Publications by authors named "Berend-Jan Bosch"

81 Publications

Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein.

Proc Natl Acad Sci U S A 2017 10 18;114(40):E8508-E8517. Epub 2017 Sep 18.

Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands;

Middle East respiratory syndrome coronavirus (MERS-CoV) targets the epithelial cells of the respiratory tract both in humans and in its natural host, the dromedary camel. Virion attachment to host cells is mediated by 20-nm-long homotrimers of spike envelope protein S. The N-terminal subunit of each S protomer, called S1, folds into four distinct domains designated S1 through S1 Binding of MERS-CoV to the cell surface entry receptor dipeptidyl peptidase 4 (DPP4) occurs via S1 We now demonstrate that in addition to DPP4, MERS-CoV binds to sialic acid (Sia). Initially demonstrated by hemagglutination assay with human erythrocytes and intact virus, MERS-CoV Sia-binding activity was assigned to S subdomain S1 When multivalently displayed on nanoparticles, S1 or S1 bound to human erythrocytes and to human mucin in a strictly Sia-dependent fashion. Glycan array analysis revealed a preference for α2,3-linked Sias over α2,6-linked Sias, which correlates with the differential distribution of α2,3-linked Sias and the predominant sites of MERS-CoV replication in the upper and lower respiratory tracts of camels and humans, respectively. Binding is hampered by Sia modifications such as 5--glycolylation and (7,)9--acetylation. Depletion of cell surface Sia by neuraminidase treatment inhibited MERS-CoV entry of Calu-3 human airway cells, thus providing direct evidence that virus-Sia interactions may aid in virion attachment. The combined observations lead us to propose that high-specificity, low-affinity attachment of MERS-CoV to sialoglycans during the preattachment or early attachment phase may form another determinant governing the host range and tissue tropism of this zoonotic pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1712592114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5635925PMC
October 2017

Cell Attachment Domains of the Porcine Epidemic Diarrhea Virus Spike Protein Are Key Targets of Neutralizing Antibodies.

J Virol 2017 06 26;91(12). Epub 2017 May 26.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs, resulting in significant economic losses to the swine industry worldwide. Current vaccination approaches against this emerging coronavirus are only partially effective, though natural infection protects pigs against reinfection and provides lactogenic immunity to suckling piglets. The viral spike (S) glycoprotein, responsible for receptor binding and cell entry, is the major target for neutralizing antibodies. However, knowledge of antibody epitopes, their nature and location in the spike structure, and the mechanisms by which the antibodies interfere with infection is scarce. Here we describe the generation and characterization of 10 neutralizing and nonneutralizing mouse monoclonal antibodies raised against the S1 receptor binding subunit of the S protein. By expression of different S1 protein fragments, six antibody epitope classes distributed over the five structural domains of the S1 subunit were identified. Characterization of antibodies for cross-reactivity and cross-neutralization revealed antigenic differences among PEDV strains. The epitopes of potent neutralizing antibodies segregated into two epitope classes and mapped within the N-terminal sialic acid binding domain and in the more C-terminal receptor binding domain. Antibody neutralization escape mutants displayed single amino acid substitutions that impaired antibody binding and neutralization and defined the locations of the epitopes. Our observations picture the antibody epitope landscape of the PEDV S1 subunit and reveal that its cell attachment domains are key targets of neutralizing antibodies. Porcine epidemic diarrhea virus (PEDV), an emerging porcine coronavirus, causes an economically important enteric disease in pigs. Effective PEDV vaccines for disease control are currently lacking. The spike (S) glycoprotein on the virion surface is the key player in virus cell entry and, therefore, the main target of neutralizing antibodies. To understand the antigenic landscape of the PEDV spike protein, we developed monoclonal antibodies against the spike protein's S1 receptor binding region and characterized their epitopes, neutralizing activity, and cross-reactivity toward multiple PEDV strains. Epitopes of antibodies segregated into six epitope classes dispersed over the multidomain S1 structure. Monoclonal antibodies revealed antigenic variability in B-cell epitopes between PEDV strains. The epitopes of neutralizing antibodies mapped to two distinct domains in S1 that are involved in binding to carbohydrate and proteinaceous cell surface molecules, respectively, indicating the importance of these cell attachment sites on the PEDV spike protein in eliciting a protective humoral immune response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00273-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446644PMC
June 2017

Aminopeptidase N is not required for porcine epidemic diarrhea virus cell entry.

Virus Res 2017 05 28;235:6-13. Epub 2017 Mar 28.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Electronic address:

Porcine epidemic diarrhea virus (PEDV) is an emerging pathogenic coronavirus that causes a significant economic burden to the swine industry. The virus infects the intestinal epithelium and causes villous atrophy, resulting in diarrhea and dehydration. Interaction of the viral spike (S) surface glycoprotein - through its S1 subunit - with the host cell receptor is the first step in infection and the main determinant for virus tropism. As for several other alphacoronaviruses including the porcine transmissible gastroenteritis virus (TGEV) and the human coronavirus 229E (HCoV-229E), the aminopeptidase N (APN) protein was reported to be a functional receptor for PEDV. In this study we examined the role of APN as a receptor. We show that overexpression of porcine APN renders MDCK cells susceptible to TGEV, but not to PEDV. Consistently, unlike TGEV-S1, PEDV-S1 exhibited no binding to cell-surface expressed APN or to a soluble version of APN. Moreover, preincubation of these viruses with soluble APN or pretreatment of APN expressing ST cells with soluble TGEV-S1 blocked TGEV infection, but had no effect on infection by PEDV. The combined observations indicated that APN is not required for PEDV infection. To definitively prove this conclusion, we applied CRISPR/Cas9 genome engineering to knock out APN expression in PEDV-susceptible porcine (ST) and human cell lines (Huh7 and HeLa). As a consequence these cells no longer bound TGEV-S1 and HCoV-229E-S1 at their surface and were resistant to infection by the corresponding viruses. However, genetic ablation of APN expression had no effect on their infectability by PEDV, demonstrating that APN is not essential for PEDV cell entry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2017.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114539PMC
May 2017

Crucial steps in the structure determination of a coronavirus spike glycoprotein using cryo-electron microscopy.

Protein Sci 2017 01 18;26(1):113-121. Epub 2016 Oct 18.

Department of Biochemistry, University of Washington, Seattle, Washington, 98195.

The tremendous pandemic potential of coronaviruses was demonstrated twice in the last 15 years by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. Despite their biomedical importance, coronavirus S glycoproteins have proven difficult targets for structural characterization, precluding high-resolution studies of the biologically relevant trimer. Recent technological developments in single particle cryo-electron microscopy allowed us to determine the first structure of a coronavirus S glycoprotein trimer which provided a framework to understand the mechanisms of viral entry and suggested potential inhibition strategies for this family of viruses. Here, we describe the key factors that enabled this breakthrough.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.3048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192993PMC
January 2017

Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy.

Nat Struct Mol Biol 2016 Oct 12;23(10):899-905. Epub 2016 Sep 12.

Department of Biochemistry, University of Washington, Seattle, Washington, USA.

The threat of a major coronavirus pandemic urges the development of strategies to combat these pathogens. Human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that can cause severe lower-respiratory-tract infections requiring hospitalization. We report here the 3.4-Å-resolution cryo-EM reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which mediates entry into host cells and is the main target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass spectrometry, provides a structural framework to understand the accessibility to antibodies. The structure reveals the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.3293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515730PMC
October 2016

Cellular entry of the porcine epidemic diarrhea virus.

Virus Res 2016 12 15;226:117-127. Epub 2016 Jun 15.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Electronic address:

Porcine epidemic diarrhea virus (PEDV), a coronavirus discovered more than 40 years ago, regained notoriety recently by its devastating outbreaks in East Asia and the Americas, causing substantial economic losses to the swine husbandry. The virus replicates extensively and almost exclusively in the epithelial cells of the small intestine resulting in villus atrophy, malabsorption and severe diarrhea. Cellular entry of this enveloped virus is mediated by the large spike (S) glycoprotein, trimers of which mediate virus attachment to the target cell and subsequent membrane fusion. The S protein has a multidomain architecture and has been reported to bind to carbohydrate (sialic acid) and proteinaceous (aminopeptidase N) cell surface molecules. PEDV propagation in vitro requires the presence of trypsin(-like) proteases in the culture medium, which capacitates the fusion function of the S protein. Here we review the current data on PEDV entry into its host cell, including therein our new observations regarding the functional role of the sialic acid binding activity of the S protein in virus infection. Moreover, we summarize the recent progress on the proteolytic activation of PEDV S proteins, and discuss factors that may determine tissue tropism of PEDV in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2016.05.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114534PMC
December 2016

Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer.

Nature 2016 Mar 8;531(7592):114-117. Epub 2016 Feb 8.

Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a mouse coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature16988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5018210PMC
March 2016

Budded baculovirus particle structure revisited.

J Invertebr Pathol 2016 Feb 29;134:15-22. Epub 2015 Dec 29.

Laboratory of Virology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands. Electronic address:

Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jip.2015.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127228PMC
February 2016

Crimean-Congo Hemorrhagic Fever Virus Subunit Vaccines Induce High Levels of Neutralizing Antibodies But No Protection in STAT1 Knockout Mice.

Vector Borne Zoonotic Dis 2015 Dec;15(12):759-64

2 Department of Microbiology and Immunology, University of Texas Medical Branch , Galveston, Texas.

Crimean-Congo hemorrhagic fever virus is a tick-borne bunyavirus of the Nairovirus genus that causes hemorrhagic fever in humans with high case fatality. Here, we report the development of subunit vaccines and their efficacy in signal transducer and activator of transcription 1 (STAT1) knockout mice. Ectodomains of the structural glycoproteins Gn and Gc were produced using a Drosophila insect cell-based expression system. A single vaccination of STAT129 mice with adjuvanted Gn or Gc ectodomains induced neutralizing antibody responses, which were boosted by a second vaccination. Despite these antibody responses, mice were not protected from a CCHFV challenge infection. These results suggest that neutralizing antibodies against CCHFV do not correlate with protection of STAT1 knockout mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/vbz.2015.1855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643766PMC
December 2015

Occupational Exposure to Dromedaries and Risk for MERS-CoV Infection, Qatar, 2013-2014.

Emerg Infect Dis 2015 Aug;21(8):1422-5

We determined the presence of neutralizing antibodies to Middle East respiratory syndrome coronavirus in persons in Qatar with and without dromedary contact. Antibodies were only detected in those with contact, suggesting dromedary exposure as a risk factor for infection. Findings also showed evidence for substantial underestimation of the infection in populations at risk in Qatar.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2108.150481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4517733PMC
August 2015

A Single Point Mutation Creating a Furin Cleavage Site in the Spike Protein Renders Porcine Epidemic Diarrhea Coronavirus Trypsin Independent for Cell Entry and Fusion.

J Virol 2015 Aug 13;89(15):8077-81. Epub 2015 May 13.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

The emerging porcine epidemic diarrhea virus (PEDV) requires trypsin supplementation to activate its S protein for membrane fusion and virus propagation in cell culture. By substitution of a single amino acid in the S protein, we created a recombinant PEDV with an artificial furin protease cleavage site N terminal of the putative fusion peptide (PEDV-SFCS). PEDV-SFCS exhibited trypsin-independent cell-cell fusion and was able to replicate in culture cells independently of trypsin, though to low titer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00356-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505673PMC
August 2015

Identification of protein receptors for coronaviruses by mass spectrometry.

Methods Mol Biol 2015 ;1282:165-82

Department of Viroscience, Erasmus Medical Center, 2040, Rotterdam, CA, 3000, The Netherlands.

As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation approach with Fc-tagged viral spike proteins followed by mass spectrometry to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-2438-7_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7121102PMC
November 2015

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

J Virol 2015 Apr 4;89(8):4434-48. Epub 2015 Feb 4.

Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Unlabelled: In addition to transporting ions, the multisubunit Na(+),K(+)-ATPase also functions by relaying cardiotonic steroid (CTS)-binding-induced signals into cells. In this study, we analyzed the role of Na(+),K(+)-ATPase and, in particular, of its ATP1A1 α subunit during coronavirus (CoV) infection. As controls, the vesicular stomatitis virus (VSV) and influenza A virus (IAV) were included. Using gene silencing, the ATP1A1 protein was shown to be critical for infection of cells with murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and VSV but not with IAV. Lack of ATP1A1 did not affect virus binding to host cells but resulted in inhibited entry of MHV and VSV. Consistently, nanomolar concentrations of the cardiotonic steroids ouabain and bufalin, which are known not to affect the transport function of Na(+),K(+)-ATPase, inhibited infection of cells with MHV, FIPV, Middle East respiratory syndrome (MERS)-CoV, and VSV, but not IAV, when the compounds were present during virus inoculation. Cardiotonic steroids were shown to inhibit entry of MHV at an early stage, resulting in accumulation of virions close to the cell surface and, as a consequence, in reduced fusion. In agreement with an early block in infection, the inhibition of VSV by CTSs could be bypassed by low-pH shock. Viral RNA replication was not affected when these compounds were added after virus entry. The antiviral effect of ouabain could be relieved by the addition of different Src kinase inhibitors, indicating that Src signaling mediated via ATP1A1 plays a crucial role in the inhibition of CoV and VSV infections.

Importance: Coronaviruses (CoVs) are important pathogens of animals and humans, as demonstrated by the recent emergence of new human CoVs of zoonotic origin. Antiviral drugs targeting CoV infections are lacking. In the present study, we show that the ATP1A1 subunit of Na(+),K(+)-ATPase, an ion transporter and signaling transducer, supports CoV infection. Targeting ATP1A1 either by gene silencing or by low concentrations of the ATP1A1-binding cardiotonic steroids ouabain and bufalin resulted in inhibition of infection with murine, feline, and MERS-CoVs at an early entry stage. Infection with the control virus VSV was also inhibited. Src signaling mediated by ATP1A1 was shown to play a crucial role in the inhibition of virus entry by ouabain and bufalin. These results suggest that targeting the Na(+),K(+)-ATPase using cardiotonic steroids, several of which are FDA-approved compounds, may be an attractive therapeutic approach against CoV and VSV infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.03274-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442369PMC
April 2015

MERS coronavirus neutralizing antibodies in camels, Eastern Africa, 1983-1997.

Emerg Infect Dis 2014 Dec;20(12):2093-5

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 camel-exporting countries, Sudan and Somalia, suggesting long-term virus circulation in these animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2012.141026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4257824PMC
December 2014

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

PLoS Pathog 2014 Nov 6;10(11):e1004502. Epub 2014 Nov 6.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1004502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4223067PMC
November 2014

Transmission of MERS-coronavirus in household contacts.

N Engl J Med 2014 Aug;371(9):828-35

From the Institute of Virology, University of Bonn Medical Center, Bonn (C.D., B.M., M.A.M., V.M.C., A.S.), and Euroimmun, Lübeck (E.L.) - both in Germany; Global Center for Mass Gatherings Medicine, Ministry of Health (M.A.-M., R.F.A., A.M. Assiri, A.I.Z., Z.A.M.), Prince Sultan Military Medical City (A.M. Albarrak), and Alfaisal University (Z.A.M.), Riyadh, Johns Hopkins Aramco Healthcare, Dhahran (J.A.A.-T.), and Regional Laboratory, Ministry of Health, Jeddah (R.H., H.M.) and Riyadh (W.H.) - all in Saudi Arabia; Indiana University School of Medicine, Indianapolis (J.A.A.-T.); the Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands (B.J.B.); and the Division of Infection and Immunity, University College London (UCL), and National Institute for Health Research Biomedical Research Centre, UCL Hospitals, London (A.I.Z.).

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 patients with MERS-CoV infection and their 280 household contacts. The median time from the onset of symptoms in index patients to the latest blood sampling in contact patients was 17.5 days (range, 5 to 216; mean, 34.4). Probable cases of secondary transmission were identified on the basis of reactivity in two reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays with independent RNA extraction from throat swabs or reactivity on enzyme-linked immunosorbent assay against MERS-CoV S1 antigen, supported by reactivity on recombinant S-protein immunofluorescence and demonstration of neutralization of more than 50% of the infectious virus seed dose on plaque-reduction neutralization testing.

Results: Among the 280 household contacts of the 26 index patients, there were 12 probable cases of secondary transmission (4%; 95% confidence interval, 2 to 7). Of these cases, 7 were identified by means of RT-PCR, all in samples obtained within 14 days after the onset of symptoms in index patients, and 5 were identified by means of serologic analysis, all in samples obtained 13 days or more after symptom onset in index patients. Probable cases of secondary transmission occurred in 6 of 26 clusters (23%). Serologic results in contacts who were sampled 13 days or more after exposure were similar to overall study results for combined RT-PCR and serologic testing.

Conclusions: The rate of secondary transmission among household contacts of patients with MERS-CoV infection has been approximately 5%. Our data provide insight into the rate of subclinical transmission of MERS-CoV in the home.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1056/NEJMoa1405858DOI Listing
August 2014

Antibodies against MERS coronavirus in dromedary camels, Kenya, 1992-2013.

Emerg Infect Dis 2014 Aug;20(8):1319-22

Dromedary camels are a putative source for human infections with Middle East respiratory syndrome coronavirus. We showed that camels sampled in different regions in Kenya during 1992-2013 have antibodies against this virus. High densities of camel populations correlated with increased seropositivity and might be a factor in predicting long-term virus maintenance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2008.140596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111164PMC
August 2014

Geographic distribution of MERS coronavirus among dromedary camels, Africa.

Emerg Infect Dis 2014 Aug;20(8):1370-4

These authors contributed equally to this article.

We found serologic evidence for the circulation of Middle East respiratory syndrome coronavirus among dromedary camels in Nigeria, Tunisia, and Ethiopia. Circulation of the virus among dromedaries across broad areas of Africa may indicate that this disease is currently underdiagnosed in humans outside the Arabian Peninsula.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2008.140590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111168PMC
August 2014

Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of β-galactosidase.

PLoS One 2014 15;9(7):e101762. Epub 2014 Jul 15.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101762PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4099126PMC
March 2015

A recombinant rabies vaccine expressing the trimeric form of the glycoprotein confers enhanced immunogenicity and protection in outbred mice.

Vaccine 2014 Aug 21;32(36):4644-50. Epub 2014 Jun 21.

Department of Viroscience, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam The Netherlands.

Rabies is a disease characterized by an invariably lethal encephalitis of viral origin that can be controlled by preventive vaccination programs of wildlife, domestic animals and humans in areas with a high risk of exposure. Currently available vaccines are expensive, cumbersome to produce and require intensive immunization and booster schemes to induce and maintain protective immunity. In the present study, we describe the development of candidate recombinant subunit rabies vaccines based on the glycoprotein G of the prototype rabies virus (RABV-G) expressed either as a monomer (RABV-mG) or in its native trimeric configuration (RABV-tG), with or without Matrix-M™ adjuvant. Immunogenicity and protective efficacy of the respective candidate vaccines were tested in outbred NIH Swiss albino mice. The RABV-tG candidate vaccine proved to be superior to the RABV-mG vaccine candidate both in terms of immunogenicity and efficacy. The relatively poor immunogenicity of the RABV-mG vaccine candidate was greatly improved by the addition of the adjuvant. A single, low dose of RABV-tG in combination with Matrix-M™ induced high levels of high avidity neutralizing antibodies and protected all mice against challenge with a lethal dose of RABV. Consequently RABV-tG used in combination with Matrix-M™ is a promising vaccine candidate that overcomes the limitations of currently used vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vaccine.2014.06.058DOI Listing
August 2014

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

Emerg Infect Dis 2014 Jun;20(6):1012-5

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 transmission. Camels may act as a direct source of human MERS-CoV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2006.140402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036761PMC
June 2014

Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

J Virol 2014 Jul 7;88(14):7952-61. Epub 2014 May 7.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands

Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00297-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097775PMC
July 2014

Membrane ectopeptidases targeted by human coronaviruses.

Curr Opin Virol 2014 Jun 22;6:55-60. Epub 2014 Apr 22.

Department of Viroscience, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands. Electronic address:

Six coronaviruses, including the recently identified Middle East respiratory syndrome coronavirus, are known to target the human respiratory tract causing mild to severe disease. Their interaction with receptors expressed on cells located in the respiratory tract is an essential first step in the infection. Thus far three membrane ectopeptidases, dipeptidyl peptidase 4 (DPP4), angiotensin-converting enzyme 2 (ACE2) and aminopeptidase N (APN), have been identified as entry receptors for four human-infecting coronaviruses. Although the catalytic activity of the ACE2, APN, and DPP4 peptidases is not required for virus entry, co-expression of other host proteases allows efficient viral entry. In addition, evolutionary conservation of these receptors may permit interspecies transmissions. Because of the physiological function of these peptidase systems, pathogenic host responses may be potentially amplified and cause acute respiratory distress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.coviro.2014.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072739PMC
June 2014

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

Emerg Infect Dis 2014 Apr;20(4):552-9

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3201/eid2004.131746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966379PMC
April 2014

Identification and characterization of a proteolytically primed form of the murine coronavirus spike proteins after fusion with the target cell.

J Virol 2014 May 19;88(9):4943-52. Epub 2014 Feb 19.

Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.

Unlabelled: Enveloped viruses carry highly specialized glycoproteins that catalyze membrane fusion under strict spatial and temporal control. To prevent premature activation after biosynthesis, viral class I fusion proteins adopt a locked conformation and require proteolytic cleavage to render them fusion-ready. This priming step may occur during virus exit from the infected cell, in the extracellular milieu or during entry at or in the next target cell. Proteolytic processing of coronavirus spike (S) fusion proteins during virus entry has been suggested but not yet formally demonstrated, while the nature and functionality of the resulting subunit is still unclear. We used a prototype coronavirus--mouse hepatitis virus (MHV)--to develop a conditional biotinylation assay that enables the specific identification and biochemical characterization of viral S proteins on virions that mediated membrane fusion with the target cell. We demonstrate that MHV S proteins are indeed cleaved upon virus endocytosis, and we identify a novel processing product S2* with characteristics of a fusion-active subunit. The precise cleavage site and the enzymes involved remain to be elucidated.

Importance: Virus entry determines the tropism and is a crucial step in the virus life cycle. We developed an approach to characterize structural components of virus particles after entering new target cells. A prototype coronavirus was used to illustrate how the virus fusion machinery can be controlled.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.03451-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993802PMC
May 2014

Adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus.

J Virol 2014 Feb 20;88(3):1834-8. Epub 2013 Nov 20.

Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands.

Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02935-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911594PMC
February 2014

Inhibition of Middle East respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody.

J Virol 2013 Dec 25;87(24):13892-9. Epub 2013 Sep 25.

Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, Hongo, Bunkyo-ku, Tokyo, Japan.

We identified the domains of CD26 involved in the binding of Middle East respiratory syndrome coronavirus (MERS-CoV) using distinct clones of anti-CD26 monoclonal antibodies (MAbs). One clone, named 2F9, almost completely inhibited viral entry. The humanized anti-CD26 MAb YS110 also significantly inhibited infection. These findings indicate that both 2F9 and YS110 are potential therapeutic agents for MERS-CoV infection. YS110, in particular, is a good candidate for immediate testing as a therapeutic modality for MERS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02448-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3838260PMC
December 2013

Spiking the MERS-coronavirus receptor.

Cell Res 2013 Sep 13;23(9):1069-70. Epub 2013 Aug 13.

Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, the Netherlands.

A novel coronavirus, the Middle East respiratory syndrome coronavirus, recently emerged through zoonotic transmission, causing a severe lower respiratory tract infection in humans. In two recent papers, one published in Cell Research, the crystal structure of the viral receptor-binding domain in complex with the host CD26/dipeptidyl peptidase 4 receptor has now been characterized.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cr.2013.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760624PMC
September 2013

Manipulation of the porcine epidemic diarrhea virus genome using targeted RNA recombination.

PLoS One 2013 2;8(8):e69997. Epub 2013 Aug 2.

Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai, PR China.

Porcine epidemic diarrhea virus (PEDV) causes severe economic losses in the swine industry in China and other Asian countries. Infection usually leads to an acute, often lethal diarrhea in piglets. Despite the impact of the disease, no system is yet available to manipulate the viral genome which has severely hampered research on this virus until today. We have established a reverse genetics system for PEDV based on targeted RNA recombination that allows the modification of the 3'-end of the viral genome, which encodes the structural proteins and the ORF3 protein. Using this system, we deleted the ORF3 gene entirely from the viral genome and showed that the ORF3 protein is not essential for replication of the virus in vitro. In addition, we inserted heterologous genes (i.e. the GFP and Renilla luciferase genes) at two positions in the viral genome, either as an extra expression cassette or as a replacement for the ORF3 gene. We demonstrated the expression of both GFP and Renilla luciferase as well as the application of these viruses by establishing a convenient and rapid virus neutralization assay. The new PEDV reverse genetics system will enable functional studies of the structural proteins and the accessory ORF3 protein and will allow the rational design and development of next generation PEDV vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069997PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732256PMC
April 2014