Peptide-Based Membrane Fusion Inhibitors Targeting HCoV-229E Spike Protein HR1 and HR2 Domains

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Shuai Xia

2018-02-01

Full Text Available Human coronavirus 229E (HCoV-229E infection in infants, elderly people, and immunocompromised patients can cause severe disease, thus calling for the development of effective and safe therapeutics to treat it. Here we reported the design, synthesis and characterization of two peptide-based membrane fusion inhibitors targeting HCoV-229E spike protein heptad repeat 1 (HR1 and heptad repeat 2 (HR2 domains, 229E-HR1P and 229E-HR2P, respectively. We found that 229E-HR1P and 229E-HR2P could interact to form a stable six-helix bundle and inhibit HCoV-229E spike protein-mediated cell-cell fusion with IC50 of 5.7 and 0.3 µM, respectively. 229E-HR2P effectively inhibited pseudotyped and live HCoV-229E infection with IC50 of 0.5 and 1.7 µM, respectively. In a mouse model, 229E-HR2P administered intranasally could widely distribute in the upper and lower respiratory tracts and maintain its fusion-inhibitory activity. Therefore, 229E-HR2P is a promising candidate for further development as an antiviral agent for the treatment and prevention of HCoV-229E infection.

Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

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Berkhout Ben

2006-12-01

Full Text Available Abstract Background The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU in Salisbury, UK. Results All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%. Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates carry a single ORF, 660 nt in size, encoding a single protein of 219 amino acids, which is a homologue of the ORF3 proteins encoded by HCoV-NL63 and PEDV. Conclusion Thus, the genome organization of the group 1b coronaviruses HCoV-NL63, PEDV and HCoV-229E is identical. It is possible that extensive culturing of the HCoV-229E laboratory strain resulted in truncation of ORF4. This may indicate that the protein is not essential in cell culture, but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo.

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

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

2012-01-01

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

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

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

2012-04-01

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

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

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Sjoerd H E van den Worm

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

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

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Foula Vassilara

2018-01-01

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

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

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

2012-07-01

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

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.

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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

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

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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.

A rare cause of acute flaccid paralysis: Human coronaviruses

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

2015-01-01

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

Prolonged Shedding of Human Coronavirus in Hematopoietic Cell Transplant Recipients: Risk Factors and Viral Genome Evolution.

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Ogimi, Chikara; Greninger, Alexander L; Waghmare, Alpana A; Kuypers, Jane M; Shean, Ryan C; Xie, Hu; Leisenring, Wendy M; Stevens-Ayers, Terry L; Jerome, Keith R; Englund, Janet A; Boeckh, Michael

2017-07-15

Recent data suggest that human coronavirus (HCoV) pneumonia is associated with significant mortality in hematopoietic cell transplant (HCT) recipients. Investigation of risk factors for prolonged shedding and intrahost genome evolution may provide critical information for development of novel therapeutics. We retrospectively reviewed HCT recipients with HCoV detected in nasal samples by polymerase chain reaction (PCR). HCoV strains were identified using strain-specific PCR. Shedding duration was defined as time between first positive and first negative sample. Logistic regression analyses were performed to evaluate factors for prolonged shedding (≥21 days). Metagenomic next-generation sequencing (mNGS) was conducted when ≥4 samples with cycle threshold values of Genome changes were consistent with the expected molecular clock of HCoV. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

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Wang, Shiu-Mei [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Huang, Kuo-Jung [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Wang, Chin-Tien, E-mail: chintien@ym.edu.tw [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China)

2014-01-20

Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments. - Highlights: • BST2 knockdown expression results in enhanced HCoV-229E egress. • HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. • HCoV-229E infection at high MOI can significantly downregulate HeLa BST2 and rescue HIV-1 egress.

An Opportunistic Pathogen Afforded Ample Opportunities: Middle East Respiratory Syndrome Coronavirus

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Ian M. Mackay

2017-12-01

Full Text Available The human coronaviruses (CoV include HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, some of which have been known for decades. The severe acute respiratory syndrome (SARS CoV briefly emerged into the human population but was controlled. In 2012, another novel severely human pathogenic CoV—the Middle East Respiratory Syndrome (MERS-CoV—was identified in the Kingdom of Saudi Arabia; 80% of over 2000 human cases have been recorded over five years. Targeted research remains key to developing control strategies for MERS-CoV, a cause of mild illness in its camel reservoir. A new therapeutic toolbox being developed in response to MERS is also teaching us more about how CoVs cause disease. Travel-related cases continue to challenge the world’s surveillance and response capabilities, and more data are needed to understand unexplained primary transmission. Signs of genetic change have been recorded, but it remains unclear whether there is any impact on clinical disease. How camels came to carry the virus remains academic to the control of MERS. To date, human-to-human transmission has been inefficient, but virus surveillance, characterisation, and reporting are key to responding to any future change. MERS-CoV is not currently a pandemic threat; it is spread mainly with the aid of human habit and error.

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

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

2014-01-01

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

Vacuolating encephalitis in mice infected by human coronavirus OC43

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Jacomy, Helene; Talbot, Pierre J.

2003-01-01

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

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

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

2013-08-01

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

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

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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.

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

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Jasper Fuk-Woo Chan

2013-07-01

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

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

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

2018-04-18

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

Plaque assay for human coronavirus NL63 using human colon carcinoma cells

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Drosten Christian

2008-11-01

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

Coronavirus Infection and Diversity in Bats in the Australasian Region.

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

2016-03-01

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

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

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Berkhout Ben

2005-02-01

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

Genetic Characteristics of Coronaviruses from Korean Bats in 2016.

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

2018-01-01

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

Coronavirus infections in horses in Saudi Arabia and Oman.

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

2017-12-01

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

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

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

2013-12-01

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

Etiology and clinical characterization of respiratory virus infections in adult patients attending an emergency department in Beijing.

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Xiaoyan Yu

Full Text Available BACKGROUND: Acute respiratory tract infections (ARTIs represent a serious global health burden. To date, few reports have addressed the prevalence of respiratory viruses (RVs in adults with ARTIs attending an emergency department (ED. Therefore, the potential impact of respiratory virus infections on such patients remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To determine the epidemiological and clinical profiles of common and recently discovered respiratory viruses in adults with ARTIs attending an ED in Beijing, a 1-year consecutive study was conducted from May, 2010, to April, 2011. Nose and throat swab samples from 416 ARTI patients were checked for 13 respiratory viruses using multiple reverse transcription polymerase chain reaction(RT-PCR assays for common respiratory viruses, including influenza viruses (Flu A, B, and adenoviruses (ADVs, picornaviruses (PICs, respiratory syncytial virus (RSV, parainfluenza viruses (PIVs 1-3, combined with real-time RT-PCR for human metapneumovirus (HMPV and human coronaviruses (HCoVs, -OC43, -229E, -NL63, and -HKU1. Viral pathogens were detected in 52.88% (220/416 of patient samples, and 7.21% (30/416 of patients tested positive for more than one virus. PICs (17.79% were the dominant agents detected, followed by FluA (16.11%, HCoVs (11.78%, and ADV (11.30%. HMPV, PIVs, and FluB were also detected (<3%, but not RSV. The total prevalence and the dominant virus infections detected differed significantly between ours and a previous report. Co-infection rates were high for HCoV-229E (12/39, 30.76%, PIC (22/74, 29.73%, ADV (12/47, 25.53% and FluA (15/67, 22.39%. Different patterns of clinical symptoms were associated with different respiratory viruses. CONCLUSIONS: The pattern of RV involvement in adults with ARTIs attending an ED in China differs from that previously reported. The high prevalence of viruses (PIC, FluA, HCoVs and ADV reported here strongly highlight the need for the development of safe and

Diversity and Evolutionary Histories of Human Coronaviruses NL63 and 229E Associated with Acute Upper Respiratory Tract Symptoms in Kuala Lumpur, Malaysia.

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Al-Khannaq, Maryam Nabiel; Ng, Kim Tien; Oong, Xiang Yong; Pang, Yong Kek; Takebe, Yutaka; Chook, Jack Bee; Hanafi, Nik Sherina; Kamarulzaman, Adeeba; Tee, Kok Keng

2016-05-04

The human alphacoronaviruses HCoV-NL63 and HCoV-229E are commonly associated with upper respiratory tract infections (URTI). Information on their molecular epidemiology and evolutionary dynamics in the tropical region of southeast Asia however is limited. Here, we analyzed the phylogenetic, temporal distribution, population history, and clinical manifestations among patients infected with HCoV-NL63 and HCoV-229E. Nasopharyngeal swabs were collected from 2,060 consenting adults presented with acute URTI symptoms in Kuala Lumpur, Malaysia, between 2012 and 2013. The presence of HCoV-NL63 and HCoV-229E was detected using multiplex polymerase chain reaction (PCR). The spike glycoprotein, nucleocapsid, and 1a genes were sequenced for phylogenetic reconstruction and Bayesian coalescent inference. A total of 68/2,060 (3.3%) subjects were positive for human alphacoronavirus; HCoV-NL63 and HCoV-229E were detected in 45 (2.2%) and 23 (1.1%) patients, respectively. A peak in the number of HCoV-NL63 infections was recorded between June and October 2012. Phylogenetic inference revealed that 62.8% of HCoV-NL63 infections belonged to genotype B, 37.2% was genotype C, while all HCoV-229E sequences were clustered within group 4. Molecular dating analysis indicated that the origin of HCoV-NL63 was dated to 1921, before it diverged into genotype A (1975), genotype B (1996), and genotype C (2003). The root of the HCoV-229E tree was dated to 1955, before it diverged into groups 1-4 between the 1970s and 1990s. The study described the seasonality, molecular diversity, and evolutionary dynamics of human alphacoronavirus infections in a tropical region. © The American Society of Tropical Medicine and Hygiene.

Evidence supporting a zoonotic origin of human coronavirus strain NL63.

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Huynh, Jeremy; Li, Shimena; Yount, Boyd; Smith, Alexander; Sturges, Leslie; Olsen, John C; Nagel, Juliet; Johnson, Joshua B; Agnihothram, Sudhakar; Gates, J Edward; Frieman, Matthew B; Baric, Ralph S; Donaldson, Eric F

2012-12-01

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.

Detection and full genome characterization of two beta CoV viruses related to Middle East respiratory syndrome from bats in Italy.

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Moreno, Ana; Lelli, Davide; de Sabato, Luca; Zaccaria, Guendalina; Boni, Arianna; Sozzi, Enrica; Prosperi, Alice; Lavazza, Antonio; Cella, Eleonora; Castrucci, Maria Rita; Ciccozzi, Massimo; Vaccari, Gabriele

2017-12-19

Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii. Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB). Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs. This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better

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

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

2016-02-01

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

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

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

2008-01-01

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

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

NARCIS (Netherlands)

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

2004-01-01

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

About Coronavirus

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

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

Science.gov (United States)

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

2013-01-01

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

Human coronavirus NL63, France

NARCIS (Netherlands)

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

2005-01-01

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

[Nosocomial infections due to human coronaviruses in the newborn].

Science.gov (United States)

Gagneur, A; Legrand, M C; Picard, B; Baron, R; Talbot, P J; de Parscau, L; Sizun, J

2002-01-01

Human coronaviruses, with two known serogroups named 229-E and OC-43, are enveloped positive-stranded RNA viruses. The large RNA is surrounded by a nucleoprotein (protein N). The envelop contains 2 or 3 glycoproteins: spike protein (or protein S), matrix protein (or protein M) and a hemagglutinin (or protein HE). Their pathogen role remains unclear because their isolation is difficult. Reliable and rapid methods as immunofluorescence with monoclonal antibodies and reverse transcription-polymerase chain reaction allow new researches on epidemiology. Human coronaviruses can survive for as long as 6 days in suspension and 3 hours after drying on surfaces, suggesting that they could be a source of hospital-acquired infections. Two prospective studies conducted in a neonatal and paediatric intensive care unit demonstrated a significant association of coronavirus-positive nasopharyngal samples with respiratory illness in hospitalised preterm neonates. Positive samples from staff suggested either a patient-to-staff or a staff-to-patient transmission. No cross-infection were observed from community-acquired respiratory-syncitial virus or influenza-infected children to neonates. Universal precautions with hand washing and surface desinfection could be proposed to prevent coronavirus transmission.

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

Science.gov (United States)

1992-05-12

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

On the biased nucleotide composition of the human coronavirus RNA genome

NARCIS (Netherlands)

Berkhout, Ben; van Hemert, Formijn

2015-01-01

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

Epidemiology of human coronavirus NL63 infection among hospitalized patients with pneumonia in Taiwan.

Science.gov (United States)

Huang, Su-Hua; Su, Mei-Chi; Tien, Ni; Huang, Chien-Jhen; Lan, Yu-Ching; Lin, Chen-Sheng; Chen, Chao-Hsien; Lin, Cheng-Wen

2017-12-01

Human coronavirus (HCoV) NL63 is recognized in association with upper or lower respiratory tract illnesses in children. This study surveyed the prevalence of HCoV-NL63 and influenza viruses in patients with influenza-like illness in Taiwan during 2010-2011. Throat samples from 107 hospitalized patients with pneumonia and 175 outpatients with influenza-like illness were examined using real-time polymerase chain reaction assays with virus-specific primers, and then virus-positive specimens were confirmed by sequencing the polymerase chain reaction products. HCoV-NL63 infection was identified in 8.4% (9/107) of hospitalized patients with pneumonia, but not found in outpatients with influenza-like illness. Age distribution of HCoV-NL63 infection in hospitalized patients with pneumonia indicated that the group aged 16-25 years (20%) had the highest positive rate compared with the other groups, and exhibited a similar age-specific pattern to influenza A/H1N1 infection, but not influenza A/H3N2 and B infections in hospitalized patients. Seasonal prevalence of HCoV-NL63 infection was late winter, overlapping the highest peak of the influenza A/H1N1 epidemic during December 2010 to March 2011 in Taiwan. Co-infection of HCoV-NL63 and influenza A/H1N1 was detected in three hospitalized patients. Clinical manifestation analysis indicated that the main symptoms for HCoV-NL63 infection included fever (88.9%), cough (77.8%), and pneumonia (100%). Co-infection caused significantly higher rates of breathing difficulties, cough, and sore throat than those of single infection with HCoV-NL63 and influenza A/H1N1. Phylogenetic analysis indicated a low level of heterogeneity between Taiwan and global HCoV-NL63 strains. Understanding epidemiology of HCoV-NL63 in Taiwan provides an insight for worldwide surveillance of HCoV-NL63 infection. Copyright © 2015. Published by Elsevier B.V.

Patterns of Human Respiratory Viruses and Lack of MERS-Coronavirus in Patients with Acute Upper Respiratory Tract Infections in Southwestern Province of Saudi Arabia

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Ahmed A. Abdulhaq

2017-01-01

Full Text Available We undertook enhanced surveillance of those presenting with respiratory symptoms at five healthcare centers by testing all symptomatic outpatients between November 2013 and January 2014 (winter time. Nasal swabs were collected from 182 patients and screened for MERS-CoV as well as other respiratory viruses using RT-PCR and multiplex microarray. A total of 75 (41.2% of these patients had positive viral infection. MERS-CoV was not detected in any of the samples. Human rhinovirus (hRV was the most detected pathogen (40.9% followed by non-MERS-CoV human coronaviruses (19.3%, influenza (Flu viruses (15.9%, and human respiratory syncytial virus (hRSV (13.6%. Viruses differed markedly depending on age in which hRV, Flu A, and hCoV-OC43 were more prevalent in adults and RSV, hCoV-HKU1, and hCoV-NL63 were mostly restricted to children under the age of 15. Moreover, coinfection was not uncommon in this study, in which 17.3% of the infected patients had dual infections due to several combinations of viruses. Dual infections decreased with age and completely disappeared in people older than 45 years. Our study confirms that MERS-CoV is not common in the southwestern region of Saudi Arabia and shows high diversity and prevalence of other common respiratory viruses. This study also highlights the importance and contribution of enhanced surveillance systems for better infection control.

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

OpenAIRE

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

2004-01-01

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

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

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

2010-03-04

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

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

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Alain Le Coupanec

Full Text Available Human coronaviruses (HCoV are respiratory pathogens that may be associated with the development of neurological diseases, in view of their neuroinvasive and neurotropic properties. The viral spike (S glycoprotein is a major virulence factor for several coronavirus species, including the OC43 strain of HCoV (HCoV-OC43. In an attempt to study the role of this protein in virus spread within the central nervous system (CNS and neurovirulence, as well as to identify amino acid residues important for such functions, we compared the sequence of the S gene found in the laboratory reference strain HCoV-OC43 ATCC VR-759 to S sequences of viruses detected in clinical isolates from the human respiratory tract. We identified one predominant mutation at amino acid 758 (from RRSR↓ G758 to RRSR↓R758, which introduces a putative furin-like cleavage (↓ site. Using a molecular cDNA infectious clone to generate a corresponding recombinant virus, we show for the first time that such point mutation in the HCoV-OC43 S glycoprotein creates a functional cleavage site between the S1 and S2 portions of the S protein. While the corresponding recombinant virus retained its neuroinvasive properties, this mutation led to decreased neurovirulence while potentially modifying the mode of virus spread, likely leading to a limited dissemination within the CNS. Taken together, these results are consistent with the adaptation of HCoV-OC43 to the CNS environment, resulting from the selection of quasi-species harboring mutations that lead to amino acid changes in viral genes, like the S gene in HCoV-OC43, which may contribute to a more efficient establishment of a less pathogenic but persistent CNS infection. This adaptative mechanism could potentially be associated with human encephalitis or other neurological degenerative pathologies.

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

Directory of Open Access Journals (Sweden)

Yunjeong Kim

2016-03-01

Full Text Available Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP, can arise through mutation of FECV to FIP virus (FIPV. The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for

Coronavirus spike-receptor interactions

NARCIS (Netherlands)

Mou, H.

2015-01-01

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

Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

Energy Technology Data Exchange (ETDEWEB)

Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

2011-09-28

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

Nation-wide surveillance of human acute respiratory virus infections between 2013 and 2015 in Korea.

Science.gov (United States)

Kim, Jeong-Min; Jung, Hee-Dong; Cheong, Hyang-Min; Lee, Anna; Lee, Nam-Joo; Chu, Hyuk; Lee, Joo-Yeon; Kim, Sung Soon; Choi, Jang-Hoon

2018-07-01

The prevalence of eight respiratory viruses detected in patients with acute respiratory infections (ARIs) in Korea was investigated through analysis of data recorded by the Korea Influenza and Respiratory Viruses Surveillance System (KINRESS) from 2013 to 2015. Nasal aspirate and throat swabs specimens were collected from 36 915 patients with ARIs, and viral nucleic acids were detected by real-time (reverse-transcription) polymerase chain reaction for eight respiratory viruses, including human respiratory syncytial viruses (HRSVs), influenza viruses (IFVs), human parainfluenza viruses (HPIVs), human coronaviruses (HCoVs), human rhinovirus (HRV), human adenovirus (HAdV), human bocavirus (HBoV), and human metapneumovirus (HMPV). The overall positive rate of patient specimens was 49.4% (18 236/36 915), 5% of which carried two or more viruses simultaneously. HRV (15.6%) was the most predominantly detected virus, followed by IFVs (14.6%), HAdV (7.5%), HPIVs (5.8%), HCoVs (4.2%), HRSVs (3.6%), HBoV (1.9%), and HMPV (1.6%). Most of the ARIs were significantly correlated with clinical symptoms of fever, cough, and runny nose. Although HRV and HAdV were frequently detected throughout the year in patients, other respiratory viruses showed apparent seasonality. HRSVs and IFVs were the major causative agents of acute respiratory diseases in infants and young children. Overall, this study demonstrates a meaningful relationship between viral infection and typical manifestations of known clinical features as well as seasonality, age distribution, and co-infection among respiratory viruses. Therefore, these data could provide useful information for public health management and to enhance patient care for primary clinicians. © 2018 Wiley Periodicals, Inc.

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

International Nuclear Information System (INIS)

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

2003-01-01

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

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

NARCIS (Netherlands)

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

2012-01-01

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

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

Science.gov (United States)

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

2017-06-02

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

Longitudinal Surveillance of Betacoronaviruses in Fruit Bats in Yunnan Province, China During 2009-2016.

Science.gov (United States)

Luo, Yun; Li, Bei; Jiang, Ren-Di; Hu, Bing-Jie; Luo, Dong-Sheng; Zhu, Guang-Jian; Hu, Ben; Liu, Hai-Zhou; Zhang, Yun-Zhi; Yang, Xing-Lou; Shi, Zheng-Li

2018-02-01

Previous studies indicated that fruit bats carry two betacoronaviruses, BatCoV HKU9 and BatCoV GCCDC1. To investigate the epidemiology and genetic diversity of these coronaviruses, we conducted a longitudinal surveillance in fruit bats in Yunnan province, China during 2009-2016. A total of 59 (10.63%) bat samples were positive for the two betacorona-viruses, 46 (8.29%) for HKU9 and 13 (2.34%) for GCCDC1, or closely related viruses. We identified a novel HKU9 strain, tentatively designated as BatCoV HKU9-2202, by sequencing the full-length genome. The BatCoV HKU9-2202 shared 83% nucleotide identity with other BatCoV HKU9 stains based on whole genome sequences. The most divergent region is in the spike protein, which only shares 68% amino acid identity with BatCoV HKU9. Quantitative PCR revealed that the intestine was the primary infection organ of BatCoV HKU9 and GCCDC1, but some HKU9 was also detected in the heart, kidney, and lung tissues of bats. This study highlights the importance of virus surveillance in natural reservoirs and emphasizes the need for preparedness against the potential spill-over of these viruses to local residents living near bat caves.

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

NARCIS (Netherlands)

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

2014-01-01

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

Culturing the Unculturable: Human Coronavirus HKU1 Infects, Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

NARCIS (Netherlands)

Pyrc, Krzysztof; Sims, Amy C.; Dijkman, Ronald; Jebbink, Maarten; Long, Casey; Deming, Damon; Donaldson, Eric; Vabret, Astrid; Baric, Ralph; van der Hoek, Lia; Pickles, Raymond

2010-01-01

Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is

Feline and canine coronaviruses: common genetic and pathobiological features.

Science.gov (United States)

Le Poder, Sophie

2011-01-01

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

Feline and Canine Coronaviruses: Common Genetic and Pathobiological Features

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Sophie Le Poder

2011-01-01

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

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

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Susanne Pfefferle

2011-10-01

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

Coronavirus infection, ER stress and Apoptosis

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TO SING eFUNG

2014-06-01

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

A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

Science.gov (United States)

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

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

Science.gov (United States)

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

2017-12-01

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

Coronaviruses in brain tissue from patients with multiple sclerosis

DEFF Research Database (Denmark)

Dessau, R B; Lisby, G; Frederiksen, J L

2001-01-01

Brain tissue from 25 patients with clinically definite multiple sclerosis (MS) and as controls brain tissue from 36 patients without neurological disease was tested for the presence of human coronaviral RNA. Four PCR assays with primers specific for N-protein of human coronavirus strain 229E...... and three PCR assays with primers specific for the nucleocapsid protein of human coronavirus strain OC43 were performed. Sporadic positive PCR assays were observed in both patients and controls in some of the PCR assays. However, these results were not reproducible and there was no difference...... in the proportion of positive signals from the MS patients compared to controls. Evidence for a chronic infection with the human coronaviruses strain 229E or OC43 in brain tissue from patients with MS or controls has not been found in this study....

Involvement of Autophagy in Coronavirus Replication

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Paul Britton

2012-11-01

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

Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

Science.gov (United States)

Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

2017-06-15

Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

Suppression of Coronavirus Replication by Cyclophilin Inhibitors

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Takashi Sasaki

2013-05-01

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

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

Science.gov (United States)

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

2017-03-01

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

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

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David M Patrick

2006-01-01

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

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

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I. N. Zhilinskaya

2017-01-01

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

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

Czech Academy of Sciences Publication Activity Database

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

2015-01-01

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

Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus

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Harbison Carole E

2007-02-01

Full Text Available Abstract Background Coronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS, and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV. Feline aminopeptidase N (fAPN serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV, canine coronavirus, transmissible gastroenteritis virus (TGEV, and human coronavirus 229E (HCoV-229E. A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047–2056. Results Here we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41, but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 ( Conclusion We conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.

Transmission of MERS-coronavirus in household contacts

NARCIS (Netherlands)

Drosten, Christian; Meyer, Benjamin; Müller, Marcel A; Corman, Victor M; Al-Masri, Malak; Hossain, Raheela; Madani, Hosam; Sieberg, Andrea; Bosch, Berend Jan|info:eu-repo/dai/nl/273306049; Lattwein, Erik; Alhakeem, Raafat F; Assiri, Abdullah M; Hajomar, Waleed; Albarrak, Ali M; Al-Tawfiq, Jaffar A; Zumla, Alimuddin I; Memish, Ziad A

2014-01-01

BACKGROUND: Strategies to contain the Middle East respiratory syndrome coronavirus (MERS-CoV) depend on knowledge of the rate of human-to-human transmission, including subclinical infections. A lack of serologic tools has hindered targeted studies of transmission. METHODS: We studied 26 index

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

Science.gov (United States)

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

2013-11-28

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

MERS-coronavirus: From discovery to intervention

NARCIS (Netherlands)

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

2017-01-01

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

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

DEFF Research Database (Denmark)

Chagnon, F; Lamarre, A; Lachance, C

1998-01-01

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

Viral etiologies of hospitalized acute lower respiratory infection patients in China, 2009-2013.

Directory of Open Access Journals (Sweden)

Luzhao Feng

Full Text Available BACKGROUND: Acute lower respiratory infections (ALRIs are an important cause of acute illnesses and mortality worldwide and in China. However, a large-scale study on the prevalence of viral infections across multiple provinces and seasons has not been previously reported from China. Here, we aimed to identify the viral etiologies associated with ALRIs from 22 Chinese provinces. METHODS AND FINDINGS: Active surveillance for hospitalized ALRI patients in 108 sentinel hospitals in 24 provinces of China was conducted from January 2009-September 2013. We enrolled hospitalized all-age patients with ALRI, and collected respiratory specimens, blood or serum collected for diagnostic testing for respiratory syncytial virus (RSV, human influenza virus, adenoviruses (ADV, human parainfluenza virus (PIV, human metapneumovirus (hMPV, human coronavirus (hCoV and human bocavirus (hBoV. We included 28,369 ALRI patients from 81 (of the 108 sentinel hospitals in 22 (of the 24 provinces, and 10,387 (36.6% were positive for at least one etiology. The most frequently detected virus was RSV (9.9%, followed by influenza (6.6%, PIV (4.8%, ADV (3.4%, hBoV (1.9, hMPV (1.5% and hCoV (1.4%. Co-detections were found in 7.2% of patients. RSV was the most common etiology (17.0% in young children aged <2 years. Influenza viruses were the main cause of the ALRIs in adults and elderly. PIV, hBoV, hMPV and ADV infections were more frequent in children, while hCoV infection was distributed evenly in all-age. There were clear seasonal peaks for RSV, influenza, PIV, hBoV and hMPV infections. CONCLUSIONS: Our findings could serve as robust evidence for public health authorities in drawing up further plans to prevent and control ALRIs associated with viral pathogens. RSV is common in young children and prevention measures could have large public health impact. Influenza was most common in adults and influenza vaccination should be implemented on a wider scale in China.

Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease

Science.gov (United States)

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

2012-01-01

Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

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

NARCIS (Netherlands)

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

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

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

Science.gov (United States)

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

2018-03-02

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

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

Science.gov (United States)

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

2012-04-01

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

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

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Elodie Monchatre-Leroy

2017-11-01

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

Clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in HIV-infected and HIV-uninfected South African children.

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Marta C Nunes

Full Text Available Advances in molecular diagnostics have implicated newly-discovered respiratory viruses in the pathogenesis of pneumonia. We aimed to determine the prevalence and clinical characteristics of human bocavirus (hBoV, human rhinovirus (hRV, polyomavirus-WU (WUPyV and -KI (KIPyV and human coronaviruses (CoV-OC43, -NL63, -HKU1 and -229E among children hospitalized with lower respiratory tract infections (LRTI.Multiplex real-time reverse-transcriptase polymerase chain reaction was undertaken on archived nasopharyngeal aspirates from HIV-infected and -uninfected children (<2 years age hospitalized for LRTI, who had been previously investigated for respiratory syncytial virus, human metapneumovirus, parainfluenza I-III, adenovirus and influenza A/B.At least one of these viruses were identified in 274 (53.0% of 517 and in 509 (54.0% of 943 LRTI-episodes in HIV-infected and -uninfected children, respectively. Human rhinovirus was the most prevalent in HIV-infected (31.7% and -uninfected children (32.0%, followed by CoV-OC43 (12.2% and hBoV (9.5% in HIV-infected; and by hBoV (13.3% and WUPyV (11.9% in HIV-uninfected children. Polyomavirus-KI (8.9% vs. 4.8%; p = 0.002 and CoV-OC43 (12.2% vs. 3.6%; p<0.001 were more prevalent in HIV-infected than -uninfected children. Combined with previously-tested viruses, respiratory viruses were identified in 60.9% of HIV-infected and 78.3% of HIV-uninfected children. The newly tested viruses were detected at high frequency in association with other respiratory viruses, including previously-investigated viruses (22.8% in HIV-infected and 28.5% in HIV-uninfected children.We established that combined with previously-investigated viruses, at least one respiratory virus was identified in the majority of HIV-infected and HIV-uninfected children hospitalized for LRTI. The high frequency of viral co-infections illustrates the complexities in attributing causality to specific viruses in the aetiology of LRTI and may indicate a

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

International Nuclear Information System (INIS)

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

2003-01-01

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

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Coronavirus Attachment and Replication

Science.gov (United States)

1988-03-28

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

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

NARCIS (Netherlands)

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

2008-01-01

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

Mosaic Evolution of the Severe Acute Respiratory Syndrome Coronavirus

Science.gov (United States)

Stavrinides, John; Guttman, David S.

2004-01-01

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

Tissue Distribution of the MERS-Coronavirus Receptor in Bats

NARCIS (Netherlands)

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

2017-01-01

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

Tissue Distribution of the MERS-Coronavirus Receptor in Bats

NARCIS (Netherlands)

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

2017-01-01

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

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

NARCIS (Netherlands)

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

1998-01-01

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

New Epidemiological and Clinical Signatures of 18 Pathogens from Respiratory Tract Infections Based on a 5-Year Study.

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Xiaohong Liao

Full Text Available Respiratory tract infections (RTIs are a heavy burden on society. However, due to the complex etiology of RTIs, the clinical diagnosis, treatment, and prevention of these infections remain challenging, especially in developing countries.To determine the epidemiological and clinical characteristics of 18 respiratory pathogens, we analyzed 12,502 patients with acute respiratory infections (ARIs by performing polymerase chain reaction (PCR on patient pharyngeal swabs.Samples positive for at least 1 pathogen were obtained from 48.42% of the total patients. Of these pathogen-positive patients, 17.99% were infected with more than 1 pathogen. Of the 18 pathogens analyzed, four were detected with a positive detection rate (PDR > 5%: influenza A virus (IAV > respiratory syncytial virus (RSV >Mycoplasma pneumoniae (MP > human coronavirus (HCoV. The pathogens with the 4 highest co-infection rates (CIRs were as follows: HCoV > human bocavirus (HBoV > enterovirus (EV > parainfluenza virus (PIV. The overall positive detection rate (PDR varied significantly according to patient age, the season and year of detection, and the disease subgroup, but not according to patient sex. The individual PDRs of the pathogens followed 3 types of distributions for patient sex, 4 types of distributions for patient age, 4 types of seasonal distributions, 2 types of seasonal epidemic trends, 4 types of yearly epidemic trends, and different susceptibility distributions in the disease subgroups. Additionally, the overall CIR showed significantly different distributions according to patient sex, patient age, and the disease subgroup, whereas the CIRs of individual pathogens suggested significant preference characteristics.IAV remains the most common pathogen among the pathogens analyzed. More effort should be directed toward the prevention and control of pathogens that show a trend of increasing incidence such as HCoV, human adenovirus (ADV, and RSV. Although clinically

The role of infections and coinfections with newly identified and emerging respiratory viruses in children

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Debiaggi Maurizia

2012-10-01

Full Text Available Abstract Acute respiratory infections are a major cause of morbidity in children both in developed and developing countries. A wide range of respiratory viruses, including respiratory syncytial virus (RSV, influenza A and B viruses, parainfluenza viruses (PIVs, adenovirus, rhinovirus (HRV, have repeatedly been detected in acute lower respiratory tract infections (LRTI in children in the past decades. However, in the last ten years thanks to progress in molecular technologies, newly discovered viruses have been identified including human Metapneumovirus (hMPV, coronaviruses NL63 (HcoV-NL63 and HKU1 (HcoV-HKU1, human Bocavirus (HBoV, new enterovirus (HEV, parechovirus (HpeV and rhinovirus (HRV strains, polyomaviruses WU (WUPyV and KI (KIPyV and the pandemic H1N1v influenza A virus. These discoveries have heavily modified previous knowledge on respiratory infections mainly highlighting that pediatric population is exposed to a variety of viruses with similar seasonal patterns. In this context establishing a causal link between a newly identified virus and the disease as well as an association between mixed infections and an increase in disease severity can be challenging. This review will present an overview of newly recognized as well as the main emerging respiratory viruses and seek to focus on the their contribution to infection and co-infection in LRTIs in childhood.

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

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Anna Lundin

2014-05-01

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

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

Science.gov (United States)

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

2005-01-01

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

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

NARCIS (Netherlands)

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

2014-01-01

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

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

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Nathalie Kin

2015-05-01

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

Cross host transmission in the emergence of MERS coronavirus

NARCIS (Netherlands)

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

2016-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Li, Fang (UMM)

2008-09-23

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

A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

Science.gov (United States)

To, Kelvin K W; Tse, Herman; Chan, Wan-Mui; Choi, Garnet K Y; Zhang, Anna J X; Sridhar, Siddharth; Wong, Sally C Y; Chan, Jasper F W; Chan, Andy S F; Woo, Patrick C Y; Lau, Susanna K P; Lo, Janice Y C; Chan, Kwok-Hung; Cheng, Vincent C C; Yuen, Kwok-Yung

2014-12-01

Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be

A novel psittacine adenovirus identified during an outbreak of avian chlamydiosis and human psittacosis: zoonosis associated with virus-bacterium coinfection in birds.

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Kelvin K W To

2014-12-01

Full Text Available Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1 was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs with sequence similarity to known adenoviral genes, and six additional ORFs at the 3' end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3-54.0% for the DNA polymerase, 64.6-70.7% for the penton protein, and 66.1-74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention

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

Science.gov (United States)

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

2016-01-01

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes. PMID:27519799

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

Science.gov (United States)

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

2017-01-01

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

Detection of Coronaviruses in Bats of Various Species in Italy

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Maria B. Boniotti

2013-10-01

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

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

International Nuclear Information System (INIS)

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

2009-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2009-08-01

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

Coronavirus 229E-related pneumonia in immunocompromised patients.

Science.gov (United States)

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

2003-10-01

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

Transmission of Middle East respiratory syndrome coronavirus ...

African Journals Online (AJOL)

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

BIOLOGICAL CLONING OF A BOVINE CORONAVIRUS ISOLATE

OpenAIRE

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

2008-01-01

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

Amylmetacresol/2,4-dichlorobenzyl alcohol, hexylresorcinol, or carrageenan lozenges as active treatments for sore throat

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Morokutti-Kurz M

2017-02-01

Full Text Available Martina Morokutti-Kurz, Christine Graf, Eva Prieschl-Grassauer Marinomed Biotechnologie GmbH, Vienna, Austria Abstract: Up to 80% of sore throats are caused by viruses. Several over the counter products are available which provide symptomatic, not causal relief. For such lozenges, containing the antiseptics and local anesthetics amylmetacresol (AMC and 2,4-dichlorobenzyl alcohol (DCBA or hexylresorcinol (HR, recently an additional virucidal effect was published. Therefore, we tested a set of Strepsils® lozenges, containing either HR (Max [#2] or AMC/DCBA (Original [#3], Extra Strong [#4], Warm [#5], Orange and Vitamin C [#6], Sugar free Lemon [#7], Children/Strawberry [#8] and Soothing Honey and Lemon [#9] for their antiviral efficiency against representatives of respiratory viruses known to cause sore throat: human rhinovirus (HRV 1a, HRV8, influenza virus A H1N1n, Coxsackievirus A10, and human coronavirus (hCoV OC43. The lozenges were tested head to head with Coldamaris® lozenges (#1, which contain the patented antiviral iota-carrageenan. None of the tested AMC/DCBA or HR containing lozenges shows any antiviral effectiveness against HRV8 at the tested concentrations, whereas all are moderately active against HRV1a. Only lozenge #5 shows any activity against hCoV OC43 and Coxsackievirus A10 at the tested concentrations. Similarly, only lozenge #3 is moderately active against influenza A H1N1n virus. The data indicates that neither the isolated effect of the active ingredients nor the pH but rather one or more of the excipients of the specific formulations are responsible for the antiviral effect of some of the AMC/DCBA or HR containing lozenges. In contrast, carrageenan-containing lozenges are highly active against all viruses tested. In another experiment, we showed that binding and inactivation of virus particles by iota-carrageenan are fast and highly effective. During the residence time of the lozenge in the mouth, the viral titer is

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

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Yutaka Terada

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

Canine coronaviruses: Epidemiology, evolution and pathobiology

NARCIS (Netherlands)

Decaro, N.

2009-01-01

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

Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

Science.gov (United States)

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

2013-01-01

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

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

Science.gov (United States)

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

2018-02-01

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

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

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K.-Y. Hwa

2008-01-01

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

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

Science.gov (United States)

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

2011-06-01

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

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

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Emmie de Wit

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

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

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Blom Nikolaj

2004-06-01

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

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

Science.gov (United States)

Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

2017-02-01

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

Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

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Mark W. Jackwood

2011-09-01

Full Text Available Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

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

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Júlia Vergara-Alert

2017-06-01

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

Spike Protein Fusion Peptide and Feline Coronavirus Virulence

Science.gov (United States)

Chang, Hui-Wen; Egberink, Herman F.; Halpin, Rebecca; Spiro, David J.

2012-01-01

Coronaviruses are well known for their potential to change their host or tissue tropism, resulting in unpredictable new diseases and changes in pathogenicity; severe acute respiratory syndrome and feline coronaviruses, respectively, are the most recognized examples. Feline coronaviruses occur as 2 pathotypes: nonvirulent feline enteric coronaviruses (FECVs), which replicate in intestinal epithelium cells, and lethal feline infectious peritonitis viruses (FIPVs), which replicate in macrophages. Evidence indicates that FIPV originates from FECV by mutation, but consistent distinguishing differences have not been established. We sequenced the full genome of 11 viruses of each pathotype and then focused on the single most distinctive site by additionally sequencing hundreds of viruses in that region. As a result, we identified 2 alternative amino acid differences in the putative fusion peptide of the spike protein that together distinguish FIPV from FECV in >95% of cases. By these and perhaps other mutations, the virus apparently acquires its macrophage tropism and spreads systemically. PMID:22709821

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

NARCIS (Netherlands)

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

2014-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary

Unraveling the Mysteries of Middle East Respiratory Syndrome Coronavirus

Centers for Disease Control (CDC) Podcasts

2014-03-11

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

Characterization of novel human respiratory viruses

NARCIS (Netherlands)

Dijkman, R.

2011-01-01

Wereldwijd komen vier humane coronavirussen (HCoVs) voor, waaronder NL63 en 229E. NL63 werd in 2004 ontdekt in het AMC en veroorzaakt de kinderziekte pseudokroep; 229E is een verkoudheidsvirus. Waarschijnlijk veroorzaken beide virussen vergelijkbare symptomen bij volwassenen. Er is weinig bekend

Evaluation of a multiplex real-time PCR assay for the detection of respiratory viruses in clinical specimens.

Science.gov (United States)

Rheem, Insoo; Park, Joowon; Kim, Tae-Hyun; Kim, Jong Wan

2012-11-01

In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea). Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses. The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%. The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR.

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

Directory of Open Access Journals (Sweden)

Monica K Borucki

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

Coronavirus infection in mink (Mustela vison). Serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus

DEFF Research Database (Denmark)

Have, P; Moving, V; Svansson, V

1992-01-01

Antibodies to a transmissible gastroenteritis virus (TGEV)-related coronavirus have been demonstrated in mink sera by indirect immunofluorescence, peroxidase-linked antibody assays and immunoblotting. This is the first serological evidence of a specific coronavirus infection in mink. The putative...

Comparative properties of feline coronaviruses in vitro.

OpenAIRE

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

1987-01-01

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

Prevalence of Korean cats with natural feline coronavirus infections

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Lee Myoung-Heon

2011-09-01

Full Text Available Abstract Background Feline coronavirus is comprised of two pathogenic biotypes consisting of feline infectious peritonitis virus (FIPV and feline enteric coronavirus (FECV, which are both divided into two serotypes. To examine the prevalence of Korean cats infected with feline coronavirus (FCoV type I and II, fecal samples were obtained from 212 cats (107 pet and 105 feral in 2009. Results Fourteen cats were FCoV-positive, including infections with type I FCoV (n = 8, type II FCoV (n = 4, and types I and II co-infection (n = 2. Low seroprevalences (13.7%, 29/212 of FCoV were identified in chronically ill cats (19.3%, 16/83 and healthy cats (10.1%, 13/129. Conclusions Although the prevalence of FCoV infection was not high in comparison to other countries, there was a higher prevalence of type I FCoV in Korean felines. The prevalence of FCoV antigen and antibody in Korean cats are expected to gradually increase due to the rising numbers of stray and companion cats.

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

Science.gov (United States)

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

2017-11-17

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

Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein

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Gary R. Whittaker

2012-06-01

Full Text Available Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes—A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

Feline coronavirus: Insights into viral pathogenesis based on the spike protein structure and function.

Science.gov (United States)

Jaimes, Javier A; Whittaker, Gary R

2018-04-01

Feline coronavirus (FCoV) is an etiological agent that causes a benign enteric illness and the fatal systemic disease feline infectious peritonitis (FIP). The FCoV spike (S) protein is considered the viral regulator for binding and entry to the cell. This protein is also involved in FCoV tropism and virulence, as well as in the switch from enteric disease to FIP. This regulation is carried out by spike's major functions: receptor binding and virus-cell membrane fusion. In this review, we address important aspects in FCoV genetics, replication and pathogenesis, focusing on the role of S. To better understand this, FCoV S protein models were constructed, based on the human coronavirus NL63 (HCoV-NL63) S structure. We describe the specific structural characteristics of the FCoV S, in comparison with other coronavirus spikes. We also revise the biochemical events needed for FCoV S activation and its relation to the structural features of the protein. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2016-02-01

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

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

Directory of Open Access Journals (Sweden)

Nerea Irigoyen

2016-02-01

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

Clinical and virological factors associated with gastrointestinal symptoms in patients with acute respiratory infection: a two-year prospective study in general practice medicine.

Science.gov (United States)

Minodier, Laetitia; Masse, Shirley; Capai, Lisandru; Blanchon, Thierry; Ceccaldi, Pierre-Emmanuel; van der Werf, Sylvie; Hanslik, Thomas; Charrel, Remi; Falchi, Alessandra

2017-11-22

Gastrointestinal (GI) symptoms, such as diarrhea, vomiting, abdominal pain and nausea are not an uncommon manifestation of an acute respiratory infection (ARI). We therefore evaluated clinical and microbiological factors associated with the presence of GI symptoms in patients consulting a general practitioner (GP) for ARI. Nasopharyngeal swabs, stool specimens and clinical data from patients presenting to GPs with an ARI were prospectively collected during two winter seasons (2014-2016). Samples were tested by quantitative real-time PCR for 12 respiratory pathogen groups and for 12 enteric pathogens. Two hundred and four of 331 included patients (61.6%) were positive for at least one respiratory pathogen. Sixty-nine stools (20.8%) were positive for at least one pathogen (respiratory and/or enteric). GI symptoms were more likely declared in case of laboratory confirmed-enteric infection (adjusted odds ratio (aOR) = 3.2; 95% confidence interval [CI] [1.2-9.9]; p = 0.02) or human coronavirus (HCoV) infection (aOR = 2.7; [1.2-6.8]; p = 0.02). Consumption of antipyretic medication before the consultation seemed to reduce the risk of developing GI symptoms for patients with laboratory-confirmed influenza (aOR = 0.3; [0.1-0.6]; p = 0.002). The presence of GI symptoms in ARI patients could not be explained by the detection of respiratory pathogens in stools. However, the detection of enteric pathogens in stool samples could explained by the presence of GI symptoms in some of ARI cases. The biological mechanisms explaining the association between the presence of HCoVs in nasopharynx and GI symptoms need to be explored.

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

DEFF Research Database (Denmark)

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

2008-01-01

, the crystal structure of HLA-B*1501 in complex with a SARS coronavirus-derived nonapeptide (VQQESSFVM) has been determined at high resolution (1.87 A). The peptide is deeply anchored in the B and F pockets, but with the Glu4 residue pointing away from the floor in the peptide-binding groove, making...

Crystal structure of Middle East respiratory syndrome coronavirus helicase.

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Wei Hao

2017-06-01

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

The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

NARCIS (Netherlands)

Bosch, B.J.

2004-01-01

The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion

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

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Sanchita Bhadra

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-09-23

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

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

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Eveline Kindler

2017-02-01

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

Comparison of automated microarray detection with real-time PCR assays for detection of respiratory viruses in specimens obtained from children.

Science.gov (United States)

Raymond, Frédéric; Carbonneau, Julie; Boucher, Nancy; Robitaille, Lynda; Boisvert, Sébastien; Wu, Whei-Kuo; De Serres, Gaston; Boivin, Guy; Corbeil, Jacques

2009-03-01

Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions.

Comparison of Automated Microarray Detection with Real-Time PCR Assays for Detection of Respiratory Viruses in Specimens Obtained from Children▿

Science.gov (United States)

Raymond, Frédéric; Carbonneau, Julie; Boucher, Nancy; Robitaille, Lynda; Boisvert, Sébastien; Wu, Whei-Kuo; De Serres, Gaston; Boivin, Guy; Corbeil, Jacques

2009-01-01

Respiratory virus infections are a major health concern and represent the primary cause of testing consultation and hospitalization for young children. We developed and compared two assays that allow the detection of up to 23 different respiratory viruses that frequently infect children. The first method consisted of single TaqMan quantitative real-time PCR assays in a 96-well-plate format. The second consisted of a multiplex PCR followed by primer extension and microarray hybridization in an integrated molecular diagnostic device, the Infiniti analyzer. Both of our assays can detect adenoviruses of groups A, B, C, and E; coronaviruses HKU1, 229E, NL63, and OC43; enteroviruses A, B, C, and D; rhinoviruses of genotypes A and B; influenza viruses A and B; human metapneumoviruses (HMPV) A and B, human respiratory syncytial viruses (HRSV) A and B; and parainfluenza viruses of types 1, 2, and 3. These tests were used to identify viruses in 221 nasopharyngeal aspirates obtained from children hospitalized for respiratory tract infections. Respiratory viruses were detected with at least one of the two methods in 81.4% of the 221 specimens: 10.0% were positive for HRSV A, 38.0% for HRSV B, 13.1% for influenzavirus A, 8.6% for any coronaviruses, 13.1% for rhinoviruses or enteroviruses, 7.2% for adenoviruses, 4.1% for HMPV, and 1.5% for parainfluenzaviruses. Multiple viral infections were found in 13.1% of the specimens. The two methods yielded concordant results for 94.1% of specimens. These tests allowed a thorough etiological assessment of respiratory viruses infecting children in hospital settings and would assist public health interventions. PMID:19158263

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

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Konstantin Pervushin

2009-07-01

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

Pitfalls in interpretation of CT-values of RT-PCR in children with acute respiratory tract infections.

Science.gov (United States)

Wishaupt, Jérôme O; Ploeg, Tjeerd van der; Smeets, Leo C; Groot, Ronald de; Versteegh, Florens G A; Hartwig, Nico G

2017-05-01

The relation between viral load and disease severity in childhood acute respiratory tract infections (ARI) is not fully understood. To assess the clinical relevance of the relation between viral load, determined by cycle threshold (CT) value of real-time reverse transcription-polymerase chain reaction assays and disease severity in children with single- and multiple viral ARI. 582 children with ARI were prospectively followed and tested for 15 viruses. Correlations were calculated between CT values and clinical parameters. In single viral ARI, statistically significant correlations were found between viral loads of Respiratory Syncytial Virus (RSV) and hospitalization and between viral loads of Human Coronavirus (HCoV) and a disease severity score. In multiple-viral ARI, statistically significant correlations between viral load and clinical parameters were found. In RSV-Rhinovirus (RV) multiple infections, a low viral load of RV was correlated with a high length of hospital stay and a high duration of extra oxygen use. The mean CT value for RV, HCoV and Parainfluenza virus was significantly lower in single- versus multiple infections. Although correlations between CT values and clinical parameters in patients with single and multiple viral infection were found, the clinical importance of these findings is limited because individual differences in host-, viral and laboratory factors complicate the interpretation of statistically significant findings. In multiple infections, viral load cannot be used to differentiate between disease causing virus and innocent bystanders. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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Markus Hoffmann

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

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

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Qing-Ye Zhuang

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

Coronavirus-like particles in laboratory rabbits with different syndromes in The Netherlands (Coronavirus-like particles in rabbits).

NARCIS (Netherlands)

A.D.M.E. Osterhaus (Albert); J.S. Teppema; G. van Steenis (Bert)

1982-01-01

textabstractVirus-like particles were identified from the plasma of rabbits which developed pleural effusion disease after inoculation with different strains of Treponema pallidum. These particles were considered coronavirus-like on the basis of their size, morphology, and buoyant density. Clinical

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

Science.gov (United States)

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

2011-06-01

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

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

NARCIS (Netherlands)

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

2013-01-01

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

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

Science.gov (United States)

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

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

Science.gov (United States)

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

2016-10-01

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

Dynamics of the coronavirus replicative structures

NARCIS (Netherlands)

Hagemeijer, M.C.

2011-01-01

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

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

Science.gov (United States)

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

2014-04-01

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

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

Science.gov (United States)

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

2016-11-28

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

Epidemiological investigation of Middle East respiratory syndrome coronavirus in dromedary camel farms linked with human infection in Abu Dhabi Emirate, United Arab Emirates.

Science.gov (United States)

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.

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

Science.gov (United States)

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

2014-04-01

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

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

DEFF Research Database (Denmark)

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

1998-01-01

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

Coronavirus infection of polarized epithelial cells

NARCIS (Netherlands)

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

1995-01-01

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

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

Science.gov (United States)

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

2013-12-01

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

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

Science.gov (United States)

Harrath, Rafik; Duhier, Faisel M Abu

2018-04-16

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

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

Directory of Open Access Journals (Sweden)

Kèvin Knoops

2008-09-01

Full Text Available Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV, replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm, and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

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

Science.gov (United States)

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

2018-01-01

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

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

Science.gov (United States)

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

2011-10-01

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

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

DEFF Research Database (Denmark)

Liu, Lihong; Hägglund, Sara; Hakhverdyan, Mikhayil

2006-01-01

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

Co-circulation of three camel coronavirus species and recombination of MERS-CoVs in Saudi Arabia.

Science.gov (United States)

Sabir, Jamal S M; Lam, Tommy T-Y; Ahmed, Mohamed M M; Li, Lifeng; Shen, Yongyi; Abo-Aba, Salah E M; Qureshi, Muhammd I; Abu-Zeid, Mohamed; Zhang, Yu; Khiyami, Mohammad A; Alharbi, Njud S; Hajrah, Nahid H; Sabir, Meshaal J; Mutwakil, Mohammed H Z; Kabli, Saleh A; Alsulaimany, Faten A S; Obaid, Abdullah Y; Zhou, Boping; Smith, David K; Holmes, Edward C; Zhu, Huachen; Guan, Yi

2016-01-01

Outbreaks of Middle East respiratory syndrome (MERS) raise questions about the prevalence and evolution of the MERS coronavirus (CoV) in its animal reservoir. Our surveillance in Saudi Arabia in 2014 and 2015 showed that viruses of the MERS-CoV species and a human CoV 229E-related lineage co-circulated at high prevalence, with frequent co-infections in the upper respiratory tract of dromedary camels. viruses of the betacoronavirus 1 species, we found that dromedary camels share three CoV species with humans. Several MERS-CoV lineages were present in camels, including a recombinant lineage that has been dominant since December 2014 and that subsequently led to the human outbreaks in 2015. Camels therefore serve as an important reservoir for the maintenance and diversification of the MERS-CoVs and are the source of human infections with this virus. Copyright © 2016, American Association for the Advancement of Science.

Feline Coronavirus 3c Protein: A Candidate for a Virulence Marker?

Directory of Open Access Journals (Sweden)

A. S. Hora

2016-01-01

Full Text Available Feline infectious peritonitis virus (FIPV is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP, whereas feline enteric coronavirus (FECV is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a–c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account.

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

Science.gov (United States)

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

2007-12-01

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

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

Science.gov (United States)

Millet, Jean Kaoru; Whittaker, Gary R.

2014-01-01

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

Cytoplasmic tail of coronavirus spike protein has intracellular

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/042/02/0231-0244. Keywords. Coronavirus spike protein trafficking; cytoplasmic tail signal; endoplasmic reticulum–Golgi intermediate complex; lysosome. Abstract. Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is ...

Molecular and pathological identification of feline coronavirus type I

African Journals Online (AJOL)

DR. NJ TONUKARI

2012-06-05

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

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

Science.gov (United States)

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

2010-05-01

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

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

Science.gov (United States)

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

2016-01-01

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

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

OpenAIRE

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

2012-01-01

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

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

OpenAIRE

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

2012-01-01

Please cite this paper as: Szczawinska‐Poplonyk et al. (2012) Fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency. Influenza and Other Respiratory Viruses DOI: 10.1111/irv.12059. Coronaviruses have been demonstrated to contribute substantially to respiratory tract infections among the child population. Though infected children commonly present mild upper airway symptoms, in high‐risk patients with underlying conditions, particularl...

Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core

International Nuclear Information System (INIS)

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

2005-01-01

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

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

Science.gov (United States)

2013-01-01

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

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

Science.gov (United States)

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

1982-07-01

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

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

Directory of Open Access Journals (Sweden)

Christine Winter

2013-07-01

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

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

International Nuclear Information System (INIS)

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

2007-01-01

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

Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

NARCIS (Netherlands)

Cong, Yingying; Kriegenburg, Franziska; de Haan, Cornelis A. M.; Reggiori, Fulvio

2017-01-01

Coronaviruses (CoV) are enveloped viruses and rely on their nucleocapsid N protein to incorporate the positive-stranded genomic RNA into the virions. CoV N proteins form oligomers but the mechanism and relevance underlying their multimerization remain to be fully understood. Using in vitro pull-down

Dromedary camels and the transmission of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

Science.gov (United States)

Hemida, Maged G; Elmoslemany, Ahmed; Al-Hizab, Fahad; Alnaeem, Abdulmohsen; Almathen, Faisal; Faye, Bernard; Chu, Daniel KW; Perera, Ranawaka A; Peiris, Malik

2015-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is an existential threat to global public health. The virus has been repeatedly detected in dromedary camels (Camelus dromedarius). Adult animals in many countries in the Middle East as well as in North and East Africa showed high (>90%) sero-prevalence to the virus. MERS-CoV isolated from dromedaries is genetically and phenotypically similar to viruses from humans. We summarise current understanding of the ecology of MERS-CoV in animals and transmission at the animal-human interface. We review aspects of husbandry, animal movements and trade and the use and consumption of camel dairy and meat products in the Middle East that may be relevant to the epidemiology of MERS. We also highlight the gaps in understanding the transmission of this virus in animals and from animals to humans. PMID:26256102

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

International Nuclear Information System (INIS)

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

2005-01-01

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

Diagnostic Methods for Feline Coronavirus: A Review

Directory of Open Access Journals (Sweden)

Saeed Sharif

2010-01-01

Full Text Available Feline coronaviruses (FCoVs are found throughout the world. Infection with FCoV can result in a diverse range of signs from clinically inapparent infections to a highly fatal disease called feline infectious peritonitis (FIP. FIP is one of the most serious viral diseases of cats. While there is neither an effective vaccine, nor a curative treatment for FIP, a diagnostic protocol for FCoV would greatly assist in the management and control of the virus. Clinical findings in FIP are non-specific and not helpful in making a differential diagnosis. Haematological and biochemical abnormalities in FIP cases are also non-specific. The currently available serological tests have low specificity and sensitivity for detection of active infection and cross-react with FCoV strains of low pathogenicity, the feline enteric coronaviruses (FECV. Reverse transcriptase polymerase chain reaction (RT-PCR has been used to detect FCoV and is rapid and sensitive, but results must be interpreted in the context of clinical findings. At present, a definitive diagnosis of FIP can be established only by histopathological examination of biopsies. This paper describes and compares diagnostic methods for FCoVs and includes a brief account of the virus biology, epidemiology, and pathogenesis.

Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

NARCIS (Netherlands)

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

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

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

Science.gov (United States)

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

Novel coronavirus and astrovirus in Delaware Bay shorebirds.

Directory of Open Access Journals (Sweden)

Kirsi S Honkavuori

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

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

Czech Academy of Sciences Publication Activity Database

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

2010-01-01

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

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

NARCIS (Netherlands)

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

2014-01-01

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

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

Directory of Open Access Journals (Sweden)

Tomoki Yoshikawa

2010-01-01

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

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

OpenAIRE

2011-01-01

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

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

Directory of Open Access Journals (Sweden)

Atkins John F

2010-01-01

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

Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of NADPH Sensor HSCARG and Upregulation of NF-κB Signaling

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Yi-Hsuan Wu

2015-12-01

Full Text Available Glucose-6-phosphate dehydrogenase (G6PD-deficient cells are highly susceptible to viral infection. This study examined the mechanism underlying this phenomenon by measuring the expression of antiviral genes—tumor necrosis factor alpha (TNF-α and GTPase myxovirus resistance 1 (MX1—in G6PD-knockdown cells upon human coronavirus 229E (HCoV-229E and enterovirus 71 (EV71 infection. Molecular analysis revealed that the promoter activities of TNF-α and MX1 were downregulated in G6PD-knockdown cells, and that the IκB degradation and DNA binding activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide phosphate (NADPH sensor and negative regulator of NF-κB, was upregulated in G6PD-knockdown cells with decreased NADPH/NADP+ ratio. Treatment of G6PD-knockdown cells with siRNA against HSCARG enhanced the DNA binding activity of NF-κB and the expression of TNF-α and MX1, but suppressed the expression of viral genes; however, the overexpression of HSCARG inhibited the antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG, resulting in increased expression of TNF-α and MX1 and reduced viral gene expression upon virus infection. Our findings suggest that the increased susceptibility of the G6PD-knockdown cells to viral infection was due to impaired NF-κB signaling and antiviral response mediated by HSCARG.

Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus.

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Guangyu Zhao

Full Text Available The Middle East Respiratory Syndrome Coronavirus (MERS-CoV causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. For the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of MERS-CoV infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against MERS-CoV infection. In this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hDPP4, the receptor for MERS-CoV. After intranasal inoculation with MERS-CoV, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. In addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. Importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. Taken together, this study characterizes the tropism of MERS-CoV upon infection. Importantly, this hDPP4-expressing transgenic mouse model will be applicable for studying the pathogenesis of MERS-CoV infection and investigating the efficacy of vaccines and antiviral agents designed to combat MERS-CoV infection.

European Surveillance for Pantropic Canine Coronavirus

Science.gov (United States)

Cordonnier, Nathalie; Demeter, Zoltan; Egberink, Herman; Elia, Gabriella; Grellet, Aurélien; Le Poder, Sophie; Mari, Viviana; Martella, Vito; Ntafis, Vasileios; von Reitzenstein, Marcela; Rottier, Peter J.; Rusvai, Miklos; Shields, Shelly; Xylouri, Eftychia; Xu, Zach; Buonavoglia, Canio

2013-01-01

Highly virulent pantropic canine coronavirus (CCoV) strains belonging to subtype IIa were recently identified in dogs. To assess the distribution of such strains in Europe, tissue samples were collected from 354 dogs that had died after displaying systemic disease in France (n = 92), Hungary (n = 75), Italy (n = 69), Greece (n = 87), The Netherlands (n = 27), Belgium (n = 4), and Bulgaria (n = 1). A total of 124 animals tested positive for CCoV, with 33 of them displaying the virus in extraintestinal tissues. Twenty-four CCoV strains (19.35% of the CCoV-positive dogs) detected in internal organs were characterized as subtype IIa and consequently assumed to be pantropic CCoVs. Sequence and phylogenetic analyses of the 5′ end of the spike protein gene showed that pantropic CCoV strains are closely related to each other, with the exception of two divergent French viruses that clustered with enteric strains. PMID:23100349

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

Science.gov (United States)

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

2014-12-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-07-21

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

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

NARCIS (Netherlands)

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

2014-01-01

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

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

Directory of Open Access Journals (Sweden)

Ricardo Piñeros

2015-05-01

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

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

Directory of Open Access Journals (Sweden)

I-Jung Liu

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

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

NARCIS (Netherlands)

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

2003-01-01

textabstractIn March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2006-04-01

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

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

DEFF Research Database (Denmark)

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

2003-01-01

current worldwide distribution. The concerted efforts of a globally united scientific community have led to the independent isolation and identification of a novel coronavirus from SARS patients by several groups. The extraordinarily rapid isolation of a causative agent of this newly emerged infectious...

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

Science.gov (United States)

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

2013-12-12

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

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

OpenAIRE

Narayanan, Krishna; Makino, Shinji

2001-01-01

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

Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.

Science.gov (United States)

Arvia, Rosaria; Corcioli, Fabiana; Ciccone, Nunziata; Della Malva, Nunzia; Azzi, Alberta

2015-12-01

Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be

Feline Coronaviruses: Pathogenesis of Feline Infectious Peritonitis.

Science.gov (United States)

Tekes, G; Thiel, H-J

2016-01-01

Feline infectious peritonitis (FIP) belongs to the few animal virus diseases in which, in the course of a generally harmless persistent infection, a virus acquires a small number of mutations that fundamentally change its pathogenicity, invariably resulting in a fatal outcome. The causative agent of this deadly disease, feline infectious peritonitis virus (FIPV), arises from feline enteric coronavirus (FECV). The review summarizes our current knowledge of the genome and proteome of feline coronaviruses (FCoVs), focusing on the viral surface (spike) protein S and the five accessory proteins. We also review the current classification of FCoVs into distinct serotypes and biotypes, cellular receptors of FCoVs and their presumed role in viral virulence, and discuss other aspects of FIPV-induced pathogenesis. Our current knowledge of genetic differences between FECVs and FIPVs has been mainly based on comparative sequence analyses that revealed "discriminatory" mutations that are present in FIPVs but not in FECVs. Most of these mutations result in amino acid substitutions in the S protein and these may have a critical role in the switch from FECV to FIPV. In most cases, the precise roles of these mutations in the molecular pathogenesis of FIP have not been tested experimentally in the natural host, mainly due to the lack of suitable experimental tools including genetically engineered virus mutants. We discuss the recent progress in the development of FCoV reverse genetics systems suitable to generate recombinant field viruses containing appropriate mutations for in vivo studies. © 2016 Elsevier Inc. All rights reserved.

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

NARCIS (Netherlands)

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

2011-01-01

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

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

Science.gov (United States)

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

2018-03-20

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

A Structural analysis of M protein in coronavirus assembly and morphology

DEFF Research Database (Denmark)

W. Neuman, Benjamin; Kiss, Gabriella; H. Kunding, Andreas

2011-01-01

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy...... protein functions to promote virus assembly....

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

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Tan Yee-Joo

2005-02-01

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

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

International Nuclear Information System (INIS)

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

2009-01-01

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

Nucleocapsid gene analysis from an imported case of Middle East respiratory syndrome coronavirus, Malaysia

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Nor-Aziyah Mat-Rahim

2015-07-01

Full Text Available Objective: To describe the complete nucleocapsid (N gene region of Middle East respiratory syndrome coronavirus (MERS-CoV from imported case in Malaysia and the relations with human- and camel-derived MERS-CoV. Methods: Combination of throat and nasal swab specimens was subjected to viral RNA extraction. For screening, the extracted RNA was subjected to real-time RT-PCR targeting upstream of E gene, open reading frame 1b and open reading frame 1a. For confirmation, the RNA was subjected to RT-PCR targeting partial part of the RNA-dependent RNA polymerase and nucleocapsid, followed by amplification of complete N gene region. Nucleotide sequencing of the first Malaysian case of MERS-CoV was performed following the confirmation with real-time RT-PCR detection. Results: Initial analysis of partial RNA-dependent RNA polymerase and N gene revealed that the nucleotides had high similarity to Jeddah_1_2013 strain. Analysis of complete N gene region (1 242 nucleotides from the case showed high similarity and yet distinct to the nucleotide sequences of camel-derived MERS-CoV. Conclusions: From the finding, there are possibilities that the patient acquired the infection from zoonotic transmission from dromedary camels.

Taking forward a ‘One Health’ approach for turning the tide against the Middle East respiratory syndrome coronavirus and other zoonotic pathogens with epidemic potential

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Alimuddin Zumla

2016-06-01

Full Text Available The appearance of novel pathogens of humans with epidemic potential and high mortality rates have threatened global health security for centuries. Over the past few decades new zoonotic infectious diseases of humans caused by pathogens arising from animal reservoirs have included West Nile virus, Yellow fever virus, Ebola virus, Nipah virus, Lassa Fever virus, Hanta virus, Dengue fever virus, Rift Valley fever virus, Crimean-Congo haemorrhagic fever virus, severe acute respiratory syndrome coronavirus, highly pathogenic avian influenza viruses, Middle East Respiratory Syndrome Coronavirus, and Zika virus. The recent Ebola Virus Disease epidemic in West Africa and the ongoing Zika Virus outbreak in South America highlight the urgent need for local, regional and international public health systems to be be more coordinated and better prepared. The One Health concept focuses on the relationship and interconnectedness between Humans, Animals and the Environment, and recognizes that the health and wellbeing of humans is intimately connected to the health of animals and their environment (and vice versa. Critical to the establishment of a One Health platform is the creation of a multidisciplinary team with a range of expertise including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral phylogeneticists, and researchers to co-operate, collaborate to learn more about zoonotic spread between animals, humans and the environment and to monitor, respond to and prevent major outbreaks. We discuss the unique opportunities for Middle Eastern and African stakeholders to take leadership in building equitable and effective partnerships with all stakeholders involved in human and health systems to take forward a ‘One Health’ approach to control such zoonotic pathogens with epidemic potential.

Streptococcus sinensis may react with Lancefield group F antiserum.

Science.gov (United States)

Woo, Patrick C Y; Teng, Jade L L; Leung, Kit-wah; Lau, Susanna K P; Tse, Herman; Wong, Beatrice H L; Yuen, Kwok-yung

2004-11-01

Lancefield group F streptococci have been found almost exclusively as members of the 'Streptococcus milleri' group, although they have been reported very occasionally in some other streptococcal species. Among 302 patients with bacteraemia caused by viridans streptococci over a 6-year period, three cases were caused by Streptococcus sinensis (type strain HKU4T, HKU5 and HKU6). All three patients had infective endocarditis complicating their underlying chronic rheumatic heart diseases. Gene sequencing showed no base differences between the 16S rRNA gene sequences of HKU5 and HKU6 and that of HKU4T. All three strains were Gram-positive, non-spore-forming cocci arranged in chains. All grew on sheep blood agar as alpha-haemolytic, grey colonies of 0.5-1 mm in diameter after 24 h incubation at 37 degrees C in ambient air. Lancefield grouping revealed that HKU5 and HKU6 were Lancefield group F, but HKU4T was non-groupable with Lancefield groups A, B, C, D, F or G antisera. HKU4T was identified by the Vitek system (GPI), API system (20 STREP) and ATB system (ID32 STREP) as 99 % Streptococcus intermedius, 51.3 % S. intermedius and 99.9 % Streptococcus anginosus, respectively. Using the same tests, HKU5 was identified as 87 % Streptococcus sanguinis/Streptococcus gordonii, 59 % Streptococcus salivarius and 99.6 % S. anginosus, respectively, and HKU6 as 87 % S. sanguinis/S. gordonii, 77 % Streptococcus pneumoniae and 98.3 % S. anginosus, respectively. The present data revealed that a proportion of Lancefield group F streptococci could be S. sinensis. Lancefield group F streptococci should not be automatically reported as 'S. milleri'.

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

Science.gov (United States)

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

2015-09-08

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

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

NARCIS (Netherlands)

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

1990-01-01

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

Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro

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Jin Soo Shin

2018-05-01

Full Text Available The Middle East respiratory syndrome-coronavirus (MERS-CoV, first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK, was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.

Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro.

Science.gov (United States)

Shin, Jin Soo; Jung, Eunhye; Kim, Meehyein; Baric, Ralph S; Go, Yun Young

2018-05-24

The Middle East respiratory syndrome-coronavirus (MERS-CoV), first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK), was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.

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

Science.gov (United States)

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

2015-01-01

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

The Paradox of Feline Coronavirus Pathogenesis: A Review

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Luciana Wanderley Myrrha

2011-01-01

Full Text Available Feline coronavirus (FCoV is an enveloped single-stranded RNA virus, of the family Coronaviridae and the order Nidovirales. FCoV is an important pathogen of wild and domestic cats and can cause a mild or apparently symptomless enteric infection, especially in kittens. FCoV is also associated with a lethal, systemic disease known as feline infectious peritonitis (FIP. Although the precise cause of FIP pathogenesis remains unclear, some hypotheses have been suggested. In this review we present results from different FCoV studies and attempt to elucidate existing theories on the pathogenesis of FCoV infection.

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

Science.gov (United States)

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

2013-01-01

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

E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

International Nuclear Information System (INIS)

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

1988-01-01

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [ 3 H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating

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

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Jinzhi Tan

2009-05-01

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

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

Science.gov (United States)

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

2005-03-01

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

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

Science.gov (United States)

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

2005-08-19

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

Genome-wide analysis of codon usage bias in Bovine Coronavirus

OpenAIRE

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

2017-01-01

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

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

Science.gov (United States)

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

2011-04-01

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

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

KAUST Repository

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

2018-01-01

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

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

International Nuclear Information System (INIS)

Venkatagopalan, Pavithra; Daskalova, Sasha M.; Lopez, Lisa A.; Dolezal, Kelly A.; Hogue, Brenda G.

2015-01-01

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

Pathogenic characteristics of persistent feline enteric coronavirus infection in cats

Science.gov (United States)

Vogel, Liesbeth; Van der Lubben, Mariken; Te Lintelo, Eddie G.; Bekker, Cornelis P.J.; Geerts, Tamara; Schuijff, Leontine S.; Grinwis, Guy C.M.; Egberink, Herman F.; Rottier, Peter J.M.

2010-01-01

Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed. PMID:20663472

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-04-15

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

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

NARCIS (Netherlands)

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

2016-01-01

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

E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

Energy Technology Data Exchange (ETDEWEB)

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

1988-12-01

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with (/sup 3/H)DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating.

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

Science.gov (United States)

Zhang, Qingzhan; Yoo, Dongwan

2016-12-02

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

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

Science.gov (United States)

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

2018-05-17

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

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

OpenAIRE

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

2007-01-01

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

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

Directory of Open Access Journals (Sweden)

Lui Wing-bong

2006-02-01

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

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

Energy Technology Data Exchange (ETDEWEB)

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

2012-12-10

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

Increase in Middle East Respiratory Syndrome-Coronavirus Cases in Saudi Arabia Linked to Hospital Outbreak With Continued Circulation of Recombinant Virus, July 1–August 31, 2015

Science.gov (United States)

Assiri, Abdullah M.; Biggs, Holly M.; Abedi, Glen R.; Lu, Xiaoyan; Bin Saeed, Abdulaziz; Abdalla, Osman; Mohammed, Mutaz; Al-Abdely, Hail M.; Algarni, Homoud S.; Alhakeem, Raafat F.; Almasri, Malak M.; Alsharef, Ali A.; Nooh, Randa; Erdman, Dean D.; Gerber, Susan I.; Watson, John T.

2016-01-01

During July–August 2015, the number of cases of Middle East respiratory syndrome (MERS) reported from Saudi Arabia increased dramatically. We reviewed the 143 confirmed cases from this period and classified each based upon likely transmission source. We found that the surge in cases resulted predominantly (90%) from secondary transmission largely attributable to an outbreak at a single healthcare facility in Riyadh. Genome sequencing of MERS coronavirus from 6 cases demonstrated continued circulation of the recently described recombinant virus. A single unique frameshift deletion in open reading frame 5 was detected in the viral sequence from 1 case. PMID:27704019

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

Directory of Open Access Journals (Sweden)

Wei-Yu Liao

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

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

NARCIS (Netherlands)

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

2017-01-01

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

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

NARCIS (Netherlands)

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

2017-01-01

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

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

International Nuclear Information System (INIS)

Sardinia, L.M.

1985-01-01

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

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

Science.gov (United States)

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

2017-12-01

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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

Science.gov (United States)

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

2016-11-08

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

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

Science.gov (United States)

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

2009-06-01

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

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

International Nuclear Information System (INIS)

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

2006-01-01

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

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

OpenAIRE

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

2007-01-01

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

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

NARCIS (Netherlands)

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

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

Middle East respiratory syndrome coronavirus specific antibodies in naturally exposed Israeli llamas, alpacas and camels

Directory of Open Access Journals (Sweden)

Dan David

2018-06-01

Full Text Available Thus far, no human MERS-CoV infections have been reported from Israel. Evidence for the circulation of MERS-CoV in dromedaries has been reported from almost all the countries of the Middle East, except Israel. Therefore, we aimed to analyze MERS-CoV infection in Israeli camelids, sampled between 2012 and 2017. A total of 411 camels, 102 alpacas and 19 llamas' sera were tested for the presence of antibodies to MERS-CoV. Our findings indicate a lower MERS-CoV seropositivity among Israeli dromedaries than in the surrounding countries, and for the first time naturally infected llamas were identified. In addition, nasal swabs of 661 camels, alpacas and lamas, obtained from January 2015 to December 2017, were tested for the presence of MERS-CoV RNA. All nasal swabs were negative, indicating no evidence for MERS-CoV active circulation in these camelids during that time period. Keywords: MERS coronavirus, Antibodies, Israel, Dromedary camels, Llamas, Alpacas

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

Science.gov (United States)

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

2009-12-01

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

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

NARCIS (Netherlands)

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

2015-01-01

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

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

OpenAIRE

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

2013-01-01

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

Overview of preparedness and response for Middle East respiratory syndrome coronavirus (MERS-CoV) in Oman.

Science.gov (United States)

Al-Abaidani, I S; Al-Maani, A S; Al-Kindi, H S; Al-Jardani, A K; Abdel-Hady, D M; Zayed, B E; Al-Harthy, K S; Al-Shaqsi, K H; Al-Abri, S S

2014-12-01

Several countries in the Middle East and around 22 countries worldwide have reported cases of human infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). The exceptionally high fatality rate resulting from MERS-CoV infection in conjunction with the paucity of knowledge about this emerging virus has led to major public and international concern. Within the framework of the national acute respiratory illness surveillance, the Ministry of Health in the Sultanate of Oman has announced two confirmed cases of MERS-CoV to date. The aim of this report is to describe the epidemiological aspects of these two cases and to highlight the importance of public health preparedness and response. The absence of secondary cases among contacts of the reported cases can be seen as evidence of the effectiveness of infection prevention and control precautions as an important pillar of the national preparedness and response plan applied in the health care institutions in Oman. Copyright © 2014. Published by Elsevier Ltd.

Overview of preparedness and response for Middle East respiratory syndrome coronavirus (MERS-CoV in Oman

Directory of Open Access Journals (Sweden)

I.S. Al-Abaidani

2014-12-01

Full Text Available Several countries in the Middle East and around 22 countries worldwide have reported cases of human infection with the Middle East respiratory syndrome coronavirus (MERS-CoV. The exceptionally high fatality rate resulting from MERS-CoV infection in conjunction with the paucity of knowledge about this emerging virus has led to major public and international concern. Within the framework of the national acute respiratory illness surveillance, the Ministry of Health in the Sultanate of Oman has announced two confirmed cases of MERS-CoV to date. The aim of this report is to describe the epidemiological aspects of these two cases and to highlight the importance of public health preparedness and response. The absence of secondary cases among contacts of the reported cases can be seen as evidence of the effectiveness of infection prevention and control precautions as an important pillar of the national preparedness and response plan applied in the health care institutions in Oman.

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

Directory of Open Access Journals (Sweden)

Hui Dong

2016-12-01

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

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

OpenAIRE

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

2016-01-01

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

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

Science.gov (United States)

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

Streptobacillus hongkongensis sp. nov., isolated from patients with quinsy and septic arthritis, and emended descriptions of the genus Streptobacillus and Streptobacillus moniliformis.

Science.gov (United States)

Woo, Patrick C Y; Wu, Alan K L; Tsang, Chi-Ching; Leung, Kit-Wah; Ngan, Antonio H Y; Curreem, Shirly O T; Lam, Kwok-Wai; Chen, Jonathan H K; Chan, Jasper F W; Lau, Susanna K P

2014-09-01

Two bacterial strains, HKU33(T) and HKU34, were isolated in Hong Kong from the pus aspirated from the right peritonsillar abscess of a patient with quinsy and the left elbow joint fluid of another patient with tophaceous gout and left elbow septic arthritis, respectively. The bacteria were Gram-stain-negative, non-motile, non-spore-forming, non-haemolytic pleomorphic bacilli. They grew best on Columbia agar with 5 % defibrinated sheep blood in an anaerobic environment or aerobic environment with 5 % CO2. They also grew on chocolate agar but not on MacConkey agar. They were catalase- and cytochrome oxidase-negative. They showed a unique profile of enzyme activities distinguishable from their closely related species. Phylogenetic analysis of the complete 16S rRNA gene, and partial groEL, gyrB and recA gene sequences showed the two isolates formed a distinct branch within the family Leptotrichiaceae, being related most closely to Streptobacillus moniliformis. Hierarchical cluster analysis of mass spectra of whole-cell protein contents showed that strains HKU33(T) and HKU34 were closely related to each other, but were distinct from Streptobacillus moniliformis, Sneathia sanguinegens and 'Leptotrichia amnionii'. The DNA G+C content of strain HKU33(T) was 26.0±2.1 mol% (mean±sd; n = 3). DNA-DNA hybridization demonstrated ≤45.02 % DNA relatedness between the two isolates and Streptobacillus moniliformis CCUG 13453(T). A novel species, Streptobacillus hongkongensis sp. nov., is proposed to accommodate strains HKU33(T) and HKU34, with HKU33(T) ( = JCM 18691(T) = NCTC 13659(T) = DSM 26322(T)) designated the type strain. Emended descriptions of the genus Streptobacillus and Streptobacillus moniliformis are also given. © 2014 IUMS.

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

Science.gov (United States)

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

2013-12-01

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

Prevalence of Diabetes in the 2009 Influenza A (H1N1) and the Middle East Respiratory Syndrome Coronavirus: A Systematic Review and Meta-Analysis.

Science.gov (United States)

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, PMERS-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%; PMERS-CoV (54.4%; 95% CI: 29.4-79.5; Pprevalence 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.

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

Science.gov (United States)

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

2014-04-01

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

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

Science.gov (United States)

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

2012-11-01

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

Feline Lectin Activity Is Critical for the Cellular Entry of Feline Infectious Peritonitis Virus▿

OpenAIRE

Regan, Andrew D.; Ousterout, David G.; Whittaker, Gary R.

2010-01-01

Feline infectious peritonitis is a lethal disease of felids caused by systemic infection with a feline coronavirus. Here, we report identification and analysis of the feline homologue to the human lectin DC-SIGN and show that it is a coreceptor for virulent strains of serotype 1 and serotype 2 feline coronaviruses.

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

Science.gov (United States)

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

2013-11-01

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

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

Science.gov (United States)

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

2010-02-01

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

DoD Global Laboratory-Based Influenza Surveillance Program End-of-Year Report, 2013-2014

Science.gov (United States)

2015-07-22

Vaccine (trivalent) § Influenza Injectable Vaccine (quadrivalent) † Live Attenuated Influenza Vaccine (quadrivalent) 40 41 42 43 44 45 46 47 48... Adenovirus 1 A(H1N1)pdm09 & Coronavirus 1 A(H1N1)pdm09 & Parainfluenza 2 A(H1N1)pdm09 & RSV 2 A(H1N1)pdm09 & Rhinovirus/Enterovirus 2 A/not...subtyped 2 Other Respiratory Pathogens 1,485 Adenovirus 86 Chlamydophila pneumoniae 32 Coronavirus 230 Human

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

Science.gov (United States)

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

2007-01-01

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

Prevalence of diabetes in the 2009 influenza A (H1N1 and the Middle East respiratory syndrome coronavirus: a systematic review and meta-analysis

Directory of Open Access Journals (Sweden)

Alaa Badawi

2016-12-01

Full Text Available 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.

First case of human bacteraemia by Catabacter hongkongensis in Scandinavia

Directory of Open Access Journals (Sweden)

R. Kaden

2017-01-01

Full Text Available Catabacter hongkongensis was isolated and cultured from human blood for the first time in Scandinavia. The patient, an 83-year-old man from Dalarna, Sweden, recovered without antibiotic treatment, although a high mortality rate associated with C. hongkongensis infection had been reported from China, Canada and France. The genome of the strain ABBA15k was sequenced, assembled and analysed. In contrast to the type strain of the species HKU16T, no antibiotic resistance was observed in Scandinavian strain ABBA15k. The strain was deposited as CCUG 68271, and the draft genome sequence is available from the DNA Data Bank of Japan (DDBJ, the European Molecular Biology Laboratory (EMBL, and GenBank under the accession number LLYX00000000.

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

NARCIS (Netherlands)

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

2016-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2017-11-15

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

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

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

Science.gov (United States)

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

2014-12-01

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

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

Science.gov (United States)

Niederwerder, M C; Hesse, R A

2018-06-01

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

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

Science.gov (United States)

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

2006-08-01

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

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

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

2013-01-01

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

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

OpenAIRE

Kim, Ji Soo; Choi, Jeong Sil

2016-01-01

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

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

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Rinki Minakshi

2009-12-01

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

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

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Maslow, Joel N

2017-12-02

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

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

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Abdullah J. Alsahafi

2016-12-01

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

Epidemiology and clinical profile of pathogens responsible for the hospitalization of children in Sousse area, Tunisia.

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Ines Brini

Full Text Available This study aimed to identify a broad spectrum of respiratory pathogens from hospitalized and not-preselected children with acute respiratory tract infections in the Farhat Hached University-hospital of Sousse, Tunisia. Between September 2013 and December 2014, samples from 372 children aged between 1 month and 5 years were collected, and tested using multiplex real-time RT-PCR by a commercial assay for 21 respiratory pathogens. In addition, samples were screened for the presence of Streptococcus pneumoniae 16S rDNA using real-time PCR. The viral distribution and its association with clinical symptoms were statistically analyzed. Viral pathogens were detected in 342 (91.93% of the samples of which 28.76% were single positive and 63.17% had multiple infections. The most frequent detected viruses were rhinovirus (55.64%, respiratory syncytial virus A/B (33.06%, adenovirus (25.00%, coronavirus NL63, HKU1, OC43, and 229E (21.50%, and metapneumovirus A/B (16.12%. Children in the youngest age group (1-3 months exhibited the highest frequencies of infection. Related to their frequency of detection, RSV A/B was the most associated pathogen with patient's demographic situation and clinical manifestations (p<0.05. Parainfluenza virus 1-4 and parechovirus were found to increase the risk of death (p<0.05. Adenovirus was statistically associated to the manifestation of gastroenteritis (p = 0.004. Rhinovirus infection increases the duration of oxygen support (p = 0.042. Coronavirus group was statistically associated with the manifestation of bronchiolitis (p = 0.009 and laryngitis (p = 0.017. Streptococcus pneumoniae DNA was detected in 143 (38.44% of tested samples. However, only 53 samples had a concentration of C-reactive protein from equal to higher than 20 milligrams per liter, and 6 of them were single positive for Streptocuccus pneumoniae. This study confirms the high incidence of respiratory viruses in children hospitalized for acute respiratory tract

Competing for Iron: Duplication and Amplification of the isd Locus in Staphylococcus lugdunensis HKU09-01 Provides a Competitive Advantage to Overcome Nutritional Limitation.

Science.gov (United States)

Heilbronner, Simon; Monk, Ian R; Brozyna, Jeremy R; Heinrichs, David E; Skaar, Eric P; Peschel, Andreas; Foster, Timothy J

2016-08-01

Staphylococcus lugdunensis is a coagulase negative bacterial pathogen that is particularly associated with severe cases of infectious endocarditis. Unique amongst the coagulase-negative staphylococci, S. lugdunensis harbors an iron regulated surface determinant locus (isd). This locus facilitates the acquisition of heme as a source of nutrient iron during infection and allows iron limitation caused by "nutritional immunity" to be overcome. The isd locus is duplicated in S. lugdunensis HKU09-01 and we show here that the duplication is intrinsically unstable and undergoes accordion-like amplification and segregation leading to extensive isd copy number variation. Amplification of the locus increased the level of expression of Isd proteins and improved binding of hemoglobin to the cell surface of S. lugdunensis. Furthermore, Isd overexpression provided an advantage when strains were competing for a limited amount of hemoglobin as the sole source of iron. Gene duplications and amplifications (GDA) are events of fundamental importance for bacterial evolution and are frequently associated with antibiotic resistance in many species. As such, GDAs are regarded as evolutionary adaptions to novel selective pressures in hostile environments pointing towards a special importance of isd for S. lugdunensis. For the first time we show an example of a GDA that involves a virulence factor of a Gram-positive pathogen and link the GDA directly to a competitive advantage when the bacteria were struggling with selective pressures mimicking "nutritional immunity".

Origin, diversity and maturation of human antiviral antibodies analyzed by high-throughput sequencing

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Ponraj ePrabakaran

2012-08-01

Full Text Available Our understanding of how antibodies are generated and function could help develop effective vaccines and antibody-based therapeutics against viruses such as HIV-1, SARS Coronavirus (CoV, and Hendra and Nipah viruses (henipaviruses. Although broadly neutralizing antibodies (bnAbs against the HIV-1 were observed in patients, elicitation of such bnAbs remains a major challenge when compared to other viral targets. We previously hypothesized that HIV-1 could have evolved a strategy to evade the immune system due to absent or very weak binding of germline antibodies to the conserved epitopes that may not be sufficient to initiate and/or maintain an effective immune response. To further explore our hypothesis, we used the 454 sequence analysis of a large naïve library of human IgM antibodies which had been used for selecting antibodies against SARS Coronavirus (CoV receptor-binding domain (RBD, and soluble G proteins (sG of Hendra and Nipah viruses (henipaviruses. We found that the human IgM repertoires from the 454 sequencing have diverse germline usages, recombination patterns, junction diversity and a lower extent of somatic mutation. In this study, we identified germline intermediates of antibodies specific to HIV-1 and other viruses as observed in normal individuals, and compared their genetic diversity and somatic mutation level along with available structural and functional data. Further computational analysis will provide framework for understanding the underlying genetic and molecular determinants related to maturation pathways of antiviral bnAbs that could be useful for applying novel approaches to the design of effective vaccine immunogens and antibody-based therapeutics.

Genotyping coronaviruses associated with feline infectious peritonitis.

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Lewis, Catherine S; Porter, Emily; Matthews, David; Kipar, Anja; Tasker, Séverine; Helps, Christopher R; Siddell, Stuart G

2015-06-01

Feline coronavirus (FCoV) infections are endemic among cats worldwide. The majority of infections are asymptomatic or result in only mild enteric disease. However, approximately 5 % of cases develop feline infectious peritonitis (FIP), a systemic disease that is a frequent cause of death in young cats. In this study, we report the complete coding genome sequences of six FCoVs: three from faecal samples from healthy cats and three from tissue lesion samples from cats with confirmed FIP. The six samples were obtained over a period of 8 weeks at a single-site cat rescue and rehoming centre in the UK. We found amino acid differences located at 44 positions across an alignment of the six virus translatomes and, at 21 of these positions, the differences fully or partially discriminated between the genomes derived from the faecal samples and the genomes derived from the tissue lesion samples. In this study, two amino acid differences fully discriminated the two classes of genomes: these were both located in the S2 domain of the virus surface glycoprotein gene. We also identified deletions in the 3c protein ORF of genomes from two of the FIP samples. Our results support previous studies that implicate S protein mutations in the pathogenesis of FIP. © 2015 The Authors.

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

OpenAIRE

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

2015-01-01

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

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

Science.gov (United States)

Maslow, Joel N.

2017-01-01

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

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

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

2012-11-21

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

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

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Ádám Bálint

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

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

Science.gov (United States)

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

2014-01-01

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

Suppression of feline coronavirus replication in vitro by cyclosporin A

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Tanaka Yoshikazu

2012-04-01

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

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

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

2014-04-24

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

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

Science.gov (United States)

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

2018-03-07

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

Competing for Iron: Duplication and Amplification of the isd Locus in Staphylococcus lugdunensis HKU09-01 Provides a Competitive Advantage to Overcome Nutritional Limitation

Science.gov (United States)

Heilbronner, Simon; Brozyna, Jeremy R.; Heinrichs, David E.; Skaar, Eric P.; Peschel, Andreas; Foster, Timothy J.

2016-01-01

Staphylococcus lugdunensis is a coagulase negative bacterial pathogen that is particularly associated with severe cases of infectious endocarditis. Unique amongst the coagulase-negative staphylococci, S. lugdunensis harbors an iron regulated surface determinant locus (isd). This locus facilitates the acquisition of heme as a source of nutrient iron during infection and allows iron limitation caused by “nutritional immunity” to be overcome. The isd locus is duplicated in S. lugdunensis HKU09-01 and we show here that the duplication is intrinsically unstable and undergoes accordion-like amplification and segregation leading to extensive isd copy number variation. Amplification of the locus increased the level of expression of Isd proteins and improved binding of hemoglobin to the cell surface of S. lugdunensis. Furthermore, Isd overexpression provided an advantage when strains were competing for a limited amount of hemoglobin as the sole source of iron. Gene duplications and amplifications (GDA) are events of fundamental importance for bacterial evolution and are frequently associated with antibiotic resistance in many species. As such, GDAs are regarded as evolutionary adaptions to novel selective pressures in hostile environments pointing towards a special importance of isd for S. lugdunensis. For the first time we show an example of a GDA that involves a virulence factor of a Gram-positive pathogen and link the GDA directly to a competitive advantage when the bacteria were struggling with selective pressures mimicking “nutritional immunity”. PMID:27575058

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

NARCIS (Netherlands)

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

1991-01-01

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

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

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Jeremiah Athmer

2017-01-01

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

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

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Amer Alazawy

2012-11-01

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

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.

Science.gov (United States)

Sung, Heungsup; Yong, Dongeun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na

2016-09-01

Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both Phomogenizing sputum samples prior to RNA extraction.

Laboratory investigation and phylogenetic analysis of an imported Middle East respiratory syndrome coronavirus case in Greece.

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Athanasios Kossyvakis

Full Text Available Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient's history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.

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

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Jianhua Sui

2008-11-01

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

Adaptive evolution of the spike gene of SARS coronavirus: changes in positively selected sites in different epidemic groups

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He Shao-Heng

2006-10-01

Full Text Available Abstract Background It is believed that animal-to-human transmission of severe acute respiratory syndrome (SARS coronavirus (CoV is the cause of the SARS outbreak worldwide. The spike (S protein is one of the best characterized proteins of SARS-CoV, which plays a key role in SARS-CoV overcoming species barrier and accomplishing interspecies transmission from animals to humans, suggesting that it may be the major target of selective pressure. However, the process of adaptive evolution of S protein and the exact positively selected sites associated with this process remain unknown. Results By investigating the adaptive evolution of S protein, we identified twelve amino acid sites (75, 239, 244, 311, 479, 609, 613, 743, 765, 778, 1148, and 1163 in the S protein under positive selective pressure. Based on phylogenetic tree and epidemiological investigation, SARS outbreak was divided into three epidemic groups: 02–04 interspecies, 03-early-mid, and 03-late epidemic groups in the present study. Positive selection was detected in the first two groups, which represent the course of SARS-CoV interspecies transmission and of viral adaptation to human host, respectively. In contrast, purifying selection was detected in 03-late group. These indicate that S protein experiences variable positive selective pressures before reaching stabilization. A total of 25 sites in 02–04 interspecies epidemic group and 16 sites in 03-early-mid epidemic group were identified under positive selection. The identified sites were different between these two groups except for site 239, which suggests that positively selected sites are changeable between groups. Moreover, it was showed that a larger proportion (24% of positively selected sites was located in receptor-binding domain (RBD than in heptad repeat (HR1-HR2 region in 02–04 interspecies epidemic group (p = 0.0208, and a greater percentage (25% of these sites occurred in HR1–HR2 region than in RBD in 03-early

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

DEFF Research Database (Denmark)

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

2003-01-01

Health Organization (WHO). As SARS has the potential of becoming the first pandemic of the new millennium, a global warning by the WHO was issued on 12 March 2003. The disease, which is believed to have its origin in the Chinese Guangdong province, spread from Hong Kong via international airports to its...... disease constitutes an unprecedented scientific achievement. The main scope of the article is to provide the clinician with an overview of the natural history, epidemiology and clinical characteristics of SARS. On the basis of the recently published viral genome and structural features common...... to the members of the coronavirus family, a model for host cell-virus interaction and possible targets for antiviral drugs are presented. The epidemiological consequences of introducing a novel pathogen in a previously unexposed population and the origin and evolution of a new and more pathogenic strain...

Longitudinal study of age-specific pattern of coronavirus infection in Lyle's flying fox (Pteropus lylei) in Thailand.

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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

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

International Nuclear Information System (INIS)

McBride, Corrin E.; Machamer, Carolyn E.

2010-01-01

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

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

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

2015-06-01

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

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

International Nuclear Information System (INIS)

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

2007-01-01

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

Middle East respiratory syndrome coronavirus (MERS-CoV) RNA and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar, April 2014

NARCIS (Netherlands)

Reusken, C B; Farag, E A; Jonges, M; Godeke, G J; El-Sayed, A M; Pas, S D; Raj, V S; Mohran, K A; Moussa, H A; Ghobashy, H; Alhajri, F; Ibrahim, A K; Bosch, B J; Pasha, S K; Al-Romaihi, H E; Al-Thani, M; Al-Marri, S A; AlHajri, M M; Haagmans, B L; Koopmans, M P

2014-01-01

Antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) were detected in serum and milk collected according to local customs from 33 camels in Qatar, April 2014. At one location, evidence for active virus shedding in nasal secretions and/or faeces was observed for 7/12 camels; viral

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

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

2012-04-01

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

No Serologic Evidence of Middle East Respiratory Syndrome Coronavirus Infection Among Camel Farmers Exposed to Highly Seropositive Camel Herds: A Household Linked Study, Kenya, 2013.

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Munyua, Peninah; Corman, Victor Max; Bitek, Austine; Osoro, Eric; Meyer, Benjamin; Müller, Marcel A; Lattwein, Erik; Thumbi, S M; Murithi, Rees; Widdowson, Marc-Alain; Drosten, Christian; Njenga, M Kariuki

2017-06-01

AbstractHigh seroprevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) among camels has been reported in Kenya and other countries in Africa. To date, the only report of MERS-CoV seropositivity among humans in Kenya is of two livestock keepers with no known contact with camels. We assessed whether persons exposed to seropositive camels at household level had serological evidence of infection. In 2013, 760 human and 879 camel sera were collected from 275 and 85 households respectively in Marsabit County. Data on human and animal demographics and type of contact with camels were collected. Human and camel sera were tested for anti-MERS-CoV IgG using a commercial enzyme-linked immunosorbent assay (ELISA) test. Human samples were confirmed by plaque reduction neutralization test (PRNT). Logistic regression was used to identify factors associated with seropositivity. The median age of persons sampled was 30 years (range: 5-90) and 50% were males. A quarter (197/760) of the participants reported having had contact with camels defined as milking, feeding, watering, slaughtering, or herding. Of the human sera, 18 (2.4%) were positive on ELISA but negative by PRNT. Of the camel sera, 791 (90%) were positive on ELISA. On univariate analysis, higher prevalence was observed in female and older camels over 4 years of age ( P MERS-CoV infection among camel pastoralists in Marsabit County. The high seropositivity suggests that MERS-CoV or other closely related virus continues to circulate in camels and highlights ongoing potential for animal-to-human transmission.

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

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Anjeanette Roberts

2007-01-01

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

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

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Weber Friedemann

2006-03-01

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

Proteomic analysis of purified coronavirus infectious bronchitis virus particles

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Shu Dingming

2010-06-01

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

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

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Christine Burkard

2014-11-01

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

Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the First Imported MERS-CoV Case in China.

Science.gov (United States)

Lu, Roujian; Wang, Yanqun; Wang, Wenling; Nie, Kai; Zhao, Yanjie; Su, Juan; Deng, Yao; Zhou, Weimin; Li, Yang; Wang, Huijuan; Wang, Wen; Ke, Changwen; Ma, Xuejun; Wu, Guizhen; Tan, Wenjie

2015-08-13

On 26 May 2015, an imported Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in Guangdong Province, China, and found to be closely related to the MERS-CoV strain prevalent in South Korea. The full genome of the ChinaGD01 strain was sequenced and analyzed to investigate the epidemiology and evolution of MERS-CoV circulating in South Korea and China. Copyright © 2015 Lu et al.

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

NARCIS (Netherlands)

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

2017-01-01

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

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

Science.gov (United States)

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

2017-02-01

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

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

International Nuclear Information System (INIS)

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

2005-01-01

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

Coronavirus gene 7 counteracts host defenses and modulates virus virulence.

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Jazmina L G Cruz

2011-06-01

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

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

International Nuclear Information System (INIS)

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

2003-01-01

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

Conservation of nucleotide sequences for molecular diagnosis of Middle East respiratory syndrome coronavirus, 2015

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Yuki Furuse

2015-11-01

Full Text Available Infection due to the Middle East respiratory syndrome coronavirus (MERS-CoV is widespread. The present study was performed to assess the protocols used for the molecular diagnosis of MERS-CoV by analyzing the nucleotide sequences of viruses detected between 2012 and 2015, including sequences from the large outbreak in eastern Asia in 2015. Although the diagnostic protocols were established only 2 years ago, mismatches between the sequences of primers/probes and viruses were found for several of the assays. Such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. A slight modification in the primer design is suggested. Protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as MERS-CoV.

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

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

2013-10-01

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

Comparison of Bovine coronavirus-specific and Bovine respiratory syncytial virus-specific antibodies in serum versus milk samples detected by enzyme-linked immunosorbent assay.

Science.gov (United States)

Ohlson, Anna; Blanco-Penedo, Isabel; Fall, Nils

2014-01-01

Bovine coronavirus (BCV; Betacoronavirus 1) and Bovine respiratory syncytial virus (BRSV) are significant causes of enteric and respiratory disease in beef and dairy cattle throughout the world. Indirect enzyme-linked immunosorbent assays are widely used to detect serum antibodies for herd monitoring and prevalence studies. In dairy herds, milk is more readily collected than serum. Hence, in order to investigate the test agreement between serum and milk, both serum and milk samples from 105 cows in 27 dairy herds were analyzed in parallel for presence of immunoglobulin G antibodies to BCV and BRSV. The Bland-Altman analyses of data demonstrated good agreement between serum and milk antibody titers for both viruses. The results indicate milk samples are sufficient for surveillance of antibodies to BCV and BRSV.

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

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I-Chueh Huang

2011-01-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

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

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

2012-06-05

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

High prevalence of common respiratory viruses and no evidence of Middle East respiratory syndrome coronavirus in Hajj pilgrims returning to Ghana, 2013.

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Annan, Augustina; Owusu, Michael; Marfo, Kwadwo Sarfo; Larbi, Richard; Sarpong, Francisca Naana; Adu-Sarkodie, Yaw; Amankwa, Joseph; Fiafemetsi, Samuel; Drosten, Christian; Owusu-Dabo, Ellis; Eckerle, Isabella

2015-06-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 on the Arabian Peninsula and has caused severe respiratory disease with more than 800 laboratory-confirmed cases. The return of infected pilgrims to their home countries with a putative spread of MERS-CoV necessitates further surveillance. A cross sectional study of 839 adult African Hajj pilgrims returning to Accra in Ghana, West Africa, was conducted in 2013 to assess the prevalence of respiratory symptoms as well as of MERS-CoV, human rhinovirus (HRV), respiratory syncytial virus (RSV) and influenza A virus (FLU A) infection. Six hundred and fifty-one (77.6%) pilgrims had respiratory symptoms. Tests were positive for at least one of the viruses other than MERS-CoV in 179 (21.3%) of all pilgrims, with 22.4% detection in symptomatic vs. 17.6% detection in asymptomatic pilgrims. No MERS-CoV was detected, although common respiratory viruses were prevalent, with positive findings for HRV in 141 individuals (16.8%), RSV in 43 individuals (5.1%) and FLU A in 11 individuals (1.3%). Results were positive for more than one virus in 16 (1.9%) individuals, including 14 (1.7%) RSV/HRV co-infections and 2 (0.2%) FLU A/HRV co-infections. A total 146 (22.4%) of the symptomatic returnees tested positive for at least one respiratory virus compared with 33 (17.6%) of the asymptomatic pilgrims who had at least one detectable virus in their sample. The prevalence of viral respiratory infections among Hajj pilgrims in both symptomatic and asymptomatic subjects was high. Although it is reassuring that MERS-CoV was not detected in the tested population, there is a need for active surveillance of Hajj pilgrims. © 2015 John Wiley & Sons Ltd.

Dicty_cDB: Contig-U08402-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 03054189. 35 7.0 EU022525_1( EU022525 |pid:none) Turkey coronavirus isolate TCoV-54... 35 7.0 AB079608_1( AB...94702 ) RecName: Full=Intron-encoded endonuclease I-PpoI; ... 35 7.0 EU095850_1( EU095850 |pid:none) Turkey

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

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

2017-10-01

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

Bats as reservoirs of severe emerging infectious diseases.

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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.

Treatment outcomes for patients with Middle Eastern Respiratory Syndrome Coronavirus (MERS CoV) infection at a coronavirus referral center in the Kingdom of Saudi Arabia.

Science.gov (United States)

Al Ghamdi, Mohammed; Alghamdi, Khalid M; Ghandoora, Yasmeen; Alzahrani, Ameera; Salah, Fatmah; Alsulami, Abdulmoatani; Bawayan, Mayada F; Vaidya, Dhananjay; Perl, Trish M; Sood, Geeta

2016-04-21

Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV) is a poorly understood disease with no known treatments. We describe the clinical features and treatment outcomes of patients with laboratory confirmed MERS-CoV at a regional referral center in the Kingdom of Saudi Arabia. In 2014, a retrospective chart review was performed on patients with a laboratory confirmed diagnosis of MERS-CoV to determine clinical and treatment characteristics associated with death. Confounding was evaluated and a multivariate logistic regression was performed to assess the independent effect of treatments administered. Fifty-one patients had an overall mortality of 37 %. Most patients were male (78 %) with a mean age of 54 years. Almost a quarter of the patients were healthcare workers (23.5 %) and 41 % had a known exposure to another person with MERS-CoV. Survival was associated with male gender, working as a healthcare worker, history of hypertension, vomiting on admission, elevated respiratory rate, abnormal lung exam, elevated alanine transaminase (ALT), clearance of MERS-CoV on repeat PCR polymerase chain reaction (PCR) testing, and mycophenolate mofetil treatment. Survival was reduced in the presence of coronary artery disease, hypotension, hypoxemia, CXR (chest X-ray) abnormalities, leukocytosis, creatinine >1 · 5 mg/dL, thrombocytopenia, anemia, and renal failure. In a multivariate analysis of treatments administered, severity of illness was the greatest predictor of reduced survival. Care for patients with MERS-CoV remains a challenge. In this retrospective cohort, interferon beta and mycophenolate mofetil treatment were predictors of increased survival in the univariate analysis. Severity of illness was the greatest predictor of reduced survival in the multivariate analysis. Larger randomized trials are needed to better evaluate the efficacy of these treatment regimens for MERS-CoV.

Detection and genetic characterization of Canine parvovirus and Canine coronavirus strains circulating in district of Tirana in Albania.

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Cavalli, Alessandra; Desario, Costantina; Kusi, Ilir; Mari, Viviana; Lorusso, Eleonora; Cirone, Francesco; Kumbe, Ilirjan; Colaianni, Maria Loredana; Buonavoglia, Domenico; Decaro, Nicola

2014-07-01

An epidemiological survey for Canine parvovirus 2 (CPV-2) and Canine coronavirus (CCoV) was conducted in Albania. A total of 57 fecal samples were collected from diarrheic dogs in the District of Tirana during 2011-2013. The molecular assays detected 53 and 31 CPV- and CCoV-positive specimens, respectively, with mixed CPV-CCoV infections diagnosed in 28 dogs. The most frequently detected CPV type was 2a, whereas IIa was the predominant CCoV subtype. A better comprehension of the CPV-CCoV epidemiology in eastern European countries will help to assess the most appropriate vaccination strategies to prevent disease due to infections with these widespread agents of acute gastroenteritis in the dog.

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

International Nuclear Information System (INIS)

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

1992-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1992-01-01

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

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

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Juan Reguera

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

A pandemic risk assessment of middle east respiratory syndrome coronavirus (MERS-CoV in Saudi Arabia

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Saleh A. Eifan

2017-11-01

Full Text Available Since the initial emergence of Middle East respiratory syndrome coronavirus (MERS-CoV in 2012, a high incidence rate has been observed in Saudi Arabia. This suggests that the country is at continuous risk. The epidemic level of MERS-CoV infection was examined in Saudi Arabia by the Susceptible-Infectious-Recovered (SIR model using a Bayesian approach for estimation of time dependent reproduction number (R across a two-year interval (May, 2013-May, 2015 in five defined clusters, followed by sensitivity analysis of the most significant clusters. Significant MERS-CoV peaks were detected in the period between March and May of each year. Moreover, MERS-CoV infection was highlighted in western (40.8% and central (31.9% regions, followed by eastern region (20%. The temporal-based Bayesian approach indicated a sub-critical epidemic in all regions in the baseline scenario (R: 0.85–0.97. However, R potential limit was exceeded in the sensitivity analysis scenario in only central and western regions (R: 1.08–1.12 that denoted epidemic level in those regions. The impact of sporadic cases was found relatively insignificant and pinpointed to the lack of zoonotic influence on MERS-CoV transmission dynamics. The results of current study would be helpful for evaluation of future progression of MERS-CoV infections, better understanding and control interventions.

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

Science.gov (United States)

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

2005-01-01

AIM: To study whether severe acute respiratory syndrome coronavirus (SARS-CoV) could be excreted from digestive system. METHODS: Cell culture and semi-nested RT-PCR were used to detect SARS-CoV and its RNA from 21 stool and urine samples, and a kind of electropositive filter media particles was used to concentrate the virus in 10 sewage samples from two hospitals receiving SARS patients in Beijing in China. RESULTS: It was demonstrated that there was no live SARS-CoV in all samples collected, but the RNA of SARS-CoV could be detected in seven stool samples from SARS patients with any one of the symptoms of fever, malaise, cough, or dyspnea, in 10 sewage samples before disinfection and 3 samples after disinfection from the two hospitals. The RNA could not be detected in urine and stool samples from patients recovered from SARS. CONCLUSION: Nucleic acid of SARS-CoV can be excreted through the stool of patients into sewage system, and the possibility of SARS-CoV transmitting through digestive system cannot be excluded. PMID:16038039

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

Science.gov (United States)

2012-10-12

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

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

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Jaume Torres

2015-06-01

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

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

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Ruili Ma

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

Inheritance-mode specific pathogenicity prioritization (ISPP) for human protein coding genes.

Science.gov (United States)

Hsu, Jacob Shujui; Kwan, Johnny S H; Pan, Zhicheng; Garcia-Barcelo, Maria-Mercè; Sham, Pak Chung; Li, Miaoxin

2016-10-15

Exome sequencing studies have facilitated the detection of causal genetic variants in yet-unsolved Mendelian diseases. However, the identification of disease causal genes among a list of candidates in an exome sequencing study is still not fully settled, and it is often difficult to prioritize candidate genes for follow-up studies. The inheritance mode provides crucial information for understanding Mendelian diseases, but none of the existing gene prioritization tools fully utilize this information. We examined the characteristics of Mendelian disease genes under different inheritance modes. The results suggest that Mendelian disease genes with autosomal dominant (AD) inheritance mode are more haploinsufficiency and de novo mutation sensitive, whereas those autosomal recessive (AR) genes have significantly more non-synonymous variants and regulatory transcript isoforms. In addition, the X-linked (XL) Mendelian disease genes have fewer non-synonymous and synonymous variants. As a result, we derived a new scoring system for prioritizing candidate genes for Mendelian diseases according to the inheritance mode. Our scoring system assigned to each annotated protein-coding gene (N = 18 859) three pathogenic scores according to the inheritance mode (AD, AR and XL). This inheritance mode-specific framework achieved higher accuracy (area under curve  = 0.84) in XL mode. The inheritance-mode specific pathogenicity prioritization (ISPP) outperformed other well-known methods including Haploinsufficiency, Recessive, Network centrality, Genic Intolerance, Gene Damage Index and Gene Constraint scores. This systematic study suggests that genes manifesting disease inheritance modes tend to have unique characteristics. ISPP is included in KGGSeq v1.0 (http://grass.cgs.hku.hk/limx/kggseq/), and source code is available from (https://github.com/jacobhsu35/ISPP.git). mxli@hku.hkSupplementary information: Supplementary data are available at Bioinformatics online. © The Author

Experimental inoculation of equine coronavirus into Japanese draft horses.

Science.gov (United States)

Nemoto, Manabu; Oue, Yasuhiro; Morita, Yoshinori; Kanno, Toru; Kinoshita, Yuta; Niwa, Hidekazu; Ueno, Takanori; Katayama, Yoshinari; Bannai, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kondo, Takashi

2014-12-01

Recently, outbreaks associated with equine coronavirus (ECoV) have occurred in Japan and the United States. While ECoV is likely to be pathogenic to horses, it has not been shown that experimental inoculation of horses with ECoV produces clinical signs of disease. In this study, we inoculated three Japanese draft horses with an ECoV-positive diarrheic fecal sample to confirm infection after inoculation and to investigate the clinical course and virus shedding patterns of ECoV. Virus neutralization tests showed that all three horses became infected with ECoV. Two of the three horses developed clinical signs similar to those observed during ECoV outbreaks, including fever, anorexia, and gastrointestinal dysfunction. All horses excreted a large amount of virus into their feces for more than 9 days after inoculation regardless of the presence or absence of clinical signs, which suggests that feces are an important source of ECoV infection. ECoV was also detected in nasal swabs from all horses, suggesting that respiratory transmission of ECoV may occur. Both symptomatic horses developed viremia, while the asymptomatic horse did not. White blood cell counts and serum amyloid A concentrations changed relative to the clinical condition of the inoculated horses; these may be useful markers for monitoring the clinical status of horses infected with ECoV. This is the first report of induction of clinical signs of ECoV infection in horses by experimental inoculation. These clinical and virological findings should aid further investigation of the pathogenesis of ECoV.

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

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Alaa Badawi

2016-08-01

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

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

International Nuclear Information System (INIS)

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

2007-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-03-31

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

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

International Nuclear Information System (INIS)

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

2007-01-01

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

The Characteristics of Middle Eastern Respiratory Syndrome Coronavirus Transmission Dynamics in South Korea.

Science.gov (United States)

Kim, Yunhwan; Lee, Sunmi; Chu, Chaeshin; Choe, Seoyun; Hong, Saeme; Shin, Youngseo

2016-02-01

The outbreak of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) was one of the major events in South Korea in 2015. In particular, this study pays attention to formulating a mathematical model for MERS transmission dynamics and estimating transmission rates. Incidence data of MERS-CoV from the government authority was analyzed for the first aim and a mathematical model was built and analyzed for the second aim of the study. A mathematical model for MERS-CoV transmission dynamics is used to estimate the transmission rates in two periods due to the implementation of intensive interventions. Using the estimates of the transmission rates, the basic reproduction number was estimated in two periods. Due to the superspreader, the basic reproduction number was very large in the first period; however, the basic reproduction number of the second period has reduced significantly after intensive interventions. It turned out to be the intensive isolation and quarantine interventions that were the most critical factors that prevented the spread of the MERS outbreak. The results are expected to be useful to devise more efficient intervention strategies in the future.

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Science.gov (United States)

Millet, Jean Kaoru; Whittaker, Gary R.

2016-01-01

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay. PMID:28018942

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

Science.gov (United States)

Badawi, Alaa; Ryoo, Seung Gwan

2016-08-01

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

The imperative to develop a human vaccine for the Hendra virus in Australia

Science.gov (United States)

Zahoor, Bilal A.; Mudie, Lucy I.

2015-01-01

The Hendra virus (HeV) poses a significant challenge to public health in Australia. Expanding migratory patterns observed among bats and the mutation of the virus to seek and successfully infect new hosts is a significant departure from the generalized epidemiological trend. The recent discovery of equine-related infections and deaths in addition to a canine infection demonstrates the inadequacy of the current equine vaccine developed in 2012. Traditional models for controlling the spread of the vector are futile given the rapid pace at which bats' habitats are eroded. Recent ongoing zoonotic epidemics, for example, Ebola and Middle East respiratory syndrome coronavirus, demonstrate that human-to-human transmission is a distinct reality rather than an obscure possibility. The development of a human HeV vaccine is essential for the biosecurity of Australia, as part of a multipronged strategy to control HeV in Australia. PMID:26519254

Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: a descriptive study.

Science.gov (United States)

Assiri, Abdullah; Al-Tawfiq, Jaffar A; Al-Rabeeah, Abdullah A; Al-Rabiah, Fahad A; Al-Hajjar, Sami; Al-Barrak, Ali; Flemban, Hesham; Al-Nassir, Wafa N; Balkhy, Hanan H; Al-Hakeem, Rafat F; Makhdoom, Hatem Q; Zumla, Alimuddin I; Memish, Ziad A

2013-09-01

Middle East respiratory syndrome (MERS) is a new human disease caused by a novel coronavirus (CoV). Clinical data on MERS-CoV infections are scarce. We report epidemiological, demographic, clinical, and laboratory characteristics of 47 cases of MERS-CoV infections, identify knowledge gaps, and define research priorities. We abstracted and analysed epidemiological, demographic, clinical, and laboratory data from confirmed cases of sporadic, household, community, and health-care-associated MERS-CoV infections reported from Saudi Arabia between Sept 1, 2012, and June 15, 2013. Cases were confirmed as having MERS-CoV by real-time RT-PCR. 47 individuals (46 adults, one child) with laboratory-confirmed MERS-CoV disease were identified; 36 (77%) were male (male:female ratio 3·3:1). 28 patients died, a 60% case-fatality rate. The case-fatality rate rose with increasing age. Only two of the 47 cases were previously healthy; most patients (45 [96%]) had underlying comorbid medical disorders, including diabetes (32 [68%]), hypertension (16 [34%]), chronic cardiac disease (13 [28%]), and chronic renal disease (23 [49%]). Common symptoms at presentation were fever (46 [98%]), fever with chills or rigors (41 [87%]), cough (39 [83%]), shortness of breath (34 [72%]), and myalgia (15 [32%]). Gastrointestinal symptoms were also frequent, including diarrhoea (12 [26%]), vomiting (ten [21%]), and abdominal pain (eight [17%]). All patients had abnormal findings on chest radiography, ranging from subtle to extensive unilateral and bilateral abnormalities. Laboratory analyses showed raised concentrations of lactate dehydrogenase (23 [49%]) and aspartate aminotransferase (seven [15%]) and thrombocytopenia (17 [36%]) and lymphopenia (16 [34%]). Disease caused by MERS-CoV presents with a wide range of clinical manifestations and is associated with substantial mortality in admitted patients who have medical comorbidities. Major gaps in our knowledge of the epidemiology, community prevalence

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

KAUST Repository

Kandeel, Mahmoud

2018-05-17

Structural studies related to Middle East Respiratory Syndrome Coronavirus (MERS CoV) infection process are so limited. In this study, molecular dynamics (MD) simulation was carried out to unravel changes in the MERS CoV heptad repeat domains (HRs) and factors affecting fusion state HR stability. Results indicated that HR trimer is more rapidly stabilized, having stable system energy and lowest root mean square deviations (RMSDs). While trimers were the predominant active form of CoVs HR, monomers were also discovered in both of viral and cellular membranes. In order to find the differences between S2 monomer and trimer molecular dynamics, S2 monomer were modelled and subjected to MD simulation. In contrast to S2 trimer, S2 monomer was unstable, having high RMSDs with major drifts above 8 Å. Fluctuation of HR residue positions revealed major changes in the C-terminal of HR2 and the linker coil between HR1 and HR2 in both monomer and trimer. Hydrophobic residues at the “a” and “d” positions of HR helices stabilize the whole system, having minimal changes in RMSD. The global distance test and contact area difference scores support instability of MERS CoV S2 monomer. Analysis of HR1-HR2 inter-residue contacts and interaction energy revealed three different energy scales along HR helices. Two strong interaction energies were identified at the start of the HR2 helix and at the C-terminal of HR2. The identified critical residues by MD simulation and residues at a and d position of HR helix were strong stabilizers of HRs recognition.

Discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis.

Science.gov (United States)

Lorusso, Eleonora; Mari, Viviana; Losurdo, Michele; Lanave, Gianvito; Trotta, Adriana; Dowgier, Giulia; Colaianni, Maria Loredana; Zatelli, Andrea; Elia, Gabriella; Buonavoglia, Domenico; Decaro, Nicola

2017-10-31

Intra-vitam diagnosis of feline infectious peritonitis (FIP) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. With the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive FIP were collected intra-vitam for detection of feline coronavirus (FCoV) antibodies and RNA by means of indirect immunofluorescence (IIF) assay and real-time RT-PCR (qRT-PCR), respectively. In 5 effusions there was no evidence for either FCoV RNA or antibodies, 51 and 52 specimens tested positive by IIF and qRT-PCR, respectively, although antibody titres≥1:1600, which are considered highly suggestive of FIP, were detected only in 37 effusions. Three samples with high antibody levels tested negative by qRT-PCR, whereas 18 qRT-PCR positive effusions contained no or low-titre antibodies. qRT-PCR positive samples with low antibody titres mostly contained low FCoV RNA loads, although the highest antibody titres were detected in effusions with C T values>30. In conclusion, combining the two methods, i.e., antibody and RNA detection would help improving the intra-vitam diagnosis of effusive FIP. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Kong Xiangang

2011-03-01

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

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

DEFF Research Database (Denmark)

Toftaker, Ingrid; Toft, Nils; Stokstad, Maria

2018-01-01

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

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

Science.gov (United States)

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

2017-11-01

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

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

Science.gov (United States)

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

2013-01-01

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

Importance of Neutralizing Monoclonal Antibodies Targeting Multiple Antigenic Sites on the Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein To Avoid Neutralization Escape

Energy Technology Data Exchange (ETDEWEB)

Wang, Lingshu; Shi, Wei; Chappell, James D.; Joyce, M. Gordon; Zhang, Yi; Kanekiyo, Masaru; Becker, Michelle M.; van Doremalen, Neeltje; Fischer, Robert; Wang, Nianshuang; Corbett, Kizzmekia S.; Choe, Misook; Mason, Rosemarie D.; Van Galen, Joseph G.; Zhou, Tongqing; Saunders, Kevin O.; Tatti, Kathleen M.; Haynes, Lia M.; Kwong, Peter D.; Modjarrad, Kayvon; Kong, Wing-Pui; McLellan, Jason S.; Denison, Mark R.; Munster, Vincent J.; Mascola, John R.; Graham, Barney S.; Gallagher, Tom

2018-03-07

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pulmonary infection with ~35% mortality. The potential for a future pandemic originating from animal reservoirs or health care-associated events is a major public health concern. There are no vaccines or therapeutic agents currently available for MERS-CoV. Using a probe-based single B cell cloning strategy, we have identified and characterized multiple neutralizing monoclonal antibodies (MAbs) specifically binding to the receptor-binding domain (RBD) or S1 (non-RBD) regions from a convalescent MERS-CoV-infected patient and from immunized rhesus macaques. RBD-specific MAbs tended to have greater neutralizing potency than non-RBD S1-specific MAbs. Six RBD-specific and five S1-specific MAbs could be sorted into four RBD and three non-RBD distinct binding patterns, based on competition assays, mapping neutralization escape variants, and structural analysis. We determined cocrystal structures for two MAbs targeting the RBD from different angles and show they can bind the RBD only in the “out” position. We then showed that selected RBD-specific, non-RBD S1-specific, and S2-specific MAbs given prophylactically prevented MERS-CoV replication in lungs and protected mice from lethal challenge. Importantly, combining RBD- and non-RBD MAbs delayed the emergence of escape mutations in a cell-based virus escape assay. These studies identify MAbs targeting different antigenic sites on S that will be useful for defining mechanisms of MERS-CoV neutralization and for developing more effective interventions to prevent or treat MERS-CoV infections.

IMPORTANCEMERS-CoV causes a highly lethal respiratory infection for which no vaccines or antiviral therapeutic options are currently available. Based on continuing exposure from established reservoirs in dromedary camels and bats, transmission of MERS-CoV into humans and future outbreaks are expected. Using

Structural evolution of the Irtysh Shear Zone: implication for the Late Paleozoic amalgamation of multiple arc systems in Central Asia

Science.gov (United States)

Li, Pengfei; Sun, Min; Rosenbaum, Gideon

2015-04-01

The NW-SE Irtysh Shear Zone represents a major tectonic boundary in the Central Asian Orogenic Belt, recording the amalgamation history between the peri-Siberian orogenic system and the Kazakhstan orogenic system. The structural evolution and geodynamics of this shear zone is still poorly documented. Here we present new structural data complemented by chronological data in an attempt to unravel the geodynamic significance of the Irtysh Shear Zone in the context of accretion history of the Central Asian Orogenic Belt. Our results show three episodes of deformation for the shear zone. D1 foliation is locally recognized in low strain area and recorded by garnet inclusions, whereas D2 is represented by a sub-horizontal fabric and related NW-SE lineation. D3 is characterized by a transpersonal deformation event, to form a series of NW-SE mylonitic belts with sinistral kinematics, and to overprint D2 fabric forming regional-scale NW-SE upright folds. A paragneiss sample from the shear zone yielded the youngest detrital zircon peaks in the late Carboniferous, placing a maximum age constraint on the deformation, which overlaps in time with the late Paleozoic collision between the Chinese Altai and the intraoceanic arc system of the East Junggar and West Junggar. We interpret three episodes of deformation to represent orogenic thickening (D1), collapse (D2) and thickening (D3) in response to this collisional event. Sinistral shearing (D3) together with the coeval dextral shearing in the Tianshan accommodate eastward extrusion of the Kazakhstan orogenic system during the late Paleozoic amalgamation of the Central Asian Orogenic Belt. Acknowledgements: This study was financially supported by the Major Basic Research Project of the Ministry of Science and Technology of China (Grant: 2014CB440801), Hong Kong Research Grant Council (HKU705311P and HKU704712P), National Science Foundation of China (41273048, 41273012) and a HKU CRCG grant. The work is a contribution of the Joint

Cross-sectional surveillance of Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels and other mammals in Egypt, August 2015 to January 2016.

Science.gov (United States)

Ali, Mohamed; El-Shesheny, Rabeh; Kandeil, Ahmed; Shehata, Mahmoud; Elsokary, Basma; Gomaa, Mokhtar; Hassan, Naglaa; El Sayed, Ahmed; El-Taweel, Ahmed; Sobhy, Heba; Fasina, Folorunso Oludayo; Dauphin, Gwenaelle; El Masry, Ihab; Wolde, Abebe Wossene; Daszak, Peter; Miller, Maureen; VonDobschuetz, Sophie; Morzaria, Subhash; Lubroth, Juan; Makonnen, Yilma Jobre

2017-03-16

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 MERS-CoV seroprevalence (p 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. This article is copyright of The Authors, 2017.

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

International Nuclear Information System (INIS)

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

2007-01-01

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

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

Directory of Open Access Journals (Sweden)

Jean Kaoru Millet

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-01-01

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

Reliable typing of MERS-CoV variants with a small genome fragment

NARCIS (Netherlands)

S.L. Smits (Saskia); V.S. Raj (Stalin); S.D. Pas (Suzan); C.B.E.M. Reusken (Chantal); K.A. Mohran (Khaled A.); E. Farag (Elmoubasher); H.E. Al Romaihi (Hamad); M.M. AlHajri (Mohd); B.L. Haagmans (Bart); M.P.G. Koopmans D.V.M. (Marion)

2015-01-01

textabstractBackground: Middle East Respiratory Syndrome coronavirus (. MERS-CoV) is an emerging pathogen that causes lower respiratory tract infection in humans. Camels are the likely animal source for zoonotic infection, although exact transmission modes remain to be determined. Human-to-human

Chemical disinfection of non-porous inanimate surfaces experimentally contaminated with four human pathogenic viruses.

OpenAIRE

Sattar, S. A.; Springthorpe, V. S.; Karim, Y.; Loro, P.

1989-01-01

The chemical disinfection of virus-contaminated non-porous inanimate surfaces was investigated using coxsackievirus B3, adenovirus type 5, parainfluenza virus type 3 and coronavirus 229E as representatives of important nosocomial viral pathogens. A 10 microliter amount of the test virus, suspended in either faeces or mucin, was placed onto each stainless steel disk (about 1 cm in diameter) and the inoculum allowed to dry for 1 h under ambient conditions. Sixteen disinfectant formulations were...

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

Energy Technology Data Exchange (ETDEWEB)

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

2010-01-01

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

Intrahost Diversity of Feline Coronavirus: A Consensus between the Circulating Virulent/Avirulent Strains and the Internal Mutation Hypotheses?

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Aline S. Hora

2013-01-01

Full Text Available To evaluate the most controversial issue concerning current feline coronavirus (FCoV virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV in regard to the pathogenesis of feline infectious peritonitis (FIP, this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1–3 from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene.

MERS-CoV: Middle East respiratory syndrome corona virus: Can radiology be of help? Initial single center experience

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Hamimi, Ahmed

2016-01-01

Human infection with a novel coronavirus named Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia and the Middle East in September, 2012. The aim of this study was to establish the most pathognomonic radiological sign(s) to diagnose MERS CoV. Patients and methods: This is a retrospective descriptive study. All patients were subjected to serial X-ray. High resolution non-contrast CT chest was also obtained for 10 patients. The scans were reviewed fo...

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).

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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.

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

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

2009-01-01

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

Effects of human SAMHD1 polymorphisms on HIV-1 susceptibility

International Nuclear Information System (INIS)

White, Tommy E.; Brandariz-Nuñez, Alberto; Valle-Casuso, Jose Carlos; Knowlton, Caitlin; Kim, Baek; Sawyer, Sara L.; Diaz-Griffero, Felipe

2014-01-01

SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition. - Highlights: • Human SAMHD1 single-nucleotide polymorphisms block HIV-1 and HIV-2 infection. • SAMHD1 polymorphisms do not affect its ability to block LINE-1 retrotransposition. • SAMHD1 polymorphisms decrease the cellular levels of dNTPs

Effects of human SAMHD1 polymorphisms on HIV-1 susceptibility

Energy Technology Data Exchange (ETDEWEB)

White, Tommy E.; Brandariz-Nuñez, Alberto; Valle-Casuso, Jose Carlos [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, 1301 Morris Park – Price Center 501, New York, NY 10461 (United States); Knowlton, Caitlin; Kim, Baek [Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 (United States); Sawyer, Sara L. [Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712 (United States); Diaz-Griffero, Felipe, E-mail: Felipe.Diaz-Griffero@einstein.yu.edu [Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, 1301 Morris Park – Price Center 501, New York, NY 10461 (United States)

2014-07-15

SAMHD1 is a human restriction factor that prevents efficient infection of macrophages, dendritic cells and resting CD4+ T cells by HIV-1. Here we explored the antiviral activity and biochemical properties of human SAMHD1 polymorphisms. Our studies focused on human SAMHD1 polymorphisms that were previously identified as evolving under positive selection for rapid amino acid replacement during primate speciation. The different human SAMHD1 polymorphisms were tested for their ability to block HIV-1, HIV-2 and equine infectious anemia virus (EIAV). All studied SAMHD1 variants block HIV-1, HIV-2 and EIAV infection when compared to wild type. We found that these variants did not lose their ability to oligomerize or to bind RNA. Furthermore, all tested variants were susceptible to degradation by Vpx, and localized to the nuclear compartment. We tested the ability of human SAMHD1 polymorphisms to decrease the dNTP cellular levels. In agreement, none of the different SAMHD1 variants lost their ability to reduce cellular levels of dNTPs. Finally, we found that none of the tested human SAMHD1 polymorphisms affected the ability of the protein to block LINE-1 retrotransposition. - Highlights: • Human SAMHD1 single-nucleotide polymorphisms block HIV-1 and HIV-2 infection. • SAMHD1 polymorphisms do not affect its ability to block LINE-1 retrotransposition. • SAMHD1 polymorphisms decrease the cellular levels of dNTPs.

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

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

2017-10-05

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

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

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

2015-04-01

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

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

Science.gov (United States)

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

2017-08-01

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

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

International Nuclear Information System (INIS)

Wang, S.-F.; Huang, Jason C.; Lee, Y.-M.; Liu, S.-J.; Chan, Yu-Jiun; Chau, Y.-P.; Chong, P.; Chen, Y.-M.A.

2008-01-01

DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing α-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capable of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans

Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections

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Müller Marcel A

2005-02-01

Full Text Available Abstract Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

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

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Albrecht von Brunn

2007-05-01

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

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

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Sara Meazzi

2017-05-01

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

Identification of cis-acting elements on positive-strand subgenomic mRNA required for the synthesis of negative-strand counterpart in bovine coronavirus.

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Yeh, Po-Yuan; Wu, Hung-Yi

2014-07-30

It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(-)-strand] complement. However, the cis-acting elements on the positive-strand [(+)-strand] sgmRNA required for (-)-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV) sgmRNA 7 required for the synthesis of its (-)-strand counterpart by deletion mutagenesis. The major findings are as follows. (1) Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (-)-strand sgmRNA complement. (2) Deletions of the 3' untranslated region (UTR) bulged stem-loop showed no effect on (-)-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (-)-strand sgmRNA. (3) Nucleotides positioned from -15 to -34 of the sgmRNA 7 3'-terminal region are required for efficient (-)-strand sgmRNA synthesis. (4) Nucleotide species at the 3'-most position (-1) of sgmRNA 7 is correlated to the efficiency of (-)-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (-)-strand sgmRNA synthesis in BCoV.

Genetic diversity of Aspergillus species isolated from onychomycosis and Aspergillus hongkongensis sp. nov., with implications to antifungal susceptibility testing.

Science.gov (United States)

Tsang, Chi-Ching; Hui, Teresa W S; Lee, Kim-Chung; Chen, Jonathan H K; Ngan, Antonio H Y; Tam, Emily W T; Chan, Jasper F W; Wu, Andrea L; Cheung, Mei; Tse, Brian P H; Wu, Alan K L; Lai, Christopher K C; Tsang, Dominic N C; Que, Tak-Lun; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

2016-02-01

Thirteen Aspergillus isolates recovered from nails of 13 patients (fingernails, n=2; toenails, n=11) with onychomycosis were characterized. Twelve strains were identified by multilocus sequencing as Aspergillus spp. (Aspergillus sydowii [n=4], Aspergillus welwitschiae [n=3], Aspergillus terreus [n=2], Aspergillus flavus [n=1], Aspergillus tubingensis [n=1], and Aspergillus unguis [n=1]). Isolates of A. terreus, A. flavus, and A. unguis were also identifiable by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The 13th isolate (HKU49(T)) possessed unique morphological characteristics different from other Aspergillus spp. Molecular characterization also unambiguously showed that HKU49(T) was distinct from other Aspergillus spp. We propose the novel species Aspergillus hongkongensis to describe this previously unknown fungus. Antifungal susceptibility testing showed most Aspergillus isolates had low MICs against itraconazole and voriconazole, but all Aspergillus isolates had high MICs against fluconazole. A diverse spectrum of Aspergillus species is associated with onychomycosis. Itraconazole and voriconazole are probably better drug options for Aspergillus onychomycosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of new genetic variants of Betacoronaviruses in Endemic Frugivorous Bats of Madagascar.

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Razanajatovo, Norosoa H; Nomenjanahary, Lalaina A; Wilkinson, David A; Razafimanahaka, Julie H; Goodman, Steven M; Jenkins, Richard K; Jones, Julia P G; Heraud, Jean-Michel

2015-03-12

Bats are amongst the natural reservoirs of many coronaviruses (CoVs) of which some can lead to severe infection in human. African bats are known to harbor a range of pathogens (e.g., Ebola and Marburg viruses) that can infect humans and cause disease outbreaks. A recent study in South Africa isolated a genetic variant closely related to MERS-CoV from an insectivorous bat. Though Madagascar is home to 44 bat species (41 insectivorous and 3 frugivorous) of which 34 are endemic, no data exists concerning the circulation of CoVs in the island's chiropteran fauna. Certain Malagasy bats can be frequently found in close contact with humans and frugivorous bats feed in the same trees where people collect and consume fruits and are hunted and consumed as bush meat. The purpose of our study is to detect and identify CoVs from frugivorous bats in Madagascar to evaluate the risk of human infection from infected bats. Frugivorous bats belonging to three species were captured in four different regions of Madagascar. We analyzed fecal and throat swabs to detect the presence of virus through amplification of the RNA-dependent RNA polymerase (RdRp) gene, which is highly conserved in all known coronaviruses. Phylogenetic analyses were performed from positive specimens. From 351 frugivorous bats, we detected 14 coronaviruses from two endemic bats species, of which 13 viruses were identified from Pteropus rufus and one from Eidolon dupreanum, giving an overall prevalence of 4.5%. Phylogenetic analysis revealed that the Malagasy strains belong to the genus Betacoronavirus but form three distinct clusters, which seem to represent previously undescribed genetic lineages. Our findings suggest that CoVs circulate in frugivorous bats of Madagascar, demonstrating the needs to evaluate spillover risk to human populations especially for individuals that hunt and consume infected bats. Possible dispersal mechanisms as to how coronaviruses arrived on Madagascar are discussed.

Expression and function of human hemokinin-1 in human and guinea pig airways.

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Grassin-Delyle, Stanislas; Naline, Emmanuel; Buenestado, Amparo; Risse, Paul-André; Sage, Edouard; Advenier, Charles; Devillier, Philippe

2010-10-07

Human hemokinin-1 (hHK-1) and endokinins are peptides of the tachykinin family encoded by the TAC4 gene. TAC4 and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study. RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages. In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK1-and NK2-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK2-receptors, which blockade unmasked a NK1-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK1-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages. We demonstrate endogenous expression of TAC4 in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.

Expression and function of human hemokinin-1 in human and guinea pig airways

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Sage Edouard

2010-10-01

Full Text Available Abstract Background Human hemokinin-1 (hHK-1 and endokinins are peptides of the tachykinin family encoded by the TAC4 gene. TAC4 and hHK-1 expression as well as effects of hHK-1 in the lung and airways remain however unknown and were explored in this study. Methods RT-PCR analysis was performed on human bronchi to assess expression of tachykinin and tachykinin receptors genes. Enzyme immunoassay was used to quantify hHK-1, and effects of hHK-1 and endokinins on contraction of human and guinea pig airways were then evaluated, as well as the role of hHK-1 on cytokines production by human lung parenchyma or bronchi explants and by lung macrophages. Results In human bronchi, expression of the genes that encode for hHK-1, tachykinin NK1-and NK2-receptors was demonstrated. hHK-1 protein was found in supernatants from explants of human bronchi, lung parenchyma and lung macrophages. Exogenous hHK-1 caused a contractile response in human bronchi mainly through the activation of NK2-receptors, which blockade unmasked a NK1-receptor involvement, subject to a rapid desensitization. In the guinea pig trachea, hHK-1 caused a concentration-dependant contraction mainly mediated through the activation of NK1-receptors. Endokinin A/B exerted similar effects to hHK-1 on both human bronchi and guinea pig trachea, whereas endokinins C and D were inactive. hHK-1 had no impact on the production of cytokines by explants of human bronchi or lung parenchyma, or by human lung macrophages. Conclusions We demonstrate endogenous expression of TAC4 in human bronchi, the encoded peptide hHK-1 being expressed and involved in contraction of human and guinea pig airways.

Tracing Airline Travelers for a Public Health Investigation: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Infection in the United States, 2014.

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Regan, Joanna J; Jungerman, M Robynne; Lippold, Susan A; Washburn, Faith; Roland, Efrosini; Objio, Tina; Schembri, Christopher; Gulati, Reena; Edelson, Paul J; Alvarado-Ramy, Francisco; Pesik, Nicki; Cohen, Nicole J

2016-01-01

CDC routinely conducts contact investigations involving travelers on commercial conveyances, such as aircrafts, cargo vessels, and cruise ships. The agency used established systems of communication and partnerships with other federal agencies to quickly provide accurate traveler contact information to states and jurisdictions to alert contacts of potential exposure to two travelers with Middle East Respiratory Syndrome Coronavirus (MERS-CoV) who had entered the United States on commercial flights in April and May 2014. Applying the same process used to trace and notify travelers during routine investigations, such as those for tuberculosis or measles, CDC was able to notify most travelers of their potential exposure to MERS-CoV during the first few days of each investigation. To prevent the introduction and spread of newly emerging infectious diseases, travelers need to be located and contacted quickly.

Retrospective testing and case series study of porcine delta coronavirus in U.S. swine herds.

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McCluskey, Brian J; Haley, Charles; Rovira, Albert; Main, Rodger; Zhang, Yan; Barder, Sunny

2016-01-01

Porcine deltacoronavirus (PDCoV) was first reported in the United States (US) in February 2014. This was the second novel swine enteric coronavirus detected in the US since May 2013. In this study, we conducted retrospective testing of samples submitted to three veterinary diagnostic laboratories where qualifying biological samples were derived from previously submitted diagnostic case submissions from US commercial swine farms with a clinical history of enteric disease or from cases that had been previously tested for transmissible gastroenteritis virus, PEDV, or rotavirus. Overall, 2286 banked samples were tested from 27 States. Samples were collected in 3 separate years and in 17 different months. Test results revealed 4 positive samples, 3 collected in August 2013 and 1 collected in October 2013. In addition, a case series including 42 operations in 10 States was conducted through administration of a survey. Survey data collected included information on characteristics of swine operations that had experienced PDCoV clinical signs. Special emphasis was placed on obtaining descriptive estimates of biosecurity practices and disease status over time of each operation. Clinical signs of PDCoV were reported to be similar to those of PEDV. The average number of animals on each operation exhibiting clinical signs (morbidity) and the average number of case fatalities was greatest for suckling and weaned pigs. Average operation-level weaned pig morbidity was greatest in the first week of the outbreak while average operation-level suckling pig case fatality was greatest in the second week of the outbreak. The survey included questions regarding biosecurity practices for visitors and operation employees; trucks, equipment and drivers; and feed sources. These questions attempted to identify a likely pathway of introduction of PDCoV onto the operations surveyed. Published by Elsevier B.V.

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

Science.gov (United States)

Felten, Sandra; Leutenegger, Christian M; Balzer, Hans-Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman; Hartmann, Katrin

2017-08-02

Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

International Nuclear Information System (INIS)

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

2009-01-01

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

A Novel Dynamic Model Describing the Spread of the MERS-CoV and the Expression of Dipeptidyl Peptidase 4

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Siming Tang

2017-01-01

Full Text Available The Middle East respiratory syndrome (MERS coronavirus, a newly identified pathogen, causes severe pneumonia in humans. MERS is caused by a coronavirus known as MERS-CoV, which attacks the respiratory system. The recently defined receptor for MERS-CoV, dipeptidyl peptidase 4 (DPP4, is generally expressed in endothelial and epithelial cells and has been shown to be present on cultured human nonciliated bronchiolar epithelium cells. In this paper, a class of novel four-dimensional dynamic model describing the infection of MERS-CoV is given, and then global stability of the equilibria of the model is discussed. Our results show that the spread of MERS-CoV can also be controlled by decreasing the expression rate of DPP4.

Common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus.

Science.gov (United States)

Pedersen, N C; Sato, R; Foley, J E; Poland, A M

2004-04-01

The purpose of this study was to determine the origin and subsequent spread of feline calicivirus (FCV), feline herpesvirus (FHV), and feline enteric coronavirus (FECV) in cats relinquished to shelters. FCV was isolated from the oral fauces of 11% of healthy cats upon entry, and isolation rates were highest for kittens (33%). FHV shedding was very low (4%) at the time of entry and occurred mainly in juveniles. FECV shedding was also common among newly relinquished cats (33%), especially older kittens and juveniles (90%). The subsequent spread of all three viruses was rapid and efficient in the shelter environment. Fifteen percent of cats were shedding FCV, 52% FHV, and 60% FECV after 1 week. More detailed studies were done with FECV shedding, which could be accurately quantitated. The amounts of FECV shed by infected cats ranged from 10(2)to 10(16)particles/swab of feces. FECV shedding was several logs higher in young kittens with primary infection than adult cats with primary infections. The mean levels of FECV shedding among adults were the same for primary and chronic infections. Although shelters were not the primary source of these viruses for many relinquished cats, factors intrinsic to the shelter environment were critical in amplifying shedding and spread to susceptible individuals. Extrinsic factors were especially important for the spread of FHV and FECV. FHV shedding rates increased from 4% to 50% in 1 week's time. The speed and magnitude of the increase in FHV shedding suggested that there was reactivation of latent infections as well as acquisition of new infections. FECV shedding increased 10 to 1,000,000 fold in 1 week among cats that were already infected at entry, and more than one-half of initially negative cats were shedding FECV a week later. Feline calicivirus infection was the least likely to spread in the shelter. The infection rate only increased from 11 to 15% in 1 week.

Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures.

Science.gov (United States)

Essaidi-Laziosi, Manel; Brito, Francisco; Benaoudia, Sacha; Royston, Léna; Cagno, Valeria; Fernandes-Rocha, Mélanie; Piuz, Isabelle; Zdobnov, Evgeny; Huang, Song; Constant, Samuel; Boldi, Marc-Olivier; Kaiser, Laurent; Tapparel, Caroline

2018-06-01

The leading cause of acute illnesses, respiratory viruses, typically cause self-limited diseases, although severe complications can occur in fragile patients. Rhinoviruses (RVs), respiratory enteroviruses (EVs), influenza virus, respiratory syncytial viruses (RSVs), and coronaviruses are highly prevalent respiratory pathogens, but because of the lack of reliable animal models, their differential pathogenesis remains poorly characterized. We sought to compare infections by respiratory viruses isolated from clinical specimens using reconstituted human airway epithelia. Tissues were infected with RV-A55, RV-A49, RV-B48, RV-C8, and RV-C15; respiratory EV-D68; influenza virus H3N2; RSV-B; and human coronavirus (HCoV)-OC43. Replication kinetics, cell tropism, effect on tissue integrity, and cytokine secretion were compared. Viral adaptation and tissue response were assessed through RNA sequencing. RVs, RSV-B, and HCoV-OC43 infected ciliated cells and caused no major cell death, whereas H3N2 and EV-D68 induced ciliated cell loss and tissue integrity disruption. H3N2 was also detected in rare goblet and basal cells. All viruses, except RV-B48 and HCoV-OC43, altered cilia beating and mucociliary clearance. H3N2 was the strongest cytokine inducer, and HCoV-OC43 was the weakest. Persistent infection was observed in all cases. RNA sequencing highlighted perturbation of tissue metabolism and induction of a transient but important immune response at 4 days after infection. No majority mutations emerged in the viral population. Our results highlight the differential in vitro pathogenesis of respiratory viruses during the acute infection phase and their ability to persist under immune tolerance. These data help to appreciate the range of disease severity observed in vivo and the occurrence of chronic respiratory tract infections in immunocompromised hosts. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Tracking Human Immunodeficiency Virus-1 Infection in the Humanized DRAG Mouse Model

OpenAIRE

Jiae Kim; Jiae Kim; Kristina K. Peachman; Kristina K. Peachman; Ousman Jobe; Ousman Jobe; Elaine B. Morrison; Atef Allam; Atef Allam; Linda Jagodzinski; Sofia A. Casares; Mangala Rao

2017-01-01

Humanized mice are emerging as an alternative model system to well-established non-human primate (NHP) models for studying human immunodeficiency virus (HIV)-1 biology and pathogenesis. Although both NHP and humanized mice have their own strengths and could never truly reflect the complex human immune system and biology, there are several advantages of using the humanized mice in terms of using primary HIV-1 for infection instead of simian immunodeficiency virus or chimera simian/HIV. Several...

Acute respiratory viral infections in pediatric cancer patients undergoing chemotherapy

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Eliana C.A. Benites

2014-07-01

Full Text Available OBJECTIVE: to estimate the prevalence of infection by respiratory viruses in pediatric patients with cancer and acute respiratory infection (ARI and/or fever. METHODS: cross-sectional study, from January 2011 to December 2012. The secretions of nasopharyngeal aspirates were analyzed in children younger than 21 years with acute respiratory infections. Patients were treated at the Grupo em Defesa da Criança Com Câncer (Grendacc and University Hospital (HU, Jundiaí, SP. The rapid test was used for detection of influenza virus (Kit Biotrin, Inc. Ireland, and real-time multiplex polymerase chain reaction (FTD, Respiratory pathogens, multiplex Fast Trade Kit, Malta for detection of influenza virus (H1N1, B, rhinovirus, parainfluenza virus, adenovirus, respiratory syncytial virus, human parechovirus, bocavirus, metapneumovirus, and human coronavirus. The prevalence of viral infection was estimated and association tests were used (χ2 or Fisher's exact test. RESULTS: 104 samples of nasopharyngeal aspirate and blood were analyzed. The median age was 12 ± 5.2 years, 51% males, 68% whites, 32% had repeated ARIs, 32% prior antibiotic use, 19.8% cough, and 8% contact with ARIs. A total of 94.3% were in good general status. Acute lymphocytic leukemia (42.3% was the most prevalent neoplasia. Respiratory viruses were detected in 50 samples: rhinoviruses (23.1%, respiratory syncytial virus AB (8.7%, and coronavirus (6.8%. Co-detection occurred in 19% of cases with 2 viruses and in 3% of those with 3 viruses, and was more frequent between rhinovirus and coronavirus 43. Fever in neutropenic patients was observed in 13%, of which four (30.7 were positive for viruses. There were no deaths. CONCLUSIONS: the prevalence of respiratory viruses was relevant in the infectious episode, with no increase in morbidity and mortality. Viral co-detection was frequent in patients with cancer and ARIs.

Tracking Human Immunodeficiency Virus-1 Infection in the Humanized DRAG Mouse Model

Science.gov (United States)

Kim, Jiae; Peachman, Kristina K.; Jobe, Ousman; Morrison, Elaine B.; Allam, Atef; Jagodzinski, Linda; Casares, Sofia A.; Rao, Mangala

2017-01-01

Humanized mice are emerging as an alternative model system to well-established non-human primate (NHP) models for studying human immunodeficiency virus (HIV)-1 biology and pathogenesis. Although both NHP and humanized mice have their own strengths and could never truly reflect the complex human immune system and biology, there are several advantages of using the humanized mice in terms of using primary HIV-1 for infection instead of simian immunodeficiency virus or chimera simian/HIV. Several different types of humanized mice have been developed with varying levels of reconstitution of human CD45+ cells. In this study, we utilized humanized Rag1KO.IL2RγcKO.NOD mice expressing HLA class II (DR4) molecule (DRAG mice) infused with HLA-matched hematopoietic stem cells from umbilical cord blood to study early events after HIV-1 infection, since the mucosal tissues of these mice are highly enriched for human lymphocytes and express the receptors and coreceptors needed for HIV-1 entry. We examined the various tissues on days 4, 7, 14, and 21 after an intravaginal administration of a single dose of purified primary HIV-1. Plasma HIV-1 RNA was detected as early as day 7, with 100% of the animals becoming plasma RNA positive by day 21 post-infection. Single cells were isolated from lymph nodes, bone marrow, spleen, gut, female reproductive tissue, and brain and analyzed for gag RNA and strong stop DNA by quantitative (RT)-PCR. Our data demonstrated the presence of HIV-1 viral RNA and DNA in all of the tissues examined and that the virus was replication competent and spread rapidly. Bone marrow, gut, and lymph nodes were viral RNA positive by day 4 post-infection, while other tissues and plasma became positive typically between 7 and 14 days post-infection. Interestingly, the brain was the last tissue to become HIV-1 viral RNA and DNA positive by day 21 post-infection. These data support the notion that humanized DRAG mice could serve as an excellent model for studying the

Tracking Human Immunodeficiency Virus-1 Infection in the Humanized DRAG Mouse Model

Directory of Open Access Journals (Sweden)

Jiae Kim

2017-10-01

Full Text Available Humanized mice are emerging as an alternative model system to well-established non-human primate (NHP models for studying human immunodeficiency virus (HIV-1 biology and pathogenesis. Although both NHP and humanized mice have their own strengths and could never truly reflect the complex human immune system and biology, there are several advantages of using the humanized mice in terms of using primary HIV-1 for infection instead of simian immunodeficiency virus or chimera simian/HIV. Several different types of humanized mice have been developed with varying levels of reconstitution of human CD45+ cells. In this study, we utilized humanized Rag1KO.IL2RγcKO.NOD mice expressing HLA class II (DR4 molecule (DRAG mice infused with HLA-matched hematopoietic stem cells from umbilical cord blood to study early events after HIV-1 infection, since the mucosal tissues of these mice are highly enriched for human lymphocytes and express the receptors and coreceptors needed for HIV-1 entry. We examined the various tissues on days 4, 7, 14, and 21 after an intravaginal administration of a single dose of purified primary HIV-1. Plasma HIV-1 RNA was detected as early as day 7, with 100% of the animals becoming plasma RNA positive by day 21 post-infection. Single cells were isolated from lymph nodes, bone marrow, spleen, gut, female reproductive tissue, and brain and analyzed for gag RNA and strong stop DNA by quantitative (RT-PCR. Our data demonstrated the presence of HIV-1 viral RNA and DNA in all of the tissues examined and that the virus was replication competent and spread rapidly. Bone marrow, gut, and lymph nodes were viral RNA positive by day 4 post-infection, while other tissues and plasma became positive typically between 7 and 14 days post-infection. Interestingly, the brain was the last tissue to become HIV-1 viral RNA and DNA positive by day 21 post-infection. These data support the notion that humanized DRAG mice could serve as an excellent model

Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229E in bats, Ghana

Czech Academy of Sciences Publication Activity Database

Pfefferle, S.; Oppong, S.; Drexler, J. F.; Gloza-Rausch, F.; Ipsen, A.; Seebens, A.; Müller, M. A.; Annan, A.; Vallo, Peter; Adu-Sarkodie, Y.; Kruppa, T. F.; Drosten, C.

2009-01-01

Roč. 15, č. 9 (2009), s. 1377-1384 ISSN 1080-6040 Grant - others:German Ministry of Education and Research(XE) SSPE-CT-2005-022639; FP7(XE) INFRASTRUCTURES-2008-No 228292; Bundesamt für Bevölkerungsschutz und Katastrophenhilfe(DE) BBK-F-440-00-1 Institutional research plan: CEZ:AV0Z60930519 Keywords : SARS-like * phylogenetic analysis * RNA * gastroenteritis * prevalence * diversity Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 6.794, year: 2009

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

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Karen M. Asano

2009-11-01

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

Art-mediated peer-to-peer learning of empathy.

Science.gov (United States)

Potash, Jordan; Chen, Julie

2014-08-01

Making experiential art in a clinical clerkship offers opportunities for students to gain self-awareness and enhance their empathic understanding of patients. The student-created art can be further used as teaching material for other students. The graduating class of 2012 from Ajou University School of Medicine in South Korea was interested in learning about medical humanities initiatives at the Li Ka Shing Faculty of Medicine, University of Hong Kong (HKU), and made an educational visit in May 2012. As part of the core family medicine curriculum, third-year HKU medical students created poetry and art based on their experiences witnessing patients in pain and suffering. Twenty of the artworks and accompanying reflective writing were chosen for an exhibition. The visiting students viewed the exhibit and created their own art based on their emotional response to one piece selected from the exhibit. The combination of viewing art made by their peers and creating art in response resulted in empathic understanding of patient pain and suffering, and an appreciation of holistic care and the value of the doctor-patient relationship. Medical student-generated artwork has the potential to educate both students and professionals on humanistic aspects of medical care. © 2014 John Wiley & Sons Ltd.

10 CFR 1.39 - Office of Human Resources.

Science.gov (United States)

2010-01-01

... 10 Energy 1 2010-01-01 2010-01-01 false Office of Human Resources. 1.39 Section 1.39 Energy... § 1.39 Office of Human Resources. The Office of Human Resources— (a) Plans and implements NRC policies... agency's human resources; (b) Provides labor relations and personnel policy guidance and supporting...

Identification of Human Junctional Adhesion Molecule 1 as a Functional Receptor for the Hom-1 Calicivirus on Human Cells

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Stanislav V. Sosnovtsev

2017-02-01

Full Text Available The Hom-1 vesivirus was reported in 1998 following the inadvertent transmission of the animal calicivirus San Miguel sea lion virus to a human host in a laboratory. We characterized the Hom-1 strain and investigated the mechanism by which human cells could be infected. An expression library of 3,559 human plasma membrane proteins was screened for reactivity with Hom-1 virus-like particles, and a single interacting protein, human junctional adhesion molecule 1 (hJAM1, was identified. Transient expression of hJAM1 conferred susceptibility to Hom-1 infection on nonpermissive Chinese hamster ovary (CHO cells. Virus infection was markedly inhibited when CHO cells stably expressing hJAM were pretreated with anti-hJAM1 monoclonal antibodies. Cell lines of human origin were tested for growth of Hom-1, and efficient replication was observed in HepG2, HuH7, and SK-CO15 cells. The three cell lines (of hepatic or intestinal origin were confirmed to express hJAM1 on their surface, and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of the hJAM1 gene in each line abolished Hom-1 propagation. Taken together, our data indicate that entry of the Hom-1 vesivirus into these permissive human cell lines is mediated by the plasma membrane protein hJAM1 as a functional receptor.

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

Science.gov (United States)

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

2015-05-01

Scientific evidence suggests that dromedary camels are the intermediary host for the Middle East respiratory syndrome coronavirus (MERS-CoV). However, the actual number of infections in people who have had contact with camels is unknown and most index patients cannot recall any such contact. We aimed to do a nationwide serosurvey in Saudi Arabia to establish the prevalence of MERS-CoV antibodies, both in the general population and in populations of individuals who have maximum exposure to camels. In the cross-sectional serosurvey, we tested human serum samples obtained from healthy individuals older than 15 years who attended primary health-care centres or participated in a national burden-of-disease study in all 13 provinces of Saudi Arabia. Additionally, we tested serum samples from shepherds and abattoir workers with occupational exposure to camels. Samples were screened by recombinant ELISA and MERS-CoV seropositivity was confirmed by recombinant immunofluorescence and plaque reduction neutralisation tests. We used two-tailed Mann Whitney U exact tests, χ(2), and Fisher's exact tests to analyse the data. Between Dec 1, 2012, and Dec 1, 2013, we obtained individual serum samples from 10,009 individuals. Anti-MERS-CoV antibodies were confirmed in 15 (0·15%; 95% CI 0·09-0·24) of 10,009 people in six of the 13 provinces. The mean age of seropositive individuals was significantly younger than that of patients with reported, laboratory-confirmed, primary Middle Eastern respiratory syndrome (43·5 years [SD 17·3] vs 53·8 years [17·5]; p=0·008). Men had a higher antibody prevalence than did women (11 [0·25%] of 4341 vs two [0·05%] of 4378; p=0·028) and antibody prevalence was significantly higher in central versus coastal provinces (14 [0·26%] of 5479 vs one [0·02%] of 4529; p=0·003). Compared with the general population, seroprevalence of MERS-CoV antibodies was significantly increased by 15 times in shepherds (two [2·3%] of 87, p=0·0004) and by 23

Identification of Cis-Acting Elements on Positive-Strand Subgenomic mRNA Required for the Synthesis of Negative-Strand Counterpart in Bovine Coronavirus

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Po-Yuan Yeh

2014-07-01

Full Text Available It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(−-strand] complement. However, the cis-acting elements on the positive-strand [(+-strand] sgmRNA required for (−-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV sgmRNA 7 required for the synthesis of its (−-strand counterpart by deletion mutagenesis. The major findings are as follows. (1 Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (−-strand sgmRNA complement. (2 Deletions of the 3' untranslated region (UTR bulged stem-loop showed no effect on (−-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (−-strand sgmRNA. (3 Nucleotides positioned from −15 to −34 of the sgmRNA 7 3'-terminal region are required for efficient (−-strand sgmRNA synthesis. (4 Nucleotide species at the 3'-most position (−1 of sgmRNA 7 is correlated to the efficiency of (−-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (−-strand sgmRNA synthesis in BCoV.

Extensive Viable Middle East Respiratory Syndrome (MERS) Coronavirus Contamination in Air and Surrounding Environment in MERS Isolation Wards.

Science.gov (United States)

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.

Frequent Nek1 overexpression in human gliomas

Energy Technology Data Exchange (ETDEWEB)

Zhu, Jun [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Cai, Yu, E-mail: aihaozuqiu22@163.com [School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai (China); Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China); Liu, Pin [Med-X Research Institute, Shanghai Jiao Tong University, Shanghai (China); Zhao, Weiguo [Neurosurgery Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai (China)

2016-08-05

Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

Frequent Nek1 overexpression in human gliomas

International Nuclear Information System (INIS)

Zhu, Jun; Cai, Yu; Liu, Pin; Zhao, Weiguo

2016-01-01

Never in mitosis A (NIMA)-related kinase 1 (Nek1) regulates cell cycle progression to mitosis. Its expression and potential functions in human gliomas have not been studied. Here, our immunohistochemistry (IHC) assay and Western blot assay results showed that Nek1 expression was significantly upregulated in fresh and paraffin-embedded human glioma tissues. Its level in normal brain tissues was low. Nek1 overexpression in human gliomas was correlated with the proliferation marker (Ki-67), tumor grade, Karnofsky performance scale (KPS) and more importantly, patients’ poor survival. Further studies showed that Nek1 expression level was also increased in multiple human glioma cell lines (U251-MG, U87-MG, U118, H4 and U373). Significantly, siRNA-mediated knockdown of Nek1 inhibited glioma cell (U87-MG/U251-MG) growth. Nek1 siRNA also sensitized U87-MG/U251-MG cells to temozolomide (TMZ), causing a profound apoptosis induction and growth inhibition. The current study indicates Nek1 might be a novel and valuable oncotarget of glioma, it is important for glioma cell growth and TMZ-resistance. - Highlights: • Nek1 is upregulated in multiple human glioma tissues and cell lines. • Nek1 overexpression correlates with glioma grades and patients’ KPS score. • Nek1 overexpression correlates with patients’ poor overall survival. • siRNA knockdown of Nek1 inhibits glioma cell growth. • siRNA knockdown of Nek1 sensitizes human glioma cells to temozolomide.

Nothing stays ever the same. Stationary fuel cells, the revolution in domestic and industrial power supply; Nichts bleibt, wie es ist.... Revolution der Energieversorgung in der Haustechnik und Industrie mit stationaeren Brennstoffzellen

Energy Technology Data Exchange (ETDEWEB)

Anon.

2001-02-01

Helmut Kaiser Unternehmensberatung (HKU), an organisation that claims to be a leading consultant in the fields of environmental engineering, energy engineering, health and life science, investigated the current trends in stationary fuel cell applications world-wide. The study covers market and technology forecasts and an analysis of the competitive standing of suppliers world-wide. [German] Die Helmut Kaiser Unternehmensberatung (HKU), nach eigenen Angaben weltweit eines der fuehrenden Forschungs- und Beratungsunternehmen in der Umwelt-, Energietechnik sowie Gesundheit (Life Science), untersuchte in einer neuen Studie die Entwicklung der Brennstoffzellen in der stationaeren Anwendung weltweit. Die Studie umfasst Markt- und Technologieprognosen sowie eine Wettbewerbsanalyse der Anbieter weltweit. (orig.)

Canine adenovirus type 1 in a fennec fox (Vulpes zerda).

Science.gov (United States)

Choi, Jeong-Won; Lee, Hyun-Kyoung; Kim, Seong-Hee; Kim, Yeon-Hee; Lee, Kyoung-Ki; Lee, Myoung-Heon; Oem, Jae-Ku

2014-12-01

A 10-mo-old female fennec fox (Vulpes zerda) with drooling suddenly died and was examined postmortem. Histologic examination of different tissue samples was performed. Vacuolar degeneration and diffuse fatty change were observed in the liver. Several diagnostic methods were used to screen for canine parvovirus, canine distemper virus, canine influenza virus, canine coronavirus, canine parainfluenza virus, and canine adenovirus (CAdV). Only CAdV type 1 (CAdV-1) was detected in several organs (liver, lung, brain, kidney, spleen, and heart), and other viruses were not found. CAdV-1 was confirmed by virus isolation and nucleotide sequencing.

Bronchiolitis (For Parents)

Science.gov (United States)

... flu) , human metapneumovirus, adenovirus , coronavirus, and parainfluenza. Childcare attendance and exposure to cigarette smoke also can increase ... View more About Us Contact Us Partners Editorial Policy Permissions Guidelines Privacy Policy & Terms of Use Notice ...

Global analysis of differential expressed genes in ECV304 ...

African Journals Online (AJOL)

EB

Abstract. Background: Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through .... and (reverse: 5'-CAG CAC CAT CCT CCT CTT. CCT CT ..... acute respiratory syndrome (SARS) coronavirus.

Prevalence of Four Enteropathogens with Immunochromatographic Rapid Test in the Feces of Diarrheic Calves in East and Southeast of Turkey

Directory of Open Access Journals (Sweden)

Hasan Içen1, Neval Berrin Arserim2, Nurettin IŞIK3, Cumali Özkan4* and Abdullah Kaya4

2013-11-01

Full Text Available In this study, fecal specimens taken from 192 diarrheic and 14 healthy calves (2-40 days old were examined for the presence of bacterial and parasitic agents. Fecal samples from diarrheic calves with the four immunochromatographic rapid tests were 92.7% positive for four enteropathogens. The individual prevalence was 25, 21.8, 9.4 and 2.1% for Rotavirus, Cryptosporidium parvum, E. coli K99 and Coronavirus, respectively. Concomitant infections caused by two agents were 15.6% for Rotavirus+Cryptosporidium, 1.0% for Rotavirus+Coronavirus, 5.2 % for Cryptosporidium+E. coli K99, and 7.3% for Rotavirus+E. coli K99. Besides concomitant infections caused by three agents were 3.1% for Cryptosporidium +Rotavirus+E.coli K99 and 1.0%, Cryptosporidium+Rotavirus+Coronavirus. In addition one calf (1.0% was infected by combination of four agents as Cryptosporidium, Rotavirus, Coronavirus, and E. coli K99. The calculated individual prevalence was 56.9% for Rotavirus, 47.8% for C. parvum, 26.0% for E. coli K99 and 5.2% for Coronavirus. However, 88 samples were positive in smear detection for Cryptosporidium while 92 were positive in rapid test. As a result of this study it can be concluded that multiple etiologies of diarrhea can be seen and this can help in the development of a specific treatment and preventative measures for practitioners in east and southeast of Turkey.

A Highly Ordered Magnetic Field in a Crushed Pulsar Wind Nebula in G327.1-1.1

Science.gov (United States)

Ma, Yik Ki; Ng, Chi-Yung; Bucciantini, Niccolò; Gaensler, Bryan M.; Slane, Patrick O.; Temim, Tea

2015-01-01

A significant fraction of a pulsar's spin-down luminosity is in the form of a relativistic magnetized particle outflow known as a pulsar wind. Confinement of the wind by the ambient medium creates a synchrotron-emitting bubble called a pulsar wind nebula (PWN). Studies of PWNe is important for understanding the physics of relativistic shocks and particle acceleration. Simulations suggest that a PWN will be crushed by the reverse shock of its surrounding supernova remnant at an age of ~10^4 yr, resulting in a turbulent environment. However, given the short timescale of the interaction stage, only a few such systems are observed.We present radio polarization observations of the PWN in supernova remnant G327.1-1.1, taken with the Australia Telescope Compact Array. Previous works suggest that this system has recently interacted with the supernova reverse shock, providing a rare example for the study of magnetic field in a crushed PWN. We found a highly ordered magnetic field in the PWN, which is unexpected given the presumed turbulent interior of the nebula. This suggests that the magnetic pressure in the PWN could play an important role in the interaction with supernova reverse shock.The Australia Telescope Compact Array is part of the Australia Telescope National Facility which is funded by the Commonwealth of Australia for operation as a National Facility managed by CSIRO.YKM and CYN are supported by a ECS grant of the Hong Kong Government under HKU 709713P

OCCURRENCE OF SMALL HOMOLOGOUS AND COMPLEMENTARY FRAGMENTS IN HUMAN VIRUS GENOMES AND THEIR POSSIBLE ROLE

Directory of Open Access Journals (Sweden)

E. P. Kharchenko

2017-01-01

Full Text Available With computer analysis occurrence of small homologous and complementary fragments (21 nucleotides in length has been studied in genomes of 14 human viruses causing most dangerous infections. The sample includes viruses with (+ and (– single stranded RNA and DNA-containing hepatitis A virus. Analysis of occurrence of homologous sequences has shown the existence two extreme situations. On the one hand, the same virus contains homologous sequences to almost all other viruses (for example, Ebola virus, severe acute respiratory syndrome-related coronavirus, and mumps virus, and numerous homologous sequences to the same other virus (especially in severe acute respiratory syndrome-related coronavirus to Dengue virus and in Ebola virus to poliovirus. On the other hand, there are rare occurrence and not numerous homologous sequences in genomes of other viruses (rubella virus, hepatitis A virus, and hepatitis B virus. Similar situation exists for occurrence of complementary sequences. Rubella virus, the genome of which has the high content of guanine and cytosine, has no complementary sequences to almost all other viruses. Most viruses have moderate level of occurrence for homologous and complementary sequences. Autocomplementary sequences are numerous in most viruses and one may suggest that the genome of single stranded RNA viruses has branched secondary structure. In addition to possible role in recombination among strains autocomplementary sequences could be regulators of translation rate of virus proteins and determine its optimal proportion in virion assembly with genome and mRNA folding. Occurrence of small homologous and complementary sequences in RNA- and DNA-containing viruses may be the result of multiple recombinations in the past and the present and determine their adaptation and variability. Recombination may take place in coinfection of human and/or common hosts. Inclusion of homologous and complementary sequences into genome could not

Stability of human interferon-beta 1: oligomeric human interferon-beta 1 is inactive but is reactivated by monomerization.

Science.gov (United States)

Utsumi, J; Yamazaki, S; Kawaguchi, K; Kimura, S; Shimizu, H

1989-10-05

Human interferon-beta 1 is extremely stable is a low ionic strength solution of pH 2 such as 10 mM HCl at 37 degrees C. However, the presence of 0.15 M NaCl led to a remarkable loss of antiviral activity. The molecular-sieve high-performance liquid chromatography revealed that, whereas completely active human interferon-beta 1 eluted as a 25 kDa species (monomeric form), the inactivated preparation eluted primarily as a 90 kDa species (oligomeric form). The specific activity (units per mg protein) of the oligomeric form was approx. 10% of that of the monomeric form. This observation shows that oligomeric human interferon-beta 1 is apparently in an inactive form. When the oligomeric eluate was resolved by polyacrylamide gel containing sodium dodecyl sulphate (SDS), it appeared to be monomeric under non-reducing conditions. Monomerization of the oligomeric human interferon-beta 1 by treatment with 1% SDS, fully regenerated its antiviral activity. These results suggest that the inactivation of the human interferon-beta 1 preparation was caused by its oligomerization via hydrophobic interactions without the formation of intermolecular disulphide bonds. These oligomers can be dissociated by SDS to restore biological activity.

Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

DEFF Research Database (Denmark)

Søe, Kent; Andersen, Thomas Lykke; Hobolt-Pedersen, Anne-Sofie

2011-01-01

fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer....... This was documented through Q-PCR, Western blot and immunofluorescence analyses. These in vitro findings were confirmed by immunohistochemical stainings in human iliac crest biopsies. A syncytin-1 inhibitory peptide reduced the number of nuclei per osteoclast by 30%, as well as TRACP activity. From a mechanistic...

YY1 positively regulates human UBIAD1 expression

International Nuclear Information System (INIS)

Funahashi, Nobuaki; Hirota, Yoshihisa; Nakagawa, Kimie; Sawada, Natumi; Watanabe, Masato; Suhara, Yoshitomo; Okano, Toshio

2015-01-01

Vitamin K is involved in bone formation and blood coagulation. Natural vitamin K compounds are composed of the plant form phylloquinone (vitamin K 1 ) and a series of bacterial menaquionones (MK-n; vitamin K 2 ). Menadione (vitamin K 3 ) is an artificial vitamin K compound. MK-4 contains 4-isoprenyl as a side group in the 2-methyl-1,4-naphthoquinone common structure and has various bioactivities. UbiA prenyltransferase domain containing 1 (UBIAD1 or TERE1) is the menaquinone-4 biosynthetic enzyme. UBIAD1 transcript expression significantly decreases in patients with prostate carcinoma and overexpressing UBIAD1 inhibits proliferation of a tumour cell line. UBIAD1 mRNA expression is ubiquitous in mouse tissues, and higher UBIAD1 mRNA expression levels are detected in the brain, heart, kidneys and pancreas. Several functions of UBIAD1 have been reported; however, regulation of the human UBIAD1 gene has not been elucidated. Here we report cloning and characterisation of the human UBIAD1 promoter. A 5′ rapid amplification of cDNA ends analysis revealed that the main transcriptional start site was 306 nucleotides upstream of the translation initiation codon. Deletion and mutation analyses revealed the functional importance of the YY1 consensus motif. Electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that YY1 binds the UBIAD1 promoter in vitro and in vivo. In addition, YY1 small interfering RNA decreased endogenous UBIAD1 mRNA expression and UBIAD1 conversion activity. These results suggest that YY1 up-regulates UBIAD1 expression and UBIAD1 conversion activity through the UBIAD1 promoter. - Highlights: • We cloned the human UBIAD1 promoter. • The functional importance of the YY1 motif was identified in the UBIAD1 promoter. • YY1 binds the UBIAD1 promoter in vitro and in vivo. • Knockdown of YY1 significantly decreased UBIAD1 expression. • YY1 up-regulates UBIAD1 conversion activity through the UBIAD1 promoter

YY1 positively regulates human UBIAD1 expression

Energy Technology Data Exchange (ETDEWEB)

Funahashi, Nobuaki, E-mail: nfunahashi@ri.ncgm.go.jp [Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe (Japan); Department of Metabolic Disorder, Diabetes Research Center, Research Institute, National Center for Global Health and Medicine, Tokyo (Japan); Hirota, Yoshihisa [Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe (Japan); Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka (Japan); Nakagawa, Kimie; Sawada, Natumi; Watanabe, Masato [Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe (Japan); Suhara, Yoshitomo [Department of Bioscience and Engineering, Shibaura Institute of Technology, Saitama (Japan); Okano, Toshio [Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe (Japan)

2015-05-01

Vitamin K is involved in bone formation and blood coagulation. Natural vitamin K compounds are composed of the plant form phylloquinone (vitamin K{sub 1}) and a series of bacterial menaquionones (MK-n; vitamin K{sub 2}). Menadione (vitamin K{sub 3}) is an artificial vitamin K compound. MK-4 contains 4-isoprenyl as a side group in the 2-methyl-1,4-naphthoquinone common structure and has various bioactivities. UbiA prenyltransferase domain containing 1 (UBIAD1 or TERE1) is the menaquinone-4 biosynthetic enzyme. UBIAD1 transcript expression significantly decreases in patients with prostate carcinoma and overexpressing UBIAD1 inhibits proliferation of a tumour cell line. UBIAD1 mRNA expression is ubiquitous in mouse tissues, and higher UBIAD1 mRNA expression levels are detected in the brain, heart, kidneys and pancreas. Several functions of UBIAD1 have been reported; however, regulation of the human UBIAD1 gene has not been elucidated. Here we report cloning and characterisation of the human UBIAD1 promoter. A 5′ rapid amplification of cDNA ends analysis revealed that the main transcriptional start site was 306 nucleotides upstream of the translation initiation codon. Deletion and mutation analyses revealed the functional importance of the YY1 consensus motif. Electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that YY1 binds the UBIAD1 promoter in vitro and in vivo. In addition, YY1 small interfering RNA decreased endogenous UBIAD1 mRNA expression and UBIAD1 conversion activity. These results suggest that YY1 up-regulates UBIAD1 expression and UBIAD1 conversion activity through the UBIAD1 promoter. - Highlights: • We cloned the human UBIAD1 promoter. • The functional importance of the YY1 motif was identified in the UBIAD1 promoter. • YY1 binds the UBIAD1 promoter in vitro and in vivo. • Knockdown of YY1 significantly decreased UBIAD1 expression. • YY1 up-regulates UBIAD1 conversion activity through the UBIAD1

Detection of feline coronavirus in cerebrospinal fluid for diagnosis of feline infectious peritonitis in cats with and without neurological signs.

Science.gov (United States)

Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Fischer, Andrea; Matiasek, Lara A; Matiasek, Kaspar; Hartmann, Katrin

2016-02-01

The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) detecting feline coronavirus (FCoV) RNA in cerebrospinal fluid (CSF) of cats with and without neurological and/or ocular signs for the diagnosis of feline infectious peritonitis (FIP). This prospective case-control study included 34 cats. Nineteen cats had a definitive histopathological diagnosis of FIP (seven of these with neurological and/or ocular signs), and 15 cats had other diseases but similar clinical signs (three of these with neurological and/or ocular signs). Real-time RT-PCR was performed on the CSF of all cats, and sensitivity, specificity, and positive (PPV) and negative predictive values (NPV) were calculated. Real-time RT-PCR of CSF showed a specificity of 100% in diagnosing FIP, a sensitivity of 42.1%, a PPV of 100% and an NPV of 57.7%. The sensitivity of the real-time RT-PCR of CSF in cats with neurological and/or ocular signs was 85.7%. Although it is known that RT-PCR can give false positive results, especially if performed using serum or plasma, this real-time RT-PCR detecting FCoV RNA in CSF can be considered a reliable specific tool for the diagnosis of FIP. If only cats with neurological involvement are evaluated, the sensitivity of this real-time RT-PCR in CSF is also high. © ISFM and AAFP 2015.

Phylogenetic analysis of feline coronavirus strains in an epizootic outbreak of feline infectious peritonitis.

Science.gov (United States)

Barker, E N; Tasker, S; Gruffydd-Jones, T J; Tuplin, C K; Burton, K; Porter, E; Day, M J; Harley, R; Fews, D; Helps, C R; Siddell, S G

2013-01-01

Feline coronavirus (FCoV) infection is common. In a small percentage of cats, FCoV infection is associated with the fatal disease feline infectious peritonitis (FIP). Genetically distinct virulent and avirulent strains of FCoV might coexist within a cat population. To determine whether the strains of FCoV in FIP-affected cats are closely related or genetically distinct from the fecally derived strains of FCoV in contemporary-asymptomatic cats during an epizootic outbreak of FIP. Four cats euthanized because of FIP and 16 asymptomatic cats. This prospective outbreak investigation was initiated during an outbreak of FIP in cats within or rehomed from a rescue/rehoming center. Postmortem samples were collected from cats with FIP and contemporaneous fecal samples from asymptomatic cats. RNA was purified from tissue and fecal samples, FCoV gene fragments were reverse transcribed, PCR-amplified using novel primers, and sequenced. Sequences were aligned with ClustalW and compared with published FCoV sequences. FCoV RNA was detected in all 4 FIP cat postmortem samples and in 9 of the 16 fecal samples from contemporary-asymptomatic cats. Novel primers successfully amplified fragments from 4 regions of the genome for all FCoV-positive samples. Phylogenetic analysis showed that the FIP-associated strains of FCoV from the outbreak were very closely related to the fecally derived strains of FCoV from contemporary-asymptomatic cats. Sequence analysis provided no evidence that genetically distinct virulent and avirulent strains of FCoV were present during this FIP outbreak. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Dicty_cDB: AHA244 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available one) Pichia stipitis CBS 6054 chromos... 35 4.8 EU095850_1( EU095850 |pid:none) Turkey coronavirus isolate M...G10, c... 35 4.8 EU095850_2( EU095850 |pid:none) Turkey coronavirus isolate MG10, c... 35 4.8 CP000094_2730(

Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)

DEFF Research Database (Denmark)

Hornyák, Ákos; Bálint, Ádám; Farsang, Attila

2012-01-01

Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCo......V) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection...... assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from...

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

Science.gov (United States)

Kim, Ji Soo; Choi, Jeong Sil

2016-12-01

Emergency department (ED) nurses suffer from persistent stress after experiencing the traumatic event of exposure to Middle East respiratory syndrome coronavirus (MERS-CoV), which can subsequently lead to burnout. This study aimed to assess ED nurses' burnout level during an outbreak of MERS-CoV and to identify influencing factors in order to provide basic information for lowering and preventing the level of burnout. Study participants were ED nurses working in eight hospitals designated for treating MERS-CoV-infected patients in Korea. We performed multiple regression analysis to explore the factors influencing burnout. The ED nurses' burnout was affected by job stress (β=0.59, pburnout. ED nurses taking care of MERS-CoV-infected patients should be aware that burnout is higher for nurses in their divisions than nurses in other hospital departments and that job stress is the biggest influential factor of burnout. To be ready for the outbreak of emerging contagious diseases such as MERS-CoV, efforts and preparations should be made to reduce burnout. Job stress should be managed and resolved. Working conditions for mitigating job stress and systematic stress management programs should be provided, and hospital resources for the treatment of MERS-CoV need to be reinforced. Moreover, promoting support from family and friends is required. Copyright © 2016. Published by Elsevier B.V.

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

International Nuclear Information System (INIS)

Chou, Te-hui W.; Wang, Shixia; Sakhatskyy, Pavlo V.; Mboudoudjeck, Innocent; Lawrence, John M.; Huang Song; Coley, Scott; Yang Baoan; Li Jiaming; Zhu Qingyu; Lu Shan

2005-01-01

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV

mTORC1 directly phosphorylates and regulates human MAF1.

Science.gov (United States)

Michels, Annemieke A; Robitaille, Aaron M; Buczynski-Ruchonnet, Diane; Hodroj, Wassim; Reina, Jaime H; Hall, Michael N; Hernandez, Nouria

2010-08-01

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.

Regulation of human skeletal stem cells differentiation by Dlk1/Pref-1

DEFF Research Database (Denmark)

Abdallah, Basem M; Jensen, Charlotte H; Gutierrez, Gloria

2004-01-01

Dlk-1/Pref-1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref-1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells. INTRODUCTION......: Molecular control of human mesenchymal stem cell (hMSC) differentiation into osteoblasts and adipocytes is not known. In this study, we examined the role of delta-like 1/preadipocyte factor-1 (Dlk1/Pref-1) in regulating the differentiation of hMSCs. MATERIALS AND METHODS: As a model for hMSCs, we have...... was used to confirm the in vitro effect of Dlk/Pref-1 on bone formation. RESULTS: Dlk1/Pref-1 was found to be expressed in fetal and adult bone, hMSCs, and some osteoblastic cell lines. A retroviral vector containing the human Dlk1/Pref-1 cDNA was used to create a cell line (hMSC-dlk1) expressing high...

African Journal of Biotechnology - Vol 11, No 75 (2012)

African Journals Online (AJOL)

Characterisation of human coronavirus-NL63 nucleocapsid protein · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD FULL TEXT. Michael Berry, Taryn-Lee Manasse, Yee-Joo Tan, Burtram C. Fielding, 13962-13968 ...

Identification of Human H1N2 and Human-Swine Reassortant H1N2 and H1N1 Influenza A Viruses among Pigs in Ontario, Canada (2003 to 2005)†