and pilgrims from the Middle East: a prospective descriptive study for the period 2013-2015.

PubMed

Atabani, Sowsan F; Wilson, Steven; Overton-Lewis, Clare; Workman, Judith; Kidd, I Michael; Petersen, Eskild; Zumla, Alimuddin; Smit, Erasmus; Osman, Husam

2016-06-01

Over 25000 pilgrims from the UK visit Saudi Arabia every year for the Umrah and Hajj pilgrimages. The recent outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) in South Korea and the continuing reports of MERS-CoV cases from Saudi Arabia highlight the need for active surveillance for MERS-CoV in returning pilgrims or travellers from the Middle East. Public Health England Birmingham Laboratory (PHEBL) is one of a few selected UK public health laboratories responsible for MERS-CoV screening in travellers returning to the UK from the Middle East who present to hospital with severe respiratory symptoms. The results of the PHEBL MERS-CoV screening and surveillance over the past 3 years is presented. UK travellers/pilgrims who returned from the Middle East and presented to a hospital with respiratory symptoms were studied over the period February 1, 2013 to December 31, 2015. Patients with respiratory symptoms, who satisfied the Public Health England MERS-CoV case algorithm, were tested for MERS-CoV and other respiratory tract viruses on admission to hospital. Two hundred and two patients suspected of having MERS-CoV were tested. None of them had a laboratory-confirmed MERS-CoV infection. A viral aetiology was detected in half (50.3%) of the cases, with rhinoviruses, influenza A (H1N1 and H3N2), and influenza B being most frequent. Peak testing occurred following the annual Hajj season and in other periods of raised national awareness. Respiratory tract infections in travellers/pilgrims returning to the UK from the Middle East are mainly due to rhinoviruses, influenza A, and influenza B. Whilst MERS-CoV was not detected in the 202 patients studied, heightened awareness of the possibility of MERS-CoV and continuous proactive surveillance are essential to rapidly identify cases of MERS-CoV and other seasonal respiratory tract viruses such as avian influenza, in patients presenting to hospital. Early identification and isolation may prevent outbreaks in nosocomial settings. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

The clinical impact of coronavirus infection in patients with hematologic malignancies and hematopoietic stem cell transplant recipients.

PubMed

Hakki, Morgan; Rattray, Rogan M; Press, Richard D

2015-07-01

Compared to other respiratory viruses, relatively little is known about the clinical impact of coronavirus (CoV) infection after hematopoietic stem cell transplant (HSCT) or in patients with hematologic malignancies. To characterize the role of CoV in respiratory tract infections among HSCT and hematologic malignancy patients. We conducted a retrospective review of all cases of CoV infection documented by polymerase chain reaction, (PCR)-based testing on nasopharyngeal and bronchoalveolar lavage fluid samples between June 2010 and 2013. Cases of CoV infection occurring in HSCT and hematologic malignancy patients were identified and the clinical characteristics of these cases were compared to other respiratory viruses. CoV was identified in 2.6% (n=43) of all samples analyzed (n=1661) and in 6.8% of all samples testing positive for a respiratory virus (n=631). 33 of 38 (86.8%) of patients in whom CoV was identified were HSCT and hematologic malignancy patients. Among these patients, CoV was detected in 9.7% of unique infection episodes, with only rhinovirus/enterovirus (RhV/EnV) infection being more common. Group I CoV subtypes accounted for 76.3% of cases, and 57% of infections were diagnosed between December and March. CoV infection was associated with upper respiratory tract symptoms in most patients, similar to other respiratory viruses. Possible and proven lower respiratory tract disease was less common compared to other respiratory viruses except RhV/EnV. CoV is frequently detected in HSCT and hematologic malignancy patients in whom suspicion for a respiratory viral infection exists, but is less likely to progress to lower respiratory tract disease than most other respiratory viruses. Copyright © 2015 Elsevier B.V. All rights reserved.

Severe acute respiratory syndrome (SARS)

MedlinePlus

SARS; Respiratory failure - SARS ... Complications may include: Respiratory failure Liver failure Heart failure ... 366. McIntosh K, Perlman S. Coronaviruses, including severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). ...

3B11-N, a monoclonal antibody against MERS-CoV, reduces lung pathology in rhesus monkeys following intratracheal inoculation of MERS-CoV Jordan-n3/2012

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Reed F., E-mail: johnsonreed@mail.nih.gov; Bagci, Ulas; Center for Research in Computer Vision

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) was identified in 2012 as the causative agent of a severe, lethal respiratory disease occurring across several countries in the Middle East. To date there have been over 1600 laboratory confirmed cases of MERS-CoV in 26 countries with a case fatality rate of 36%. Given the endemic region, it is possible that MERS-CoV could spread during the annual Hajj pilgrimage, necessitating countermeasure development. In this report, we describe the clinical and radiographic changes of rhesus monkeys following infection with 5×10{sup 6} PFU MERS-CoV Jordan-n3/2012. Two groups of NHPs were treated with either a humanmore » anti-MERS monoclonal antibody 3B11-N or E410-N, an anti-HIV antibody. MERS-CoV Jordan-n3/2012 infection resulted in quantifiable changes by computed tomography, but limited other clinical signs of disease. 3B11-N treated subjects developed significantly reduced lung pathology when compared to infected, untreated subjects, indicating that this antibody may be a suitable MERS-CoV treatment. - Highlights: • MERS-CoV Jordan-n3/2012 challenge of rhesus monkeys results in a mild disease. • CT can be used to monitor disease progression to aid models of human disease. • Treatment with the human monoclonal antibody 3B11-N resulted in decreased disease.« less

Fabrication of β-CoV3O8 nanorods embedded in graphene sheets and their application for electrochemical charge storage electrode.

PubMed

Jeong, Gyoung Hwa; Lee, Ilbok; Lee, Donghyun; Lee, Hea-Min; Baek, Seungmin; Kwon, O-Pil; Kumta, Prashant N; Yoon, Songhun; Kim, Sang-Wook

2018-05-11

The fabrication of β-CoV 3 O 8 nanorods embedded in graphene sheets and their application as electrochemical charge storage electrodes is reported. From the surfactant treatment of raw graphite, graphene was directly prepared and its nanocomposite with β-CoV 3 O 8 nanorods distributed between graphene layers (β-CoV 3 O 8 -G) was synthesized by a hydrothermal method. When applied as an anode in lithium-ion batteries, the β-CoV 3 O 8 -G anode exhibits greatly improved charge and discharge capacities of 790 and 627 mAh · g -1 , respectively, with unexpectedly high initial efficiency of 82%. The observed discharge capacity reflected that at least 3.7 mol of Li + is selectively accumulated within the β-CoV 3 O 8 phase (Li x CoV 3 O 8 , x > 3.7), indicative of significantly improved Li + uptake when compared with aggregated β-CoV 3 O 8 nanorods. Moreover, very distinct peak plateaus and greatly advanced cycling performance are observed, showing more improved Li + storage within the β-CoV 3 O 8 phase. As a supercapacitor electrode, moreover, our composite electrode exhibits very high peak pseudocapacitances of 2.71 F · cm -2 and 433.65 F · g -1 in the β-CoV 3 O 8 phase with extremely stable cycling performance. This remarkably enhanced performance in the individual electrochemical charge storage electrodes is attributed to the novel phase formation of β-CoV 3 O 8 and its optimized nanocomposite structure with graphene, which yield fast electrical conduction through graphene, easy accessibility of ions through the open multilayer nanosheet structure, and a relaxation space between the β-CoV 3 O 8 -G.

Debate on MERS-CoV respiratory precautions: surgical mask or N95 respirators?

PubMed Central

Chung, Jasmine Shimin; Ling, Moi Lin; Seto, Wing Hong; Ang, Brenda Sze Peng; Tambyah, Paul Anantharajah

2014-01-01

Since the emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) in mid-2012, there has been controversy over the respiratory precaution recommendations in different guidelines from various international bodies. Our understanding of MERS-CoV is still evolving. Current recommendations on infection control practices are heavily influenced by the lessons learnt from severe acute respiratory syndrome. A debate on respiratory precautions for MERS-CoV was organised by Infection Control Association (Singapore) and the Society of Infectious Disease (Singapore). We herein discuss and present the evidence for surgical masks for the protection of healthcare workers from MERS-CoV. PMID:25017402

Severe acute respiratory syndrome (SARS): knowledge, attitudes, practices and sources of information among physicians answering a SARS fever hotline service.

PubMed

Deng, J-F; Olowokure, B; Kaydos-Daniels, S C; Chang, H-J; Barwick, R S; Lee, M-L; Deng, C-Y; Factor, S H; Chiang, C-E; Maloney, S A

2006-01-01

In June 2003, Taiwan introduced a severe acute respiratory syndrome (SARS) telephone hotline service to provide concerned callers with rapid access to information, advice and appropriate referral where necessary. This paper reports an evaluation of the knowledge, attitude, practices and sources of information relating to SARS among physicians who staffed the SARS fever hotline service. A retrospective survey was conducted using a self-administered postal questionnaire. Participants were physicians who staffed a SARS hotline during the SARS epidemic in Taipei, Taiwan from June 1 to 10, 2003. A response rate of 83% was obtained. All respondents knew the causative agent of SARS, and knowledge regarding SARS features and preventive practices was good. However, only 54% of respondents knew the incubation period of SARS. Hospital guidelines and news media were the major information sources. In responding to two case scenarios most physicians were likely to triage callers at high risk of SARS appropriately, but not callers at low risk. Less than half of all respondents answered both scenarios correctly. The results obtained suggest that knowledge of SARS was generally good although obtained from both medical and non-medical sources. Specific knowledge was however lacking in certain areas and this affected the ability to appropriately triage callers. Standardized education and assessment of prior knowledge of SARS could improve the ability of physicians to triage callers in future outbreaks.

Coping with future epidemics: Tai chi practice as an overcoming strategy used by survivors of severe acute respiratory syndrome (SARS) in post-SARS Hong Kong.

PubMed

Siu, Judy Yuen-Man

2016-06-01

Although SARS had been with a controversial topic for a decade at the time of this study, numerous SARS survivors had not yet physically, psychologically or socially recovered from the aftermath of SARS. Among chronically ill patients, the use of complementary and alternative medicine (CAM) is reported to be widespread. However, extremely little is known about the use of CAM by SARS survivors in the post-SARS period and even less is known about how the use of CAM is related to the unpleasant social and medical-treatment experiences of SARS survivors, their eagerness to re-establish social networks, and their awareness to prepare for future epidemics. To investigate the motivations for practising tai chi among SARS survivors in post-SARS Hong Kong. Using a qualitative approach, I conducted individual semi-structured interviews with 35 SARS survivors, who were purposively sampled from a tai chi class of a SARS-patient self-help group in Hong Kong. Health concerns and social experiences motivated the participants to practise tai chi in post-SARS Hong Kong. Experiencing health deterioration in relation to SARS-associated sequelae, coping with unpleasant experiences during follow-up biomedical treatments, a desire to regain an active role in recovery and rehabilitation, overcoming SARS-associated stigmas by establishing a new social network and preparing for potential future stigmatization and discrimination were the key motivators for them. The participants practised tai chi not only because they sought to improve their health but also because it provided a crucial social function and meaning to them. © 2014 John Wiley & Sons Ltd.

12 CFR 390.355 - Suspicious Activity Reports and other reports and statements.

Code of Federal Regulations, 2012 CFR

2012-01-01

... Activity Report on the form prescribed by the FDIC. (3) SARs required. A State savings association shall... to supervisory action. (11) Obtaining SARs. A State savings association may obtain SARs and the... SARs. SARs are confidential. Any institution or person subpoenaed or otherwise requested to disclose a...

12 CFR 390.355 - Suspicious Activity Reports and other reports and statements.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Activity Report on the form prescribed by the FDIC. (3) SARs required. A State savings association shall... to supervisory action. (11) Obtaining SARs. A State savings association may obtain SARs and the... SARs. SARs are confidential. Any institution or person subpoenaed or otherwise requested to disclose a...

Discovery of Novel Bat Coronaviruses in South China That Use the Same Receptor as Middle East Respiratory Syndrome Coronavirus.

PubMed

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

2018-07-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has represented a human health threat since 2012. Although several MERS-related CoVs that 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 1,059 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 coronavirus HKU4 , Pipistrellus pipistrellus coronavirus 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 have acquired their spike genes from a DPP4-recognizing bat coronavirus HKU4. Our study provides further evidence that bats represent the evolutionary origins of MERS-CoV. IMPORTANCE Previous studies suggested that MERS-CoV originated in bats. However, its evolutionary path from bats to humans remains unclear. In this study, we discovered 89 novel lineage C betacoronaviruses in eight bat species. We provide 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 ranges of MERS-related CoV and represents an important step toward establishing bats as the natural reservoir of MERS-CoV. These findings may lead to improved epidemiological surveillance of MERS-CoV and the prevention and control of the spread of MERS-CoV to humans. Copyright © 2018 American Society for Microbiology.

Deciphering MERS-CoV Evolution in Dromedary Camels.

PubMed

Du, Lin; Han, Guan-Zhu

2016-02-01

The emergence of the Middle East respiratory syndrome coronavirus (MERS-CoV) poses a potential threat to global public health. Many aspects of the evolution and transmission of MERS-CoV in its animal reservoir remain unclear. A recent study provides new insights into the evolution and transmission of MERS-CoV in dromedary camels. Copyright © 2015 Elsevier Ltd. All rights reserved.

12 CFR 21.11 - Suspicious Activity Report.

Code of Federal Regulations, 2011 CFR

2011-01-01

... Activity Report on the form prescribed by the OCC. (c) SARs required. A national bank shall file a SAR with... supervisory action. (j) Obtaining SARs. A national bank may obtain SARs and the Instructions from the appropriate OCC District Office listed in 12 CFR part 4. (k) Confidentiality of SARs. SARs are confidential...

12 CFR 390.355 - Suspicious Activity Reports and other reports and statements.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Activity Report on the form prescribed by the FDIC. (3) SARs required. A State savings association shall... to supervisory action. (11) Obtaining SARs. A State savings association may obtain SARs and the... SARs. SARs are confidential. Any institution or person subpoenaed or otherwise requested to disclose a...

Inactivation and safety testing of Middle East Respiratory Syndrome Coronavirus

PubMed Central

Kumar, Mia; Mazur, Steven; Ork, Britini L.; Postnikova, Elena; Hensley, Lisa E.; Jahrling, Peter B.; Johnson, Reed; Holbrook, Michael R.

2015-01-01

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a recently emerged virus that has caused a number of human infections and deaths, primarily in the Middle East. The transmission of MERS-CoV to humans has been proposed to be as a result of contact with camels, but evidence of human-to-human transmission also exists. In order to work with MERS-CoV in a laboratory setting, the US Centers for Disease Control and Prevention (CDC) has determined that MERS-CoV should be handled at a biosafety level (BSL) 3 (BSL-3) biocontainment level. Many processes and procedures used to characterize MERS-CoV and to evaluate samples from MERS-CoV infected animals are more easily and efficiently completed at BSL-2 or lower containment. In order to complete experimental work at BSL-2, demonstration or proof of inactivation is required before removal of specimens from biocontainment laboratories. In the studies presented here, we evaluated typical means of inactivating viruses prior to handling specimens at a lower biocontainment level. We found that Trizol, AVL buffer and gamma irradiation were effective at inactivating MERS-CoV, that formaldehyde-based solutions required at least 30 minutes of contact time in a cell culture system while a mixture of methanol and acetone required 60 minutes to inactivate MERS-CoV. Together, these data provide a foundation for safely inactivating MERS-CoV, and potentially other coronaviruses, prior to removal from biocontainment facilities. PMID:26190637

Korean Society for Laboratory Medicine Practice Guidelines for the Molecular Diagnosis of Middle East Respiratory Syndrome During an Outbreak in Korea in 2015.

PubMed

Ki, Chang Seok; Lee, Hyukmin; Sung, Heungsup; Kim, Sinyoung; Seong, Moon Woo; Yong, Dongeun; Kim, Jae Seok; Lee, Mi Kyung; Kim, Mi Na; Choi, Jong Rak; Kim, Jeong Ho

2016-05-01

For two months between May and July 2015, a nationwide outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) occurred in Korea. On June 3, 2015, the Korean Society for Laboratory Medicine (KSLM) launched a MERS-CoV Laboratory Response Task Force (LR-TF) to facilitate clinical laboratories to set up the diagnosis of MERS-CoV infection. Based on the WHO interim recommendations, the Centers for Disease Control and Prevention of United States guidelines for MERS-CoV laboratory testing, and other available resources, the KSLM MERS-CoV LR-TF provided the first version of the laboratory practice guidelines for the molecular diagnosis of MERS-CoV to the clinical laboratories on June 12, 2015. The guidelines described here are an updated version that includes case definition, indications for testing, specimen type and protocols for specimen collection, specimen packing and transport, specimen handling and nucleic acid extraction, molecular detection of MERS-CoV, interpretation of results and reporting, and laboratory safety. The KSLM guidelines mainly focus on the molecular diagnosis of MERS-CoV, reflecting the unique situation in Korea and the state of knowledge at the time of publication.

Middle East respiratory syndrome in children. Dental considerations.

PubMed

Al-Sehaibany, Fares S

2017-04-01

As of January 2016, 1,633 laboratory-confirmed cases of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection and 587 MERS-related deaths have been reported by the World Health Organization globally. Middle East Respiratory Syndrome Coronavirus  may occur sporadically in communities or may be transmitted within families or hospitals. The number of confirmed MERS-CoV cases among healthcare workers has been increasing. Middle East Respiratory Syndrome Coronavirus may also spread through aerosols generated during various dental treatments, resulting in transmission between patients and dentists. As MERS-CoV cases have also been reported among children, pediatric dentists are at risk of MERS-CoV infection. This review discusses MERS-CoV infection in children and healthcare workers, especially pediatric dentists, and considerations pertaining to pediatric dentistry. Although no cases of MERS-CoV transmission between a patient and a dentist have yet been reported, the risk of MERS-CoV transmission from an infected patient may be high due to the unique work environment of dentists (aerosol generation).

Evaluation of candidate vaccine approaches for MERS-CoV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Lingshu; Shi, Wei; Joyce, M. Gordon

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanismsmore » were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development.« less

Comparative Genomic Analysis MERS CoV Isolated from Humans and Camels with Special Reference to Virus Encoded Helicase.

PubMed

Alnazawi, Mohamed; Altaher, Abdallah; Kandeel, Mahmoud

2017-01-01

Middle East Respiratory Syndrome Coronavirus (MERS CoV) is a new emerging viral disease characterized by high fatality rate. Understanding MERS CoV genetic aspects and codon usage pattern is important to understand MERS CoV survival, adaptation, evolution, resistance to innate immunity, and help in finding the unique aspects of the virus for future drug discovery experiments. In this work, we provide comprehensive analysis of 238 MERS CoV full genomes comprised of human (hMERS) and camel (cMERS) isolates of the virus. MERS CoV genome shaping seems to be under compositional and mutational bias, as revealed by preference of A/T over G/C nucleotides, preferred codons, nucleotides at the third position of codons (NT3s), relative synonymous codon usage, hydropathicity (Gravy), and aromaticity (Aromo) indices. Effective number of codons (ENc) analysis reveals a general slight codon usage bias. Codon adaptation index reveals incomplete adaptation to host environment. MERS CoV showed high ability to resist the innate immune response by showing lower CpG frequencies. Neutrality evolution analysis revealed a more significant role of mutation pressure in cMERS over hMERS. Correspondence analysis revealed that MERS CoV genomes have three genetic clusters, which were distinct in their codon usage, host, and geographic distribution. Additionally, virtual screening and binding experiments were able to identify three new virus-encoded helicase binding compounds. These compounds can be used for further optimization of inhibitors.

Circulation of Alphacoronavirus, Betacoronavirus and Paramyxovirus in Hipposideros bat species in Zimbabwe.

PubMed

Bourgarel, Mathieu; Pfukenyi, Davies M; Boué, Vanina; Talignani, Loïc; Chiweshe, Ngoni; Diop, Fodé; Caron, Alexandre; Matope, Gift; Missé, Dorothée; Liégeois, Florian

2018-03-01

Bats carry a great diversity of zoonotic viruses with a high-impact on human health and livestock. Since the emergence of new coronaviruses and paramyxoviruses in humans (e.g. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Nipah virus), numerous studies clearly established that bats can maintain some of these viruses. Improving our understanding on the role of bats in the epidemiology of the pathogens they harbour is necessary to prevent cross-species spill over along the wild/domestic/human gradient. In this study, we screened bat faecal samples for the presence of Coronavirus and Paramyxovirus in two caves frequently visited by local people to collect manure and/or to hunt bats in Zimbabwe. We amplified partial RNA-dependent RNA polymerase genes of Alpha and Betacoronavirus together with the partial polymerase gene of Paramyxovirus. Identified coronaviruses were related to pathogenic human strains and the paramyxovirus belonged to the recently described Jeilongvirus genus. Our results highlighted the importance of monitoring virus circulation in wildlife, especially bats, in the context of intense human-wildlife interfaces in order to strengthen prevention measures among local populations and to implement sentinel surveillance in sites with high zoonotic diseases transmission potential. Copyright © 2018 Elsevier B.V. All rights reserved.

Successful recovery of MERS CoV pneumonia in a patient with acquired immunodeficiency syndrome: a case report.

PubMed

Shalhoub, Sarah; AlZahrani, Abdulwahab; Simhairi, Raed; Mushtaq, Adnan

2015-01-01

Middle East Respiratory Syndrome Coronavirus (MERS CoV) may cause severe pneumonia with significant morbidity and mortality, particularly in patients with multiple comorbid condition. MERS CoV pneumonia has not been previously reported in patients with Human Immunodeficiency Virus (HIV). Herein, we report a case of MERS CoV pneumonia with a successful outcome in a patient recently diagnosed with HIV. Copyright © 2014 Elsevier B.V. All rights reserved.

A Highly Immunogenic and Protective Middle East Respiratory Syndrome Coronavirus Vaccine Based on a Recombinant Measles Virus Vaccine Platform

PubMed Central

Malczyk, Anna H.; Kupke, Alexandra; Prüfer, Steffen; Scheuplein, Vivian A.; Hutzler, Stefan; Kreuz, Dorothea; Beissert, Tim; Bauer, Stefanie; Hubich-Rau, Stefanie; Tondera, Christiane; Eldin, Hosam Shams; Schmidt, Jörg; Vergara-Alert, Júlia; Süzer, Yasemin; Seifried, Janna; Hanschmann, Kay-Martin; Kalinke, Ulrich; Herold, Susanne; Sahin, Ugur; Cichutek, Klaus; Waibler, Zoe; Eickmann, Markus; Becker, Stephan

2015-01-01

ABSTRACT In 2012, the first cases of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) were identified. Since then, more than 1,000 cases of MERS-CoV infection have been confirmed; infection is typically associated with considerable morbidity and, in approximately 30% of cases, mortality. Currently, there is no protective vaccine available. Replication-competent recombinant measles virus (MV) expressing foreign antigens constitutes a promising tool to induce protective immunity against corresponding pathogens. Therefore, we generated MVs expressing the spike glycoprotein of MERS-CoV in its full-length (MERS-S) or a truncated, soluble variant of MERS-S (MERS-solS). The genes encoding MERS-S and MERS-solS were cloned into the vaccine strain MVvac2 genome, and the respective viruses were rescued (MVvac2-CoV-S and MVvac2-CoV-solS). These recombinant MVs were amplified and characterized at passages 3 and 10. The replication of MVvac2-CoV-S in Vero cells turned out to be comparable to that of the control virus MVvac2-GFP (encoding green fluorescent protein), while titers of MVvac2-CoV-solS were impaired approximately 3-fold. The genomic stability and expression of the inserted antigens were confirmed via sequencing of viral cDNA and immunoblot analysis. In vivo, immunization of type I interferon receptor-deficient (IFNAR−/−)-CD46Ge mice with 2 × 105 50% tissue culture infective doses of MVvac2-CoV-S(H) or MVvac2-CoV-solS(H) in a prime-boost regimen induced robust levels of both MV- and MERS-CoV-neutralizing antibodies. Additionally, induction of specific T cells was demonstrated by T cell proliferation, antigen-specific T cell cytotoxicity, and gamma interferon secretion after stimulation of splenocytes with MERS-CoV-S presented by murine dendritic cells. MERS-CoV challenge experiments indicated the protective capacity of these immune responses in vaccinated mice. IMPORTANCE Although MERS-CoV has not yet acquired extensive distribution, being mainly confined to the Arabic and Korean peninsulas, it could adapt to spread more readily among humans and thereby become pandemic. Therefore, the development of a vaccine is mandatory. The integration of antigen-coding genes into recombinant MV resulting in coexpression of MV and foreign antigens can efficiently be achieved. Thus, in combination with the excellent safety profile of the MV vaccine, recombinant MV seems to constitute an ideal vaccine platform. The present study shows that a recombinant MV expressing MERS-S is genetically stable and induces strong humoral and cellular immunity against MERS-CoV in vaccinated mice. Subsequent challenge experiments indicated protection of vaccinated animals, illustrating the potential of MV as a vaccine platform with the potential to target emerging infections, such as MERS-CoV. PMID:26355094

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

PubMed Central

Lugari, Adrien; Betzi, Stephane; Decroly, Etienne; Bonnaud, Emmanuel; Hermant, Aurélie; Guillemot, Jean-Claude; Debarnot, Claire; Borg, Jean-Paul; Bouvet, Mickaël; Canard, Bruno; Morelli, Xavier; Lécine, Patrick

2010-01-01

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses. PMID:20699222

Transmission of Middle East Respiratory Syndrome Coronavirus Infections in Healthcare Settings, Abu Dhabi

PubMed Central

Nguyen, Duc; Aden, Bashir; Al Bandar, Zyad; Al Dhaheri, Wafa; Abu Elkheir, Kheir; Khudair, Ahmed; Al Mulla, Mariam; El Saleh, Feda; Imambaccus, Hala; Al Kaabi, Nawal; Sheikh, Farrukh Amin; Sasse, Jurgen; Turner, Andrew; Abdel Wareth, Laila; Weber, Stefan; Al Ameri, Asma; Abu Amer, Wesal; Alami, Negar N.; Bunga, Sudhir; Haynes, Lia M.; Hall, Aron J.; Kallen, Alexander J.; Kuhar, David; Pham, Huong; Pringle, Kimberly; Tong, Suxiang; Whitaker, Brett L.; Gerber, Susan I.; Al Hosani, Farida Ismail

2016-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) infections sharply increased in the Arabian Peninsula during spring 2014. In Abu Dhabi, United Arab Emirates, these infections occurred primarily among healthcare workers and patients. To identify and describe epidemiologic and clinical characteristics of persons with healthcare-associated infection, we reviewed laboratory-confirmed MERS-CoV cases reported to the Health Authority of Abu Dhabi during January 1, 2013–May 9, 2014. Of 65 case-patients identified with MERS-CoV infection, 27 (42%) had healthcare-associated cases. Epidemiologic and genetic sequencing findings suggest that 3 healthcare clusters of MERS-CoV infection occurred, including 1 that resulted in 20 infected persons in 1 hospital. MERS-CoV in healthcare settings spread predominantly before MERS-CoV infection was diagnosed, underscoring the importance of increasing awareness and infection control measures at first points of entry to healthcare facilities. PMID:26981708

High Prevalence of Middle East Respiratory Coronavirus in Young Dromedary Camels in Jordan.

PubMed

van Doremalen, Neeltje; Hijazeen, Zaidoun S K; Holloway, Peter; Al Omari, Bilal; McDowell, Chester; Adney, Danielle; Talafha, Hani A; Guitian, Javier; Steel, John; Amarin, Nadim; Tibbo, Markos; Abu-Basha, Ehab; Al-Majali, Ahmad M; Munster, Vincent J; Richt, Juergen A

2017-02-01

Prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) was determined in 45 dromedary camels from two geographically separated herds in Jordan. Virus shedding was only detected in swabs obtained from the respiratory tract and primarily observed in camels younger than 3 years. MERS-CoV seroprevalence increased with age of camels. Bovine and sheep sera were seronegative. Phylogenetic analysis of partial S2 clustered the Jordanian MERS-CoV strains with contemporary MERS-CoV strains associated with nosocomial outbreaks.

Prophylaxis With a Middle East Respiratory Syndrome Coronavirus (MERS-CoV)–Specific Human Monoclonal Antibody Protects Rabbits From MERS-CoV Infection

PubMed Central

Houser, Katherine V.; Gretebeck, Lisa; Ying, Tianlei; Wang, Yanping; Vogel, Leatrice; Lamirande, Elaine W.; Bock, Kevin W.; Moore, Ian N.; Dimitrov, Dimiter S.; Subbarao, Kanta

2016-01-01

With >1600 documented human infections with Middle East respiratory syndrome coronavirus (MERS-CoV) and a case fatality rate of approximately 36%, medical countermeasures are needed to prevent and limit the disease. We examined the in vivo efficacy of the human monoclonal antibody m336, which has high neutralizing activity against MERS-CoV in vitro. m336 was administered to rabbits intravenously or intranasally before infection with MERS-CoV. Prophylaxis with m336 resulted in a reduction of pulmonary viral RNA titers by 40–9000-fold, compared with an irrelevant control antibody with little to no inflammation or viral antigen detected. This protection in rabbits supports further clinical development of m336. PMID:26941283

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

PubMed

Keum, Young-Sam; Jeong, Yong-Joo

2012-11-15

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

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.

PubMed

Scobey, Trevor; Yount, Boyd L; Sims, Amy C; Donaldson, Eric F; Agnihothram, Sudhakar S; Menachery, Vineet D; Graham, Rachel L; Swanstrom, Jesica; Bove, Peter F; Kim, Jeeho D; Grego, Sonia; Randell, Scott H; Baric, Ralph S

2013-10-01

Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

Prevalence of Diabetes in the 2009 Influenza A (H1N1) and the Middle East Respiratory Syndrome Coronavirus: A Systematic Review and Meta-Analysis.

PubMed

Badawi, Alaa; Ryoo, Seung Gwan

2016-12-09

Over the past two decades a number of severe acute respiratory infection outbreaks such as the 2009 influenza A (H1N1) and the Middle East respiratory syndrome coronavirus (MERS-CoV) have emerged and presented a considerable global public health threat. Epidemiologic evidence suggests that diabetic subjects are more susceptible to these conditions. However, the prevalence of diabetes in H1N1 and MERS-CoV has not been systematically described. The aim of this study is to conduct a systematic review and meta-analysis of published reports documenting the prevalence of diabetes in H1N1 and MERS-CoV and compare its frequency in the two viral conditions. Meta-analysis for the proportions of subjects with diabetes was carried out in 29 studies for H1N1 ( n =92,948) and 9 for MERS-CoV ( n =308). Average age of H1N1 patients (36.2±6.0 years) was significantly younger than that of subjects with MERS-CoV (54.3±7.4 years, P<0.05). Compared to MERS-CoV patients, subjects with H1N1 exhibited 3-fold lower frequency of cardiovascular diseases and 2- and 4-fold higher prevalence of obesity and immunosuppression, respectively. The overall prevalence of diabetes in H1N1 was 14.6% (95% CI: 12.3-17.0%; P<0.001), a 3.6-fold lower than in MERS-CoV (54.4%; 95% CI: 29.4-79.5; P<0.001). The prevalence of diabetes among H1N1 cases from Asia and North America was ~two-fold higher than those from South America and Europe. The prevalence of diabetes in MERS-CoV cases is higher than in H1N1. Regional comparisons suggest that an etiologic role of diabetes in MERS-CoV may exist distinctive from that in H1N1.

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.

PubMed

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 study on SARS awareness and health-seeking behaviour - findings from a sampled population attending National Healthcare Group Polyclinics.

PubMed

Seng, S L; Lim, P S; Ng, M Y; Wong, H B; Emmanuel, S C

2004-09-01

The study aimed to assess the effectiveness of massive SARS public education effort on SARS awareness and the conduct of those suspected of having SARS. Five hundred and ninety-three respondents attending the National Healthcare Group Polyclinics (NHGP) participated in the survey from 9 to 13 June 2003. Associations between awareness of SARS symptoms and (i) first action to be taken and (ii) mode of transportation used, if the respondent was suspected of having SARS, were analysed using Chi-square or Fisher's exact tests. Logistic regression was performed to adjust for relevant covariates. The majority (92.7%) of the respondents were aware of SARS symptoms. Television (91.6%), newspaper (65.2%) and radio (30.4%) formed the top 3 sources of information on SARS. Slightly more than half (51.6%) of those who suspect themselves of having SARS would choose to visit their primary health care doctors, while 22.7% of the respondents would go to Tan Tock Seng Hospital (TTSH). If they suspected themselves to have SARS, most (84.9%) of the 578 respondents would react appropriately by taking the SARS ambulance or driving themselves to TTSH. However, 60 respondents would nonetheless take public transport to TTSH [by taxi 8.5%, mass rapid transit (MRT) or bus 1.9%]. In particular, the retired with lower educational levels were likely to be oblivious both to the symptoms of SARS and the possible consequences of travelling by inappropriate transport. Despite more than 2 months of intensive SARS public education in Singapore, there remain important gaps in knowledge and appropriate behaviour that have to be bridged.

Extensive Viable Middle East Respiratory Syndrome (MERS) Coronavirus Contamination in Air and Surrounding Environment in MERS Isolation Wards.

PubMed

Kim, Sung-Han; Chang, So Young; Sung, Minki; Park, Ji Hoon; Bin Kim, Hong; Lee, Heeyoung; Choi, Jae-Phil; Choi, Won Suk; Min, Ji-Young

2016-08-01

The largest outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) outside the Middle East occurred in South Korea in 2015 and resulted in 186 laboratory-confirmed infections, including 36 (19%) deaths. Some hospitals were considered epicenters of infection and voluntarily shut down most of their operations after nearly half of all transmissions occurred in hospital settings. However, the ways that MERS-CoV is transmitted in healthcare settings are not well defined. We explored the possible contribution of contaminated hospital air and surfaces to MERS transmission by collecting air and swabbing environmental surfaces in 2 hospitals treating MERS-CoV patients. The samples were tested by viral culture with reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA) using MERS-CoV Spike antibody, and electron microscopy (EM). The presence of MERS-CoV was confirmed by RT-PCR of viral cultures of 4 of 7 air samples from 2 patients' rooms, 1 patient's restroom, and 1 common corridor. In addition, MERS-CoV was detected in 15 of 68 surface swabs by viral cultures. IFA on the cultures of the air and swab samples revealed the presence of MERS-CoV. EM images also revealed intact particles of MERS-CoV in viral cultures of the air and swab samples. These data provide experimental evidence for extensive viable MERS-CoV contamination of the air and surrounding materials in MERS outbreak units. Thus, our findings call for epidemiologic investigation of the possible scenarios for contact and airborne transmission, and raise concern regarding the adequacy of current infection control procedures. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Human rhinoviruses: the cold wars resume.

PubMed

Mackay, Ian M

2008-08-01

Human rhinoviruses (HRVs) are the most common cause of viral illness worldwide but today, less than half the strains have been sequenced and only a handful examined structurally. This viral super-group, known for decades, has still to face the full force of a molecular biology onslaught. However, newly identified viruses (NIVs) including human metapneumovirus and bocavirus and emergent viruses including SARS-CoV have already been exhaustively scrutinized. The clinical impact of most respiratory NIVs is attributable to one or two major strains but there are 100+ distinct HRVs and, because we have never sought them independently, we must arbitrarily divide the literature's clinical impact findings among them. Early findings from infection studies and use of inefficient detection methods have shaped the way we think of 'common cold' viruses today. To review past HRV-related studies in order to put recent HRV discoveries into context. HRV infections result in undue antibiotic prescriptions, sizable healthcare-related expenditure and exacerbation of expiratory wheezing associated with hospital admission. The finding of many divergent and previously unrecognized HRV strains has drawn attention and resources back to the most widespread and frequent infectious agent of humans; providing us the chance to seize the advantage in a decades-long cold war.

Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus

PubMed Central

Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T.; Weidmann, Manfred

2013-01-01

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV. PMID:24459611

75 FR 46912 - Draft 2010 Marine Mammal Stock Assessment Reports

Federal Register 2010, 2011, 2012, 2013, 2014

2010-08-04

... reviewed the Alaska, Atlantic, and Pacific regional marine mammal stock assessment reports (SARs) in accordance with the Marine Mammal Protection Act. SARs for marine mammals in the Alaska, Atlantic, and... SARs. DATES: Comments must be received by November 2, 2010. ADDRESSES: The 2010 draft SARs, summaries...

Risk Factors for SARS among Persons without Known Contact with SARS Patients, Beijing, China

PubMed Central

Wu, Jiang; Xu, Fujie; Zhou, Weigong; Feikin, Daniel R.; Lin, Chang-Ying; He, Xiong; Zhu, Zonghan; Liang, Wannian; Chin, Daniel P.

2004-01-01

Most cases of severe acute respiratory syndrome (SARS) have occurred in close contacts of SARS patients. However, in Beijing, a large proportion of SARS cases occurred in persons without such contact. We conducted a case-control study in Beijing that compared exposures of 94 unlinked, probable SARS patients with those of 281 community-based controls matched for age group and sex. Case-patients were more likely than controls to have chronic medical conditions or to have visited fever clinics (clinics at which possible SARS patients were separated from other patients), eaten outside the home, or taken taxis frequently. The use of masks was strongly protective. Among 31 case-patients for whom convalescent-phase (>21 days) sera were available, 26% had immunoglobulin G to SARS-associated coronavirus. Our finding that clinical SARS was associated with visits to fever clinics supports Beijing’s strategy of closing clinics with poor infection-control measures. Our finding that mask use lowered the risk for disease supports the community’s use of this strategy. PMID:15030685

Epidemiological investigation of Middle East respiratory syndrome coronavirus in dromedary camel farms linked with human infection in Abu Dhabi Emirate, United Arab Emirates.

PubMed

Muhairi, Salama Al; Hosani, Farida Al; Eltahir, Yassir M; Mulla, Mariam Al; Yusof, Mohammed F; Serhan, Wissam S; Hashem, Farouq M; Elsayed, Elsaeid A; Marzoug, Bahaaeldin A; Abdelazim, Assem S

2016-12-01

The objective of this research was to investigate the prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) infection primarily in dromedary camel farms and the relationship of those infections with infections in humans in the Emirate of Abu Dhabi. Nasal swabs from 1113 dromedary camels (39 farms) and 34 sheep (1 farm) and sputum samples from 2 MERS-CoV-infected camel farm owners and 1 MERS-CoV-infected sheep farm owner were collected. Samples from camels and humans underwent real-time reverse-transcription quantitative PCR screening to detect MERS-CoV. In addition, sequencing and phylogenetic analysis of partially characterized MERS-CoV genome fragments obtained from camels were performed. Among the 40 farms, 6 camel farms were positive for MERS-CoV; the virus was not detected in the single sheep farm. The maximum duration of viral shedding from infected camels was 2 weeks after the first positive test result as detected in nasal swabs and in rectal swabs obtained from infected calves. Three partial camel sequences characterized in this study (open reading frames 1a and 1ab, Spike1, Spike2, and ORF4b) together with the corresponding regions of previously reported MERS-CoV sequence obtained from one farm owner were clustering together within the larger MERS-CoV sequences cluster containing human and camel isolates reported for the Arabian Peninsula. Data provided further evidence of the zoonotic potential of MERS-CoV infection and strongly suggested that camels may have a role in the transmission of the virus to humans.

Occurrence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) across the Gulf Corporation Council countries: Four years update.

PubMed

Aly, Mahmoud; Elrobh, Mohamed; Alzayer, Maha; Aljuhani, Sameera; Balkhy, Hanan

2017-01-01

The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infections has become a global issue of dire concerns. MERS-CoV infections have been identified in many countries all over the world whereas high level occurrences have been documented in the Middle East and Korea. MERS-CoV is mainly spreading across the geographical region of the Middle East, especially in the Arabian Peninsula, while some imported sporadic cases were reported from the Europe, North America, Africa, and lately Asia. The prevalence of MERS-CoV infections across the Gulf Corporation Council (GCC) countries still remains unclear. Therefore, the objective of the current study was to report the prevalence of MERS-CoV in the GCC countries and to also elucidate on its demographics in the Arabian Peninsula. To date, the World Health Organization (WHO) has reported 1,797 laboratory-confirmed cases of MERS-CoV infection since June 2012, involving 687 deaths in 27 different countries worldwide. Within a time span of 4 years from June 2012 to July 2016, we collect samples form MERS-CoV infected individuals from National Guard Hospital, Riyadh, and Ministry of health Saudi Arabia and other GCC countries. Our data comprise a total of 1550 cases (67.1% male and 32.9% female). The age-specific prevalence and distribution of MERS-CoV was as follow: <20 yrs (36 cases: 3.28%), 20-39 yrs (331 cases: 30.15%), 40-59 yrs (314 cases: 28.60%), and the highest-risk elderly group aged ≥60 yrs (417 cases: 37.98%). The case distribution among GCC countries was as follows: Saudi Arabia (1441 cases: 93%), Kuwait (4 cases: 0.3%), Bahrain (1 case: 0.1%), Oman (8 cases: 0.5%), Qatar (16 cases: 1.0%), and United Arab Emirates (80 cases: 5.2%). Thus, MERS-CoV was found to be more prevalent in Saudi Arabia especially in Riyadh, where 756 cases (52.4%) were the worst hit area of the country identified, followed by the western region Makkah where 298 cases (20.6%) were recorded. This prevalence update indicates that the Arabian Peninsula, particularly Saudi Arabia, is the hardest hit region regarding the emerging MERS-CoV infections worldwide. GCC countries including Saudi Arabia now have the infrastructure in place that allows physicians and scientific community to identify and immediately respond to the potential risks posed by new outbreaks of MERS-CoV infections in the region. Given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of MERS-CoV in our region.

Characterization and Demonstration of the Value of a Lethal Mouse Model of Middle East Respiratory Syndrome Coronavirus Infection and Disease.

PubMed

Tao, Xinrong; Garron, Tania; Agrawal, Anurodh Shankar; Algaissi, Abdullah; Peng, Bi-Hung; Wakamiya, Maki; Chan, Teh-Sheng; Lu, Lu; Du, Lanying; Jiang, Shibo; Couch, Robert B; Tseng, Chien-Te K

2016-01-01

Characterized animal models are needed for studying the pathogenesis of and evaluating medical countermeasures for persisting Middle East respiratory syndrome-coronavirus (MERS-CoV) infections. Here, we further characterized a lethal transgenic mouse model of MERS-CoV infection and disease that globally expresses human CD26 (hCD26)/DPP4. The 50% infectious dose (ID50) and lethal dose (LD50) of virus were estimated to be <1 and 10 TCID50 of MERS-CoV, respectively. Neutralizing antibody developed in the surviving mice from the ID50/LD50 determinations, and all were fully immune to challenge with 100 LD50 of MERS-CoV. The tissue distribution and histopathology in mice challenged with a potential working dose of 10 LD50 of MERS-CoV were subsequently evaluated. In contrast to the overwhelming infection seen in the mice challenged with 10(5) LD50 of MERS-CoV, we were able to recover infectious virus from these mice only infrequently, although quantitative reverse transcription-PCR (qRT-PCR) tests indicated early and persistent lung infection and delayed occurrence of brain infection. Persistent inflammatory infiltrates were seen in the lungs and brain stems at day 2 and day 6 after infection, respectively. While focal infiltrates were also noted in the liver, definite pathology was not seen in other tissues. Finally, using a receptor binding domain protein vaccine and a MERS-CoV fusion inhibitor, we demonstrated the value of this model for evaluating vaccines and antivirals against MERS. As outcomes of MERS-CoV infection in patients differ greatly, ranging from asymptomatic to overwhelming disease and death, having available both an infection model and a lethal model makes this transgenic mouse model relevant for advancing MERS research. Fully characterized animal models are essential for studying pathogenesis and for preclinical screening of vaccines and drugs against MERS-CoV infection and disease. When given a high dose of MERS-CoV, our transgenic mice expressing hCD26/DPP4 viral receptor uniformly succumbed to death within 6 days, making it difficult to evaluate host responses to infection and disease. We further characterized this model by determining both the ID50 and the LD50 of MERS-CoV in order to establish both an infection model and a lethal model for MERS and followed this by investigating the antibody responses and immunity of the mice that survived MERS-CoV infection. Using the estimated LD50 and ID50 data, we dissected the kinetics of viral tissue distribution and pathology in mice challenged with 10 LD50 of virus and utilized the model for preclinical evaluation of a vaccine and drug for treatment of MERS-CoV infection. This further-characterized transgenic mouse model will be useful for advancing MERS research. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A loophole in international quarantine procedures disclosed during the SARS crisis.

PubMed

Lee, Chi-Wei; Tsai, Yen-Shuo; Wong, Tai-Wai; Lau, Chor-Chiu

2006-01-01

This study describes a loophole in the international quarantine system during the recent Asian severe acute respiratory syndrome (SARS) outbreak. Specifically, that of travelers disguising symptoms of respiratory tract infection at international airports, in order to board aircraft to return to their home countries-notwithstanding the infection risks this involves to others. High medical fees for treatment to non-residents in epidemic areas were found to be the main cause for this behaviour. This phenomenon revealed a loophole in the control mechanisms of international quarantine procedures, letting travelers carrying a highly contagious virus slip by undetected and causing possible multi-country outbreaks of communicable diseases. Clinical evidence collected from medical records at medical centers can highlight this oversight.

12 CFR 563.180 - Suspicious Activity Reports and other reports and statements.

Code of Federal Regulations, 2012 CFR

2012-01-01

....C. 1813(u) and 1818(b)(9)). (iii) SAR means a Suspicious Activity Report. (3) SARs required. A... action. (11) Obtaining SARs. A savings association or service corporation may obtain SARs and the...) Confidentiality of SARs. A SAR, and any information that would reveal the existence of a SAR, are confidential...

Redescriptions of six species of Ilyodromus Sars, 1894 (Crustacea, Ostracoda, Cyprididae) from New Zealand and Eastern Australia.

PubMed

Shearn, Rylan; Halse, Stuart; Koenders, Annette; Schön, Isa; Martens, Koen

2014-10-23

In this paper, we redescribe six species of the genus Ilyodromus Sars, 1894: I, stanleyanus (King, 1855), I. varrovillius (King, 1855), I. smaragdinus Sars, 1894, I. obtusus Sars, 1894, I. substriatus Sars, 1894 and I. viridulus (Brady, 1886) using materials stored in the Oslo museum (Norway) and (re-) described by G.O. Sars.  For each species examined, we have identified a number of additional diagnostic characters to those used by Sars and earlier authors. In particular, the length of setae, claws and segments of the antennule, antenna, sixth limb, and caudal ramus appear to be important for species delineation in the genus, as does the internal structure of the valves.

High Prevalence of Middle East Respiratory Coronavirus in Young Dromedary Camels in Jordan

PubMed Central

van Doremalen, Neeltje; Hijazeen, Zaidoun S.K.; Holloway, Peter; Al Omari, Bilal; McDowell, Chester; Adney, Danielle; Talafha, Hani A.; Guitian, Javier; Steel, John; Amarin, Nadim; Tibbo, Markos; Abu-Basha, Ehab; Al-Majali, Ahmad M.

2017-01-01

Abstract Prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) was determined in 45 dromedary camels from two geographically separated herds in Jordan. Virus shedding was only detected in swabs obtained from the respiratory tract and primarily observed in camels younger than 3 years. MERS-CoV seroprevalence increased with age of camels. Bovine and sheep sera were seronegative. Phylogenetic analysis of partial S2 clustered the Jordanian MERS-CoV strains with contemporary MERS-CoV strains associated with nosocomial outbreaks. PMID:28009529

Air pollution and case fatality of SARS in the People's Republic of China: an ecologic study

PubMed Central

Cui, Yan; Zhang, Zuo-Feng; Froines, John; Zhao, Jinkou; Wang, Hua; Yu, Shun-Zhang; Detels, Roger

2003-01-01

Background Severe acute respiratory syndrome (SARS) has claimed 349 lives with 5,327 probable cases reported in mainland China since November 2002. SARS case fatality has varied across geographical areas, which might be partially explained by air pollution level. Methods Publicly accessible data on SARS morbidity and mortality were utilized in the data analysis. Air pollution was evaluated by air pollution index (API) derived from the concentrations of particulate matter, sulfur dioxide, nitrogen dioxide, carbon monoxide and ground-level ozone. Ecologic analysis was conducted to explore the association and correlation between air pollution and SARS case fatality via model fitting. Partially ecologic studies were performed to assess the effects of long-term and short-term exposures on the risk of dying from SARS. Results Ecologic analysis conducted among 5 regions with 100 or more SARS cases showed that case fatality rate increased with the increment of API (case fatality = - 0.063 + 0.001 * API). Partially ecologic study based on short-term exposure demonstrated that SARS patients from regions with moderate APIs had an 84% increased risk of dying from SARS compared to those from regions with low APIs (RR = 1.84, 95% CI: 1.41–2.40). Similarly, SARS patients from regions with high APIs were twice as likely to die from SARS compared to those from regions with low APIs. (RR = 2.18, 95% CI: 1.31–3.65). Partially ecologic analysis based on long-term exposure to ambient air pollution showed the similar association. Conclusion Our studies demonstrated a positive association between air pollution and SARS case fatality in Chinese population by utilizing publicly accessible data on SARS statistics and air pollution indices. Although ecologic fallacy and uncontrolled confounding effect might have biased the results, the possibility of a detrimental effect of air pollution on the prognosis of SARS patients deserves further investigation. PMID:14629774

Mapping Potential Amplification and Transmission Hotspots for MERS-CoV, Kenya.

PubMed

Gikonyo, Stephen; Kimani, Tabitha; Matere, Joseph; Kimutai, Joshua; Kiambi, Stella G; Bitek, Austine O; Juma Ngeiywa, K J Z; Makonnen, Yilma J; Tripodi, Astrid; Morzaria, Subhash; Lubroth, Juan; Rugalema, Gabriel; Fasina, Folorunso Oludayo

2018-03-16

Dromedary camels have been implicated consistently as the source of Middle East respiratory syndrome coronavirus (MERS-CoV) human infections and attention to prevent and control it has focused on camels. To understanding the epidemiological role of camels in the transmission of MERS-CoV, we utilized an iterative empirical process in Geographic Information System (GIS) to identify and qualify potential hotspots for maintenance and circulation of MERS-CoV, and produced risk-based surveillance sites in Kenya. Data on camel population and distribution were used to develop camel density map, while camel farming system was defined using multi-factorial criteria including the agro-ecological zones (AEZs), production and marketing practices. Primary and secondary MERS-CoV seroprevalence data from specific sites were analyzed, and location-based prevalence matching with camel densities was conducted. High-risk convergence points (migration zones, trade routes, camel markets, slaughter slabs) were profiled and frequent cross-border camel movement mapped. Results showed that high camel-dense areas and interaction (markets and migration zones) were potential hotspot for transmission and spread. Cross-border contacts occurred with in-migrated herds at hotspot locations. AEZ differential did not influence risk distribution and plausible risk factors for spatial MERS-CoV hotspots were camel densities, previous cases of MERS-CoV, high seroprevalence and points of camel convergences. Although Kenyan camels are predisposed to MERS-CoV, no shedding is documented to date. These potential hotspots, determined using anthropogenic, system and trade characterizations should guide selection of sampling/surveillance sites, high-risk locations, critical areas for interventions and policy development in Kenya, as well as instigate further virological examination of camels.

Analysis of Spatiotemporal Characteristics of Pandemic SARS Spread in Mainland China.

PubMed

Cao, Chunxiang; Chen, Wei; Zheng, Sheng; Zhao, Jian; Wang, Jinfeng; Cao, Wuchun

2016-01-01

Severe acute respiratory syndrome (SARS) is one of the most severe emerging infectious diseases of the 21st century so far. SARS caused a pandemic that spread throughout mainland China for 7 months, infecting 5318 persons in 194 administrative regions. Using detailed mainland China epidemiological data, we study spatiotemporal aspects of this person-to-person contagious disease and simulate its spatiotemporal transmission dynamics via the Bayesian Maximum Entropy (BME) method. The BME reveals that SARS outbreaks show autocorrelation within certain spatial and temporal distances. We use BME to fit a theoretical covariance model that has a sine hole spatial component and exponential temporal component and obtain the weights of geographical and temporal autocorrelation factors. Using the covariance model, SARS dynamics were estimated and simulated under the most probable conditions. Our study suggests that SARS transmission varies in its epidemiological characteristics and SARS outbreak distributions exhibit palpable clusters on both spatial and temporal scales. In addition, the BME modelling demonstrates that SARS transmission features are affected by spatial heterogeneity, so we analyze potential causes. This may benefit epidemiological control of pandemic infectious diseases.

Analysis of Spatiotemporal Characteristics of Pandemic SARS Spread in Mainland China

PubMed Central

Cao, Chunxiang; Zheng, Sheng; Zhao, Jian; Wang, Jinfeng; Cao, Wuchun

2016-01-01

Severe acute respiratory syndrome (SARS) is one of the most severe emerging infectious diseases of the 21st century so far. SARS caused a pandemic that spread throughout mainland China for 7 months, infecting 5318 persons in 194 administrative regions. Using detailed mainland China epidemiological data, we study spatiotemporal aspects of this person-to-person contagious disease and simulate its spatiotemporal transmission dynamics via the Bayesian Maximum Entropy (BME) method. The BME reveals that SARS outbreaks show autocorrelation within certain spatial and temporal distances. We use BME to fit a theoretical covariance model that has a sine hole spatial component and exponential temporal component and obtain the weights of geographical and temporal autocorrelation factors. Using the covariance model, SARS dynamics were estimated and simulated under the most probable conditions. Our study suggests that SARS transmission varies in its epidemiological characteristics and SARS outbreak distributions exhibit palpable clusters on both spatial and temporal scales. In addition, the BME modelling demonstrates that SARS transmission features are affected by spatial heterogeneity, so we analyze potential causes. This may benefit epidemiological control of pandemic infectious diseases. PMID:27597972

How Change of Public Transportation Usage Reveals Fear of the SARS Virus in a City

PubMed Central

Wang, Kuo-Ying

2014-01-01

The outbreaks of the severe acute respiratory syndrome (SARS) epidemic in 2003 resulted in unprecedented impacts on people's daily life. One of the most significant impacts to people is the fear of contacting the SARS virus while engaging daily routine activity. Here we use data from daily underground ridership in Taipei City and daily reported new SARS cases in Taiwan to model the dynamics of the public fear of the SARS virus during the wax and wane of the SARS period. We found that for each reported new SARS case there is an immediate loss of about 1200 underground ridership (the fresh fear). These daily loss rates dissipate to the following days with an e-folding time of about 28 days, reflecting the public perception on the risk of contacting SARS virus when traveling with the underground system (the residual fear). About 50% of daily ridership was lost during the peak of the 2003 SARS period, compared with the loss of 80% daily ridership during the closure of the underground system after Typhoon Nari, the loss of 50–70% ridership due to the closure of the governmental offices and schools during typhoon periods, and the loss of 60% daily ridership during Chinese New Year holidays. PMID:24647278

Web-GIS-based SARS epidemic situation visualization

NASA Astrophysics Data System (ADS)

Lu, Xiaolin

2004-03-01

In order to research, perform statistical analysis and broadcast the information of SARS epidemic situation according to the relevant spatial position, this paper proposed a unified global visualization information platform for SARS epidemic situation based on Web-GIS and scientific virtualization technology. To setup the unified global visual information platform, the architecture of Web-GIS based interoperable information system is adopted to enable public report SARS virus information to health cure center visually by using the web visualization technology. A GIS java applet is used to visualize the relationship between spatial graphical data and virus distribution, and other web based graphics figures such as curves, bars, maps and multi-dimensional figures are used to visualize the relationship between SARS virus tendency with time, patient number or locations. The platform is designed to display the SARS information in real time, simulate visually for real epidemic situation and offer an analyzing tools for health department and the policy-making government department to support the decision-making for preventing against the SARS epidemic virus. It could be used to analyze the virus condition through visualized graphics interface, isolate the areas of virus source, and control the virus condition within shortest time. It could be applied to the visualization field of SARS preventing systems for SARS information broadcasting, data management, statistical analysis, and decision supporting.

The effect of severe acute respiratory syndrome (SARS) on emergency airway management.

PubMed

Wong, Evelyn; Ho, Khoy Kheng

2006-07-01

From early March 2003 to late May 2003, severe acute respiratory syndrome (SARS) was detected in Singapore. The increase in workload and new infection control procedures were thought to affect resuscitation and airway management. Our aim was to study the effects of wearing of personal protective equipment (PPE) and powered air-purifying respirator (PAPR) and the restriction in the number of resuscitation personnel on airway management during the SARS crisis. Data was collected prospectively through an ongoing emergency airway registry. The data was divided into three periods: (1) before PPE was instituted from 1 November 2002 to 31 March 2003; (2) during SARS (when PPE use was mandatory) from 1 April to 31 July 2003; (3) post-SARs (when PPE use was non-mandatory but encouraged) from 1 August to 31 March 2004. There was no change in patient demographics during the three periods. There were significant increases in the proportion of resuscitation cases and airway interventions during the SARS period compared to the pre-SARS period. The resident medical officer intubation rate decreased from 45.1% pre-SARS to 35.2% during SARS and 17.7% post-SARS. The complication rates were 10.5%, 9.9% and 9.4% in periods 1-3, respectively. Restriction in the number of healthcare staff attending to each patient may have influenced the department's decision to allow only the most confident or experienced personnel to manage the airway. The exposure of junior medical officers in emergency airway management during SARS and the immediate post-SARS period was decreased. This trend should be monitored further and intervention may be necessary should it continue to decline.

Intratracheal exposure of common marmosets to MERS-CoV Jordan-n3/2012 or MERS-CoV EMC/2012 isolates does not result in lethal disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Reed F., E-mail: johnsonreed@mail.nih.gov; Via, Laura E.; Kumar, Mia R.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) continues to be a threat to human health in the Middle East. Development of countermeasures is ongoing; however, an animal model that faithfully recapitulates human disease has yet to be defined. A recent study indicated that inoculation of common marmosets resulted in inconsistent lethality. Based on these data we sought to compare two isolates of MERS-CoV. We followed disease progression in common marmosets after intratracheal exposure with: MERS-CoV-EMC/2012, MERS-CoV-Jordan-n3/2012, media, or inactivated virus. Our data suggest that common marmosets developed a mild to moderate non-lethal respiratory disease, which was quantifiable by computed tomography (CT),more » with limited other clinical signs. Based on CT data, clinical data, and virological data, MERS-CoV inoculation of common marmosets results in mild to moderate clinical signs of disease that are likely due to manipulations of the marmoset rather than as a result of robust viral replication. - Highlights: • Common marmosets infected with MERS-EMC and MERS-JOR did not develop lethal disease. • Infected subjects developed transient signs of clinical disease. • CT indicated few differences between the infected and control groups. • Marmosets do not faithfully replicate human MERS pathogenesis.« less

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

PubMed Central

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; Woo, Patrick CY

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-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. PMID:26632876

Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice.

PubMed

Wang, Chong; Zheng, Xuexing; Gai, Weiwei; Wong, Gary; Wang, Hualei; Jin, Hongli; Feng, Na; Zhao, Yongkun; Zhang, Weijiao; Li, Nan; Zhao, Guoxing; Li, Junfu; Yan, Jinghua; Gao, Yuwei; Hu, Guixue; Yang, Songtao; Xia, Xianzhu

2017-04-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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.

17 CFR 229.402 - (Item 402) Executive compensation.

Code of Federal Regulations, 2014 CFR

2014-04-01

... rights (“SARs”) refers to SARs payable in cash or stock, including SARs payable in cash or stock at the... without tandem SARs (including awards that subsequently have been transferred), the aggregate grant date..., the registrant has adjusted or amended the exercise price of options or SARs previously awarded to a...

78 FR 3399 - Draft 2012 Marine Mammal Stock Assessment Reports

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-16

... mammal stock assessment reports (SARs) in accordance with the Marine Mammal Protection Act, and solicited public comment on draft 2012 SARs. Subsequently, SARs for ten stocks of marine mammals in the Atlantic... solicits public comments on revised draft 2012 SARs for these ten stocks. DATES: Comments must be received...

SARS Risk Perception, Knowledge, Precautions, and Information Sources, the Netherlands

PubMed Central

Aro, Arja R.; Oenema, Anke; de Zwart, Onno; Richardus, Jan Hendrik; Bishop, George D.

2004-01-01

Severe acute respiratory syndrome (SARS)–related risk perceptions, knowledge, precautionary actions, and information sources were studied in the Netherlands during the 2003 SARS outbreak. Although respondents were highly aware of the SARS outbreak, the outbreak did not result in unnecessary precautionary actions or fears. PMID:15496256

Histopathology of Middle East respiratory syndrome coronovirus (MERS-CoV) infection - clinicopathological and ultrastructural study.

PubMed

Alsaad, Khaled O; Hajeer, Ali H; Al Balwi, Mohammed; Al Moaiqel, Mohammed; Al Oudah, Nourah; Al Ajlan, Abdulaziz; AlJohani, Sameera; Alsolamy, Sami; Gmati, Giamal E; Balkhy, Hanan; Al-Jahdali, Hamdan H; Baharoon, Salim A; Arabi, Yaseen M

2018-02-01

The pathogenesis, viral localization and histopathological features of Middle East respiratory syndrome - coronavirus (MERS-CoV) in humans are not described sufficiently. The aims of this study were to explore and define the spectrum of histological and ultrastructural pathological changes affecting various organs in a patient with MERS-CoV infection and represent a base of MERS-CoV histopathology. We analysed the post-mortem histopathological findings and investigated localisation of viral particles in the pulmonary and extrapulmonary tissue by transmission electron microscopic examination in a 33-year-old male patient of T cell lymphoma, who acquired MERS-CoV infection. Tissue needle biopsies were obtained from brain, heart, lung, liver, kidney and skeletal muscle. All samples were collected within 45 min from death to reduce tissue decomposition and artefact. Histopathological examination showed necrotising pneumonia, pulmonary diffuse alveolar damage, acute kidney injury, portal and lobular hepatitis and myositis with muscle atrophic changes. The brain and heart were histologically unremarkable. Ultrastructurally, viral particles were localised in the pneumocytes, pulmonary macrophages, renal proximal tubular epithelial cells and macrophages infiltrating the skeletal muscles. The results highlight the pulmonary and extrapulmonary pathological changes of MERS-CoV infection and provide the first evidence of the viral presence in human renal tissue, which suggests tissue trophism for MERS-CoV in kidney. © 2017 John Wiley & Sons Ltd.

MERS-CoV Accessory ORFs Play Key Role for Infection and Pathogenesis

PubMed Central

Menachery, Vineet D.; Mitchell, Hugh D.; Cockrell, Adam S.; Gralinski, Lisa E.; Yount, Boyd L.; Graham, Rachel L.; McAnarney, Eileen T.; Douglas, Madeline G.; Scobey, Trevor; Beall, Anne; Dinnon, Kenneth; Kocher, Jacob F.; Hale, Andrew E.; Stratton, Kelly G.; Waters, Katrina M.

2017-01-01

ABSTRACT While dispensable for viral replication, coronavirus (CoV) accessory open reading frame (ORF) proteins often play critical roles during infection and pathogenesis. Utilizing a previously generated mutant, we demonstrate that the absence of all four Middle East respiratory syndrome CoV (MERS-CoV) accessory ORFs (deletion of ORF3, -4a, -4b, and -5 [dORF3-5]) has major implications for viral replication and pathogenesis. Importantly, attenuation of the dORF3-5 mutant is primarily driven by dysregulated host responses, including disrupted cell processes, augmented interferon (IFN) pathway activation, and robust inflammation. In vitro replication attenuation also extends to in vivo models, allowing use of dORF3-5 as a live attenuated vaccine platform. Finally, examination of ORF5 implicates a partial role in modulation of NF-κB-mediated inflammation. Together, the results demonstrate the importance of MERS-CoV accessory ORFs for pathogenesis and highlight them as potential targets for surveillance and therapeutic treatments moving forward. PMID:28830941

Fear of Severe Acute Respiratory Syndrome (SARS) among Health Care Workers

ERIC Educational Resources Information Center

Ho, Samuel M. Y.; Kwong-Lo, Rosalie S. Y.; Mak, Christine W. Y.; Wong, Joe S.

2005-01-01

In this study, the authors examined fear related to severe acute respiratory syndrome (SARS) among 2 samples of hospital staff in Hong Kong. Sample 1 included health care workers (n = 82) and was assessed during the peak of the SARS epidemic. Sample 2 included hospital staff who recovered from SARS (n = 97). The results show that participants in…

Prevalence and Factors Associated with Social Avoidance of Recovered SARS Patients in the Hong Kong General Population

ERIC Educational Resources Information Center

Lau, Joseph T. F.; Yang, Xilin; Wong, Eric; Tsui, H. Y.

2006-01-01

The study investigated the general population's perceived infectivity of asymptomatic and recovered severe acute respiratory syndrome (SARS) patients and factors associated with avoidance and discriminatory attitudes, including demographic background, SARS-related perceptions and emotional response to the SARS epidemic. A population-based survey…

12 CFR 563.180 - Suspicious Activity Reports and other reports and statements.

Code of Federal Regulations, 2013 CFR

2013-01-01

....C. 1813(u) and 1818(b)(9)). (iii) SAR means a Suspicious Activity Report. (3) SARs required. A... action. (11) Obtaining SARs. A savings association or service corporation may obtain SARs and the...) Confidentiality of SARs. A SAR, and any information that would reveal the existence of a SAR, are confidential...

Structural transition and orbital glass physics in near-itinerant CoV 2O 4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reig-i-Plessis, D.; Casavant, D.; Garlea, Vasile O.

2016-01-25

In this study, the ferrimagnetic spinel CoV 2O 4 has been a topic of intense recent interest, both as a frustrated insulator with unquenched orbital degeneracy and as a near-itinerant magnet which can be driven metallic with moderate applied pressure. Here, we report on our recent neutron di raction and inelastic scattering measurements on powders with minimal cation site disorder. Our main new result is the identification of a weak (Δa/a ~ 10 –4), first order structural phase transition at T* = 90 K, the same temperature where spin canting was seen in recent single crystal measurements. This transition ismore » characterized by a short-range distortion of oxygen octahedral positions, and inelastic data further establish a weak 1.25meV spin gap at low temperature. Together, these findings provide strong support for the local orbital picture and the existence of an orbital glass state at temperatures below T*.« less

Response to Emergence of Middle East Respiratory Syndrome Coronavirus, Abu Dhabi, United Arab Emirates, 2013-2014.

PubMed

Al Hosani, Farida Ismail; Pringle, Kimberly; Al Mulla, Mariam; Kim, Lindsay; Pham, Huong; Alami, Negar N; Khudhair, Ahmed; Hall, Aron J; Aden, Bashir; El Saleh, Feda; Al Dhaheri, Wafa; Al Bandar, Zyad; Bunga, Sudhir; Abou Elkheir, Kheir; Tao, Ying; Hunter, Jennifer C; Nguyen, Duc; Turner, Andrew; Pradeep, Krishna; Sasse, Jurgen; Weber, Stefan; Tong, Suxiang; Whitaker, Brett L; Haynes, Lia M; Curns, Aaron; Gerber, Susan I

2016-07-01

In January 2013, several months after Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia, Abu Dhabi, United Arab Emirates, began surveillance for MERS-CoV. We analyzed medical chart and laboratory data collected by the Health Authority-Abu Dhabi during January 2013-May 2014. Using real-time reverse transcription PCR, we tested respiratory tract samples for MERS-CoV and identified 65 case-patients. Of these patients, 23 (35%) were asymptomatic at the time of testing, and 4 (6%) showed positive test results for >3 weeks (1 had severe symptoms and 3 had mild symptoms). We also identified 6 clusters of MERS-CoV cases. This report highlights the potential for virus shedding by mildly ill and asymptomatic case-patients. These findings will be useful for MERS-CoV management and infection prevention strategies.

Beyond SARS: ethnic community organization's role in public health -- a Toronto experience.

PubMed

Weizhen Dong

2008-12-01

The SARS outbreak in Toronto was a public health crisis. It was particularly frightening to the Chinese-Canadians, because of the origin of the deadly disease. The Chinese-Canadian community organizations launched various activities to help the Chinese-Canadians as well as other Asian-Canadian communities to fight against SARS and its social side-effects. From launching the SARS Supporting Line, distributing health promotional material, disseminating SARS related information, paying tribute to frontline health workers, and promoting local business, to fundraising for SARS related research; they played an active role in easing the public's anxiety, especially for the Chinese-Canadians in the great Toronto area. The culturally diverse population brought problems as well as solutions. Ethnic groups have expertise in almost all areas, including people with leadership skills. The Toronto Chinese community's experience in combating SARS is a good example. The Chinese-Canadian community organizations' activities during the SARS outbreak demonstrate that ethnic minority organizations can play an important role in public health, especially in a public health crisis, and beyond.

Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study.

PubMed

Reusken, Chantal B E M; Haagmans, Bart L; Müller, Marcel A; Gutierrez, Carlos; Godeke, Gert-Jan; Meyer, Benjamin; Muth, Doreen; Raj, V Stalin; Smits-De Vries, Laura; Corman, Victor M; Drexler, Jan-Felix; Smits, Saskia L; El Tahir, Yasmin E; De Sousa, Rita; van Beek, Janko; Nowotny, Norbert; van Maanen, Kees; Hidalgo-Hermoso, Ezequiel; Bosch, Berend-Jan; Rottier, Peter; Osterhaus, Albert; Gortázar-Schmidt, Christian; Drosten, Christian; Koopmans, Marion P G

2013-10-01

A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, molecular clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralising antibody titres varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralisation by bovine coronavirus antibodies. MERS-CoV or a related virus has infected camel populations. Both titres and seroprevalences in sera from different locations in Oman suggest widespread infection. European Union, European Centre For Disease Prevention and Control, Deutsche Forschungsgemeinschaft. Copyright © 2013 Elsevier Ltd. All rights reserved.

Spatial Distribution of the Coefficient of Variation and Bayesian Forecast for the Paleo-Earthquakes in Japan

NASA Astrophysics Data System (ADS)

Nomura, Shunichi; Ogata, Yosihiko

2016-04-01

We propose a Bayesian method of probability forecasting for recurrent earthquakes of inland active faults in Japan. Renewal processes with the Brownian Passage Time (BPT) distribution are applied for over a half of active faults in Japan by the Headquarters for Earthquake Research Promotion (HERP) of Japan. Long-term forecast with the BPT distribution needs two parameters; the mean and coefficient of variation (COV) for recurrence intervals. The HERP applies a common COV parameter for all of these faults because most of them have very few specified paleoseismic events, which is not enough to estimate reliable COV values for respective faults. However, different COV estimates are proposed for the same paleoseismic catalog by some related works. It can make critical difference in forecast to apply different COV estimates and so COV should be carefully selected for individual faults. Recurrence intervals on a fault are, on the average, determined by the long-term slip rate caused by the tectonic motion but fluctuated by nearby seismicities which influence surrounding stress field. The COVs of recurrence intervals depend on such stress perturbation and so have spatial trends due to the heterogeneity of tectonic motion and seismicity. Thus we introduce a spatial structure on its COV parameter by Bayesian modeling with a Gaussian process prior. The COVs on active faults are correlated and take similar values for closely located faults. It is found that the spatial trends in the estimated COV values coincide with the density of active faults in Japan. We also show Bayesian forecasts by the proposed model using Markov chain Monte Carlo method. Our forecasts are different from HERP's forecast especially on the active faults where HERP's forecasts are very high or low.

Serological Evidence of MERS-CoV Antibodies in Dromedary Camels (Camelus dromedaries) in Laikipia County, Kenya

PubMed Central

Deem, Sharon L.; Fèvre, Eric M.; Kinnaird, Margaret; Browne, A. Springer; Muloi, Dishon; Godeke, Gert-Jan; Koopmans, Marion; Reusken, Chantal B.

2015-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently identified virus causing severe viral respiratory illness in people. Little is known about the reservoir in the Horn of Africa. In Kenya, where no human MERS cases have been reported, our survey of 335 dromedary camels, representing nine herds in Laikipia County, showed a high seroprevalence (46.9%) to MERS-CoV antibodies. Between herd differences were present (14.3%– 82.9%), but was not related to management type or herd isolation. Further research should focus on identifying similarity between MERS-CoV viral isolates in Kenya and clinical isolates from the Middle East and elsewhere. PMID:26473733

Chronic widespread musculoskeletal pain, fatigue, depression and disordered sleep in chronic post-SARS syndrome; a case-controlled study.

PubMed

Moldofsky, Harvey; Patcai, John

2011-03-24

The long term adverse effects of Severe Acute Respiratory Syndrome (SARS), a viral disease, are poorly understood. Sleep physiology, somatic and mood symptoms of 22 Toronto subjects, 21 of whom were healthcare workers, (19 females, 3 males, mean age 46.29 yrs.+/- 11.02) who remained unable to return to their former occupation (mean 19.8 months, range: 13 to 36 months following SARS) were compared to 7 healthy female subjects. Because of their clinical similarities to patients with fibromyalgia syndrome (FMS) these post-SARS subjects were similarly compared to 21 drug free female patients, (mean age 42.4 +/- 11.8 yrs.) who fulfilled criteria for fibromyalgia. Chronic post-SARS is characterized by persistent fatigue, diffuse myalgia, weakness, depression, and nonrestorative sleep with associated REM-related apneas/hypopneas, an elevated sleep EEG cyclical alternating pattern, and alpha EEG sleep anomaly. Post- SARS patients had symptoms of pre and post-sleep fatigue and post sleep sleepiness that were similar to the symptoms of patients with FMS, and similar to symptoms of patients with chronic fatigue syndrome. Both post-SARS and FMS groups had sleep instability as indicated by the high sleep EEG cyclical alternating pattern rate. The post-SARS group had a lower rating of the alpha EEG sleep anomaly as compared to the FMS patients. The post-SARS group also reported less pre-sleep and post-sleep musculoskeletal pain symptoms. The clinical and sleep features of chronic post-SARS form a syndrome of chronic fatigue, pain, weakness, depression and sleep disturbance, which overlaps with the clinical and sleep features of FMS and chronic fatigue syndrome.

12 CFR 563.180 - Suspicious Activity Reports and other reports and statements.

Code of Federal Regulations, 2014 CFR

2014-01-01

....C. 1813(u) and 1818(b)(9)). (iii) SAR means a Suspicious Activity Report. (3) SARs required. A... action. (11) Obtaining SARs. A savings association or service corporation may obtain SARs and the...) Confidentiality of SARs. A SAR, and any information that would reveal the existence of a SAR, are confidential...

Rapid and Effective Virucidal Activity of Povidone-Iodine Products Against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Modified Vaccinia Virus Ankara (MVA).

PubMed

Eggers, Maren; Eickmann, Markus; Zorn, Juergen

2015-12-01

Since the first case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infection was reported in 2012, the virus has infected more than 1300 individuals in 26 countries, and caused more than 480 deaths. Human-to-human transmission requires close contact, and has typically occurred in the healthcare setting. Improved global awareness, together with improved hygiene practices in healthcare facilities, has been highlighted as key strategies in controlling the spread of MERS-CoV. This study tested the in vitro efficacy of three formulations of povidone iodine (PVP-I: 4% PVP-I skin cleanser, 7.5% PVP-I surgical scrub, and 1% PVP-I gargle/mouthwash) against a reference virus (Modified vaccinia virus Ankara, MVA) and MERS-CoV. According to EN14476, a standard suspension test was used to assess virucidal activity against MVA and large volume plating was used for MERS-CoV. All products were tested under clean (0.3 g/L bovine serum albumin, BSA) and dirty conditions (3.0 g/L BSA + 3.0 mL/L erythrocytes), with application times of 15, 30, and 60 s for MVA, and 15 s for MERS-CoV. The products were tested undiluted, 1:10 and 1:100 diluted against MVA, and undiluted against MERS-CoV. A reduction in virus titer of ≥4 log10 (corresponding to an inactivation of ≥99.99%) was regarded as evidence of virucidal activity. This was achieved versus MVA and MERS-CoV, under both clean and dirty conditions, within 15 s of application of each undiluted PVP-I product. These data indicate that PVP-I-based hand wash products for potentially contaminated skin, and PVP-I gargle/mouthwash for reduction of viral load in the oral cavity and the oropharynx, may help to support hygiene measures to prevent transmission of MERS-CoV. Mundipharma Research GmbH & Co.

12 CFR 21.11 - Suspicious Activity Report.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Activity Report. (c) SARs required. A national bank shall file a SAR with the appropriate Federal law... supervisory action. (j) Obtaining SARs. A national bank may obtain SARs and the Instructions from the appropriate OCC District Office listed in 12 CFR part 4. (k) Confidentiality of SARs. A SAR, and any...

12 CFR 21.11 - Suspicious Activity Report.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Activity Report. (c) SARs required. A national bank shall file a SAR with the appropriate Federal law... supervisory action. (j) Obtaining SARs. A national bank may obtain SARs and the Instructions from the appropriate OCC District Office listed in 12 CFR part 4. (k) Confidentiality of SARs. A SAR, and any...

78 FR 33842 - Determination and Declaration Regarding Emergency Use of In Vitro Diagnostics for Detection of...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-05

... In Vitro Diagnostics for Detection of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) AGENCY... Middle East respiratory syndrome coronavirus (MERS-CoV). On the basis of this determination, she also... detection of Middle East respiratory syndrome coronavirus (MERS-CoV) pursuant to section 564 of the FD&C Act...

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

PubMed

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.

Cross-sectional surveillance of Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels and other mammals in Egypt, August 2015 to January 2016

PubMed Central

Ali, Mohamed; El-Shesheny, Rabeh; Kandeil, Ahmed; Shehata, Mahmoud; Elsokary, Basma; Gomaa, Mokhtar; Hassan, Naglaa; El Sayed, Ahmed; El-Taweel, Ahmed; Sobhy, Heba; Oludayo, Fasina Folorunso; Dauphin, Gwenaelle; El Masry, Ihab; Wolde, Abebe Wossene; Daszak, Peter; Miller, Maureen; VonDobschuetz, Sophie; Gardner, Emma; Morzaria, Subhash; Lubroth, Juan; Makonnen, Yilma Jobre

2017-01-01

A cross-sectional study was conducted in Egypt to determine the prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) in imported and resident camels and bats, as well as to assess possible transmission of the virus to domestic ruminants and equines. A total of 1,031 sera, 1,078 nasal swabs, 13 rectal swabs, and 38 milk samples were collected from 1,078 camels in different types of sites. In addition, 145 domestic animals and 109 bats were sampled. Overall, of 1,031 serologically-tested camels, 871 (84.5%) had MERS-CoV neutralising antibodies. Seroprevalence was significantly higher in imported (614/692; 88.7%) than resident camels (257/339; 5.8%) (p < 0.05). Camels from Sudan (543/594; 91.4%) had a higher seroprevalence than those from East Africa (71/98; 72.4%) (p < 0.05). Sampling site and age were also associated with MERS-CoV seroprevalence (p < 0.05). All tested samples from domestic animals and bats were negative for MERS-CoV antibodies except one sheep sample which showed a 1:640 titre. Of 1,078 camels, 41 (3.8%) were positive for MERS-CoV genetic material. Sequences obtained were not found to cluster with clade A or B MERS-CoV sequences and were genetically diverse. The presence of neutralising antibodies in one sheep apparently in contact with seropositive camels calls for further studies on domestic animals in contact with camels. PMID:28333616

Assessment of documentation requirements under DOE 5481. 1, Safety Analysis and Review System (SARS)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Browne, E.T.

1981-03-01

This report assesses the requirements of DOE Order 5481.1, Safety Analysis and Review System for DOE Operations (SARS) in regard to maintaining SARS documentation. Under SARS, all pertinent details of the entire safety analysis and review process for each DOE operation are to be traceable from the initial identification of a hazard. This report is intended to provide assistance in identifying the points in the SARS cycle at which documentation is required, what type of documentation is most appropriate, and where it ultimately should be maintained.

Lack of SARS transmission among public hospital workers, Vietnam.

PubMed

Le, Dang Ha; Bloom, Sharon A; Nguyen, Quang Hien; Maloney, Susan A; Le, Quynh Mai; Leitmeyer, Katrin C; Bach, Huy Anh; Reynolds, Mary G; Montgomery, Joel M; Comer, James A; Horby, Peter W; Plant, Aileen J

2004-02-01

The severe acute respiratory syndrome (SARS) outbreak in Vietnam was amplified by nosocomial spread within hospital A, but no transmission was reported in hospital B, the second of two designated SARS hospitals. Our study documents lack of SARS-associated coronavirus transmission to hospital B workers, despite variable infection control measures and the use of personal protective equipment.

Association between Australian Hajj Pilgrims' awareness of MERS-CoV, and their compliance with preventive measures and exposure to camels.

PubMed

Alqahtani, Amani S; Wiley, Kerrie E; Mushta, Sami M; Yamazaki, Kaoruko; BinDhim, Nasser F; Heywood, Anita E; Booy, Robert; Rashid, Harunor

2016-05-01

Through a prospective cohort study the relationship between travellers' awareness of MERS-CoV, and compliance with preventive measures and exposure to camels was evaluated among Australian Hajj pilgrims who attended Hajj in 2015. Only 28% of Australian Hajj pilgrims were aware of MERS-CoV in Saudi Arabia. Those who were aware of MERS-CoV were more likely to receive recommended vaccines [odds ratio (OR) 3.1, 95% confidence interval (CI): 1.5-5.9, P < 0.01], but there was no significant difference in avoiding camels or their raw products during Hajj between those who were aware of MERS-CoV and those who were not (OR 1.2, 95% CI: 0.3-5.2, P = 0.7). Hajj pilgrims' awareness is reflected in some of their practices but not in all. © International Society of Travel Medicine, 2016. All rights reserved. Published by Oxford University Press. For permissions, please e-mail: journals.permissions@oup.com.

Severe Acute Respiratory Syndrome (SARS) Prevention in Taiwan

ERIC Educational Resources Information Center

Liu, Hsueh-Erh

2004-01-01

Severe Acute Respiratory Syndrome (SARS) is a newly identified respiratory disease that threatened Taiwan between April 14 and July 5, 2003. Chang Gung University experienced various SARS-related episodes, such as the postponement of classes for 7 days, the reporting of probable SARS cases, and the isolation of students under Level A and B…

Rapid generation of a mouse model for Middle East respiratory syndrome

PubMed Central

Zhao, Jincun; Li, Kun; Wohlford-Lenane, Christine; Agnihothram, Sudhakar S.; Fett, Craig; Zhao, Jingxian; Gale, Michael J.; Baric, Ralph S.; Enjuanes, Luis; Gallagher, Tom; McCray, Paul B.; Perlman, Stanley

2014-01-01

In this era of continued emergence of zoonotic virus infections, the rapid development of rodent models represents a critical barrier to public health preparedness, including the testing of antivirus therapy and vaccines. The Middle East respiratory syndrome coronavirus (MERS-CoV) was recently identified as the causative agent of a severe pneumonia. Given the ability of coronavirus to rapidly adapt to new hosts, a major public health concern is that MERS-CoV will further adapt to replication in humans, triggering a pandemic. No small-animal model for this infection is currently available, but studies suggest that virus entry factors can confer virus susceptibility. Here, we show that mice were sensitized to MERS-CoV infection by prior transduction with adenoviral vectors expressing the human host-cell receptor dipeptidyl peptidase 4. Mice developed a pneumonia characterized by extensive inflammatory-cell infiltration with virus clearance occurring 6–8 d after infection. Clinical disease and histopathological changes were more severe in the absence of type-I IFN signaling whereas the T-cell response was required for virus clearance. Using these mice, we demonstrated the efficacy of a therapeutic intervention (poly I:C) and a potential vaccine [Venezuelan equine encephalitis replicon particles expressing MERS-CoV spike protein]. We also found little protective cross-reactivity between MERS-CoV and the severe acute respiratory syndrome-CoV. Our results demonstrate that this system will be useful for MERS-CoV studies and for the rapid development of relevant animal models for emerging respiratory viral infections. PMID:24599590

Repurposing a Library of Human Cathepsin L Ligands: Identification of Macrocyclic Lactams as Potent Rhodesain and Trypanosoma brucei Inhibitors.

PubMed

Giroud, Maude; Dietzel, Uwe; Anselm, Lilli; Banner, David; Kuglstatter, Andreas; Benz, Jörg; Blanc, Jean-Baptiste; Gaufreteau, Delphine; Liu, Haixia; Lin, Xianfeng; Stich, August; Kuhn, Bernd; Schuler, Franz; Kaiser, Marcel; Brun, Reto; Schirmeister, Tanja; Kisker, Caroline; Diederich, François; Haap, Wolfgang

2018-04-26

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( K i < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC 50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.

Inactivation of Ebola virus and Middle East respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet C light and methylene blue plus visible light, respectively.

PubMed

Eickmann, Markus; Gravemann, Ute; Handke, Wiebke; Tolksdorf, Frank; Reichenberg, Stefan; Müller, Thomas H; Seltsam, Axel

2018-05-06

Ebola virus (EBOV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have been identified as potential threats to blood safety. This study investigated the efficacy of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate EBOV and MERS-CoV in platelet concentrates (PCs) and plasma, respectively. PCs and plasma were spiked with high titers of cell culture-derived EBOV and MERS-CoV, treated with various light doses of ultraviolet C (UVC; THERAFLEX UV-Platelets) or methylene blue (MB) plus visible light (MB/light; THERAFLEX MB-Plasma), and assessed for residual viral infectivity. UVC reduced EBOV (≥4.5 log) and MERS-CoV (≥3.7 log) infectivity in PCs to the limit of detection, and MB/light decreased EBOV (≥4.6 log) and MERS-CoV (≥3.3 log) titers in plasma to nondetectable levels. Both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce EBOV and MERS-CoV infectivity in platelets and plasma, respectively. © 2018 AABB.

Infection control and prevention practices implemented to reduce transmission risk of Middle East respiratory syndrome-coronavirus in a tertiary care institution in Saudi Arabia.

PubMed

Butt, Taimur S; Koutlakis-Barron, Irene; AlJumaah, Suliman; AlThawadi, Sahar; AlMofada, Saleh

2016-05-01

Transmission of Middle East respiratory syndrome-coronavirus (MERS-CoV) among health care workers (HCWs) and patients has been documented with mortality rate approximating 36%. We propose advanced infection control measures (A-IC) used in conjunction with basic infection control measures (B-IC) help reduce pathogen transmission. B-IC include standard and transmission-based precautions. A-IC are initiatives implemented within our center to enhance effectiveness of B-IC. Study effectiveness of combining B-IC and A-IC to prevent transmission of MERS-CoV to HCWs. A retrospective observational study was undertaken. A-IC measures include administrative support with daily rounds; infection control risk assessment; timely screening, isolation, and specimen analysis; collaboration; epidemic planning; stockpiling; implementation of contingency plans; full personal protective equipment use for advanced airway management; use of a real-time electronic isolation flagging system; infection prevention and control team on-call protocols; pretransfer MERS-CoV testing; and education. A total of 874 real-time polymerase chain reaction MERS-CoV tests were performed during the period beginning July 1, 2013, and ending January 31, 2015. Six hundred ninety-four non-HCWs were tested, of these 16 tested positive for MERS-CoV and their infection was community acquired. Sixty-nine percent of the confirmed MERS-CoV-positive cases were men, with an average age of 56 years (range, 19-84 years). Of the total tested for MERS-CoV, 180 individuals were HCWs with zero positivity. Adhering to a combination of B-IC and A-IC reduces the risk of MERS-CoV transmission to HCWs. Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

The experience of SARS-related stigma at Amoy Gardens.

PubMed

Lee, Sing; Chan, Lydia Y Y; Chau, Annie M Y; Kwok, Kathleen P S; Kleinman, Arthur

2005-11-01

Severe Acute Respiratory Syndrome (SARS) possesses characteristics that render it particularly prone to stigmatization. SARS-related stigma, despite its salience for public health and stigma research, has had little examination. This study combines survey and case study methods to examine subjective stigma among residents of Amoy Gardens (AG), the first officially recognized site of community outbreak of SARS in Hong Kong. A total of 903 residents of AG completed a self-report questionnaire derived from two focus groups conducted toward the end of the 3-month outbreak. Case studies of two residents who lived in Block E, the heart of the SARS epidemic at AG, complement the survey data. Findings show that stigma affected most residents and took various forms of being shunned, insulted, marginalized, and rejected in the domains of work, interpersonal relationships, use of services and schooling. Stigma was also associated with psychosomatic distress. Residents' strategies for diminishing stigma varied with gender, age, education, occupation, and proximity to perceived risk factors for SARS such as residential location, previous SARS infection and the presence of ex-SARS household members. Residents attributed stigma to government mismanagement, contagiousness of the mysterious SARS virus, and alarmist media reporting. Stigma clearly decreased, but never completely disappeared, after the outbreak. The findings confirm and add to existing knowledge on the varied origins, correlates, and impacts of stigma. They also highlight the synergistic roles of inconsistent health policy responses and risk miscommunication by the media in rapidly amplifying stigma toward an unfamiliar illness. While recognizing the intrinsically stigmatizing nature of public health measures to control SARS, we recommend that a consistent inter-sectoral approach is needed to minimize stigma and to make an effective health response to future outbreaks.

SARS-related perceptions in Hong Kong.

PubMed

Lau, Joseph T F; Yang, Xilin; Pang, Ellie; Tsui, H Y; Wong, Eric; Wing, Yun Kwok

2005-03-01

To understand different aspects of community responses related to severe acute respiratory syndrome (SARS), 2 population-based, random telephone surveys were conducted in June 2003 and January 2004 in Hong Kong. More than 70% of respondents would avoid visiting hospitals or mainland China to avoid contracting SARS. Most respondents believed that SARS could be transmitted through droplets, fomites, sewage, and animals. More than 90% believed that public health measures were efficacious means of prevention; 40.4% believed that SARS would resurge in Hong Kong; and approximately equals 70% would then wear masks in public places. High percentages of respondents felt helpless, horrified, and apprehensive because of SARS. Approximately 16% showed signs of posttraumatic symptoms, and approximately equals 40% perceived increased stress in family or work settings. The general public in Hong Kong has been very vigilant about SARS but needs to be more psychologically prepared to face a resurgence of the epidemic.

SARS-related Perceptions in Hong Kong

PubMed Central

Yang, Xilin; Pang, Ellie; Tsui, H.Y.; Wong, Eric; Wing, Yun Kwok

2005-01-01

To understand different aspects of community responses related to severe acute respiratory syndrome (SARS), 2 population-based, random telephone surveys were conducted in June 2003 and January 2004 in Hong Kong. More than 70% of respondents would avoid visiting hospitals or mainland China to avoid contracting SARS. Most respondents believed that SARS could be transmitted through droplets, fomites, sewage, and animals. More than 90% believed that public health measures were efficacious means of prevention; 40.4% believed that SARS would resurge in Hong Kong; and ≈70% would then wear masks in public places. High percentages of respondents felt helpless, horrified, and apprehensive because of SARS. Approximately 16% showed signs of posttraumatic symptoms, and ≈40% perceived increased stress in family or work settings. The general public in Hong Kong has been very vigilant about SARS but needs to be more psychologically prepared to face a resurgence of the epidemic. PMID:15757557

A Support Group for Home-Quarantined College Students Exposed to SARS: Learning from Practice

ERIC Educational Resources Information Center

Pan, Peter J. D.; Chang, Shih-Hua; Yu, Yen-Yen

2005-01-01

This article is an initial description of a meaningful and valuable clinical experience in interacting with SARS home-quarantined college students in a support group in Taiwan. Information about SARS and home quarantine, the tasks of the Counseling Centers and group work after the SARS outbreak, the support group for home-quarantined members, the…

Healthcare Worker Seroconversion in SARS Outbreak

PubMed Central

Ooi, Eng-Eong; Tan, Hiang-Khoon; Ong, Kong-Wee; Sil, Bijon Kumar; Teo, Melissa; Ng, Timothy; Soo, Khee-Chee

2004-01-01

Serum samples were obtained from healthcare workers 5 weeks after exposure to an outbreak of severe acute respiratory syndrome (SARS). A sensitive dot blot enzyme-linked immunosorbent assay, complemented by a specific neutralization test, shows that only persons in whom probable SARS was diagnosed had specific antibodies and suggests that subclinical SARS is not an important feature of the disease. PMID:15030691

Longitudinal study of age-specific pattern of coronavirus infection in Lyle's flying fox (Pteropus lylei) in Thailand.

PubMed

Wacharapluesadee, Supaporn; Duengkae, Prateep; Chaiyes, Aingorn; Kaewpom, Thongchai; Rodpan, Apaporn; Yingsakmongkon, Sangchai; Petcharat, Sininat; Phengsakul, Patcharakiti; Maneeorn, Pattarapol; Hemachudha, Thiravat

2018-02-20

Bats are natural reservoirs for several highly pathogenic and novel viruses including coronaviruses (CoVs) (mainly Alphacoronavirus and Betacoronavirus). Lyle's flying fox (Pteropus lylei)'s roosts and foraging sites are usually in the proximity to humans and animals. Knowledge about age-specific pattern of CoV infection in P. lylei, prevalence, and viral shedding at roosts and foraging sites may have an impact on infection-age-structure model to control CoV outbreak. P. lylei bats were captured monthly during January-December 2012 for detection of CoV at three areas in Chonburi province; two human dwellings, S1 and S2, where few fruit trees were located with an open pig farm, 0.6 km and 5.5 km away from the bat roost, S3. Nested RT-PCR of RNA-dependent RNA polymerase (RdRp) gene from rectal swabs was used for CoV detection. The strain of CoV was confirmed by sequencing and phylogenetic analysis. CoV infection was found in both juveniles and adult bats between May and October (January, in adults only and April, in juveniles only). Of total rectal swab positives (68/367, 18.5%), ratio was higher in bats captured at S1 (11/44, 25.0%) and S2 (35/99, 35.4%) foraging sites than at roost (S3) (22/224, 9.8%). Juveniles (forearm length ≤ 136 mm) were found with more CoV infection than adults at all three sites; S1 (9/24, 37.5% vs 2/20, 10%), S2 (22/49, 44.9% vs 13/50, 26.0%), and S3 (10/30, 33.3% vs 12/194, 6.2%). The average BCI of CoV infected bats was significantly lower than uninfected bats. No gender difference related to infection was found at the sites. Phylogenetic analysis of conserved RdRp gene revealed that the detected CoVs belonged to group D betacoronavirus (n = 64) and alphacoronavirus (n = 4). The fact that CoV infection and shedding was found in more juvenile than adult bats may suggest transmission from mother during peripartum period. Whether viral reactivation during parturition period or stress is responsible in maintaining transmission in the bat colony needs to be explored.

Pharmacokinetics of the Antiviral Lectin Griffithsin Administered by Different Routes Indicates Multiple Potential Uses.

PubMed

Barton, Christopher; Kouokam, J Calvin; Hurst, Harrell; Palmer, Kenneth E

2016-12-17

Griffithsin (GRFT) is a red alga-derived lectin with demonstrated broad spectrum antiviral activity against enveloped viruses, including severe acute respiratory syndrome-Coronavirus (SARS-CoV), Japanese encephalitis virus (JEV), hepatitis C virus (HCV), and herpes simplex virus-2 (HSV-2). However, its pharmacokinetic profile remains largely undefined. Here, Sprague Dawley rats were administered a single dose of GRFT at 10 or 20 mg/kg by intravenous, oral, and subcutaneous routes, respectively, and serum GRFT levels were measured at select time points. In addition, the potential for systemic accumulation after oral dosing was assessed in rats after 10 daily treatments with GRFT (20 or 40 mg/kg). We found that parenterally-administered GRFT in rats displayed a complex elimination profile, which varied according to administration routes. However, GRFT was not orally bioavailable, even after chronic treatment. Nonetheless, active GRFT capable of neutralizing HIV-Env pseudoviruses was detected in rat fecal extracts after chronic oral dosing. These findings support further evaluation of GRFT for pre-exposure prophylaxis against emerging epidemics for which specific therapeutics are not available, including systemic and enteric infections caused by susceptible enveloped viruses. In addition, GRFT should be considered for antiviral therapy and the prevention of rectal transmission of HIV-1 and other susceptible viruses.