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Putative Receptor Binding Domain of Bat-Derived Coronavirus HKU9 Spike Protein: Evolution of Betacoronavirus Receptor Binding Motifs

  • Canping Huang
    Canping Huang
    National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing 102206, China
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  • , 
  • Jianxun Qi
    Jianxun Qi
    CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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  • , 
  • Guangwen Lu
    Guangwen Lu
    West China Hospital Emergency Department (WCHED), State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan 610041, China
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  • , 
  • Qihui Wang
    Qihui Wang
    CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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  • , 
  • Yuan Yuan
    Yuan Yuan
    School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province 230026, China
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  • , 
  • Ying Wu
    Ying Wu
    CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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  • Yanfang Zhang
    Yanfang Zhang
    CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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  • , 
  • Jinghua Yan
    Jinghua Yan
    CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
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  • , and 
  • George F. Gao*
    George F. Gao
    National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing 102206, China
    CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
    School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province 230026, China
    Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
    Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China
    *Telephone: 86-10-64807688. Fax: 86-10-64807882. E-mail: [email protected] or [email protected]
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Cite this: Biochemistry 2016, 55, 43, 5977–5988
Publication Date (Web):October 3, 2016
https://doi.org/10.1021/acs.biochem.6b00790
Copyright © 2016 American Chemical Society
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Abstract

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The suggested bat origin for Middle East respiratory syndrome coronavirus (MERS-CoV) has revitalized the studies of other bat-derived coronaviruses with respect to interspecies transmission potential. Bat coronavirus (BatCoV) HKU9 is an important betacoronavirus (betaCoV) that is phylogenetically affiliated with the same genus as MERS-CoV. The bat surveillance data indicated that BatCoV HKU9 has been widely spreading and circulating in bats. This highlights the necessity of characterizing the virus for its potential to cross species barriers. The receptor binding domain (RBD) of the coronavirus spike (S) protein recognizes host receptors to mediate virus entry and is therefore a key factor determining the viral tropism and transmission capacity. In this study, the putative S RBD of BatCoV HKU9 (HKU9-RBD), which is homologous to other betaCoV RBDs that have been structurally and functionally defined, was characterized via a series of biophysical and crystallographic methods. By using surface plasmon resonance, we demonstrated that HKU9-RBD binds to neither SARS-CoV receptor ACE2 nor MERS-CoV receptor CD26. We further determined the atomic structure of HKU9-RBD, which as expected is composed of a core and an external subdomain. The core subdomain fold resembles those of other betaCoV RBDs, whereas the external subdomain is structurally unique with a single helix, explaining the inability of HKU9-RBD to react with either ACE2 or CD26. Via comparison of the available RBD structures, we further proposed a homologous intersubdomain binding mode in betaCoV RBDs that anchors the external subdomain to the core subdomain. The revealed RBD features would shed light on the evolution route of betaCoV.

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This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

Coronaviruses are large, enveloped, and positive-stranded RNA viruses that can infect birds, animals, and humans. (1,2) Taxonomically, these viruses are affiliated with the Coronaviridae family within the Nidovirales order. (1,3) Ever since the 1930s when the first coronavirus of infectious bronchitis virus was isolated in chickens, (4) coronaviruses have expanded into four genera, Alpha-, Beta-, Gamma-, (3) and Deltacoronavirus. (5,6) Of these, betacoronaviruses (betaCoVs) have attracted attention worldwide because of their pathogenic capacity and potential to cause a global pandemic of human infections (7,8) and the widespread existence of an enormous number of species in bats. (6,9−11) In 2002 and 2003, one representative betaCoV, the severe acute respiratory syndrome coronavirus (SARS-CoV), first emerged in China (12−15) and then rapidly spread to other countries, leading to >8000 cases of infection and >800 deaths. (7) In 2012, another betaCoV, named the Middle East respiratory syndrome coronavirus (MERS-CoV), (16) was identified first in Saudi Arabia. (17,18) Despite the global efforts trying to control its transmission, MERS-CoV still spreads to affect multiple countries in the Middle East, Europe, North America, and Asia, causing 1800 confirmed infections and at least 640 deaths as of June 23rd, 2016 (based on the latest statistical data released by the World Health Organization (8)). Meanwhile, a human-infective betaCoV of HKU1 was isolated from a patient with respiratory disease in Hong Kong. (19) These unexpected outbreaks of betaCoV infection have posed a severe threat to global public health and led to enormous socioeconomic disruptions.
Phylogenetically, betaCoVs can be further categorized into four (A–D) evolutionary lineages/subgroups. (1,3) SARS-CoV is a typical lineage B member, while MERS-CoV is grouped in lineage C. (20) Despite belonging to different subgroups, these two betaCoVs likely share similar interspecies transmission routes by “jumping” from their natural host(s) to an intermediate adaptive animal(s) and finally to humans. (21) Current evidence clearly shows that SARS-CoV originated from bats (9,22,23) and possibly adapted in civets or raccoon dogs (24) before it infected humans. Given the close phylogenetic relationship between MERS-CoV and a variety of bat-derived coronaviruses (BatCoV) (e.g., HKU4, HKU5, (10,25) and those recently identified in the Middle East, Africa, Europe, and Asia (26−31)), it is widely accepted that the current MERS epidemic represents another bat-to-human transmission event related to a betaCoV, though its intermediate host is shown, this time, to be dromedaries. (32,33) Notably, two recent studies reported that BatCoV HKU4 could recognize human CD26, the MERS-CoV receptor, (34) as a functional entry receptor, (35,36) indicating its potential adaptation for human infection. These continuously occurring yet unpredictable events of betaCoVs repeatedly crossing species barriers highlight the pressing necessity of studies of other members of the genus for the characteristics relevant to interspecies transmission. (21)
The coronavirus spike (S) protein, which is located on the envelope surface of the virion, functions to mediate receptor recognition and membrane fusion (1) and is therefore a key factor determining the virus tropism for a specific species. (21,37) In most cases, coronaviral S will be further cleaved into S1 and S2 subunits, and the receptor binding capacity is allocated to the S1 subunit. (1) The receptor binding domain (RBD) of betaCoV that directly engages the receptor is commonly located in the C-terminal half of S1 [C-terminal domain (CTD)] such as in SARS-CoV, (38) MERS-CoV, (39,40) and BatCoV HKU4, (35) though in rare cases such as with mouse hepatitis virus (MHV), (41) the RBD region was identified in the S1 N-terminal domain (NTD). We previously characterized structurally the MERS-CoV RBD (MERS-RBD) as a relatively independent entity composed of a core and an external subdomain. (39) The latter subdomain, which is topologically an insertion between two scaffold strands of the core subdomain, presents a flat four-stranded β-sheet surface for contacting the CD26 receptor. (39) A similar topological arrangement of the core and external subdomains into a structural unit for receptor engagement was also observed in the SARS-CoV RBD (SARS-RBD). (38) Nevertheless, the SARS-RBD exhibits a unique loop-dominated external fold to recognize human angiotensin converting enzyme 2 (ACE2) (42) as a receptor. These observations indicate that the homologous RBD regions of betaCoVs represent a key determinant in receptor adaptation and cross-species transmission. (21)
BatCoV HKU9 is a representative betaCoV of lineage D. (11) The virus was first identified in bats in 2007 by next-generation sequencing (NGS). (11) Though the isolation of live viruses has been unsuccessful thus far, its genomes are widespread in different bat species. (43−46) As its interspecies transmission potential is worrisome, the features of its S protein, especially of the homologous RBD region (HKU9-RBD), remain unknown. This would be an indispensable step in understanding the pathogenesis of BatCoV HKU9. In addition, the atomic structure of HKU9-RBD would provide requisite information for understanding the evolution of betaCoVs. It is notable that MERS-RBD and SARS-RBD share a conserved core structure but differ in the external fold for engaging different receptors. (21,38,39) Sequence features of betaCoV RBDs clearly indicate that this scheme of subdomain arrangement might be expanded to the whole Betacoronavirus genus, regardless of the species. This notion was supported by our recent study of the BatCoV HKU4 RBD (HKU4-RBD) which exhibits a structure that quite resembles that of MERS-RBD. (35)
In this study, we reported the structural and functional characterization of HKU9-RBD. The determined structure as expected contains a core subdomain homologous to those observed in other betaCoV RBD structures and an external subdomain that is mainly α-helical. This unique structural feature explains its inability to react with either human CD26 or ACE2, which is easily observed in our surface plasmon resonance (SPR) assay. Via comparison of available RBD structures, we further showed that the detailed interactions, anchoring the external subdomain to the core subdomain, share similar patterns in betaCoV RBDs. We believe the observed core/external interacting mode represents another structural feature in the S that is reserved during the evolution of betaCoVs, in addition to the conservation in the fold for the core subdomain. Our study therefore further supports the notion that betaCoV S originates from the same ancestor and divergently evolves mainly in the RBD external region to engage variant receptors, thereby preparing for potential interspecies transmission.

Materials and Methods

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Plasmid Construction

The plasmids used for protein expression were individually constructed by insertion of the coding sequences for HKU9-RBD (S residues S355–N521, GenBank accession number EF065513), MERS-RBD (S residues E367–Y606, GenBank accession number JX869050), SARS-RBD (S residues R306–F527, GenBank accession number NC_004718), human CD26 (residues S39–P766, GenBank accession number NP_001926), and human ACE2 (residues S19–D615, GenBank accession number BAJ21180) into the EcoRI and XhoI restriction sites of a previously modified pFastBac1 vector (47) that was engineered to include an N-terminal gp67 signal peptide coding sequence. For each protein, an engineered C-terminal hexahistidine tag was utilized to facilitate protein purification. To prepare mouse IgG Fc fragment (mFc)-fused proteins, the coding sequences of MERS-RBD, SARS-RBD, and HKU9-RBD were fused with the mFc sequence and then introduced into the pCAGGS vector. (35)

Protein Expression and Purification

The proteins used for crystallization and SPR analysis were prepared with the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer’s instructions. (48) In brief, the verified pFastBac1 recombinant plasmid was transformed into the DH10Bac competent cells to generate the recombinant bacmid. The bacmid was then extracted and transfected into Sf9 cells to prepare the baculovirus stocks. Sf9 cells were further used to amplify the baculoviruses, while High5 cells were used to express the protein.
The cell culture of High5 was collected 48 h postinfection. In total, 4 L of cell culture of each protein was collected and centrifuged at 6500 rpm for 1.5 h to remove cell debris. After the samples had been filtered with a 0.22 μm membrane, the supernatant was passed through two 5 mL HisTrap HP columns (GE Healthcare) to capture the individual protein of interest. For MERS-RBD, SARS-RBD, human CD26, and human ACE2, the bound proteins were detached from HisTrap with 20, 50, and 300 mM imidazole in 20 mM Tris-HCl and 150 mM NaCl buffer (pH 8.0). After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) determination, fractions detached with 300 mM imidazole were pooled and further purified with a Superdex 200 column (GE Healthcare). For HKU9-RBD, the bound proteins were detached from HisTrap with 20, 50, and 300 mM imidazole in 20 mM HEPES and 150 mM NaCl buffer (pH 7.0). Fractions detached with 50 and 300 mM imidazole were pooled and dialyzed overnight against 5 L of 20 mM HEPES and 150 mM NaCl buffer (pH 7.0) to remove imidazole. The dialysates were concentrated and further purified with a Superdex 200 column (GE Healthcare). Each protein was stored in the buffer that was used for purification.
To prepare mFc-fused proteins with the mammalian cell expression system, the recombinant pCAGGS plasmids were confirmed with Sanger sequencing and then prepared with the EndoFree Maxi Plasmid Kit (Tiangen, Beijing, China). Each recombinant plasmid was transfected into 293T cells with 50 μg of plasmid DNA per T75 plate using polyethylimine (PEI, Polysciences Inc.). After being incubated for 5 h, the transfected cells were washed with PBS twice and then replaced with DMEM without serum. The cells were maintained for 3 days, and the supernatant was harvested and replaced with fresh DMEM medium and then maintained for an additional 4 days. The harvested supernatants were pooled and concentrated and then mixed with 2 volumes of 20 mM trisodium phosphate (pH 7.0). The mixture was passed through a 5 mL HiTrap Protein A HP prepacked column (GE Healthcare) to capture the individual protein of interest. After removal of impure proteins with 20 mM trisodium phosphate (pH 7.0), the bound protein was detached from the column with 100 mM glycine (pH 3.0). Each fraction was neutralized with 1 M Tris-HCl (pH 9.0). After SDS–PAGE determination, the detached fractions with the protein of interest were pooled and concentrated. The buffer of each protein was then changed to PBS (pH 7.0) for further experiments.

SPR Assay

The BIAcore experiments were performed at 25 °C using a BIAcore 3000 or BIAcore T100 machine with CM5 chips (GE Healthcare). For all the measurements, an HBS-EP buffer consisting of 10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% (v/v) Tween 20 was used, and all proteins were exchanged into this buffer in advance. First, the HKU9-RBD, MERS-RBD, and SARS-RBD proteins expressed in insect cells were used for the SPR assay using a BIAcore 3000 machine. BSA (negative control), HKU9-RBD, MERS-RBD, and SARS-RBD proteins were immobilized on the chip at ∼1000 response units (RU), according to the manufacturer’s amine coupling chemistry protocol (GE Healthcare). Gradient concentrations of human CD26 (0, 19.5 to 5000 nM) or human ACE2 (0, 39 to 625 nM) were then passed over the chip surface. After each cycle, the sensor surface was regenerated via a short treatment with 10 mM NaOH. The equilibrium dissociation constants (binding affinity and KD values) were analyzed using BIA evaluation (BIAcore software). To exclude the possibility that HKU9-RBD could be nonfunctional because of immobilization or could be missing some important post-translational modifications on the protein, we purified the mFc-fused HKU9-RBD proteins in mammalian cells and assessed the abilities to bind CD26 or ACE2 proteins using a captured SPR method by a BIAcore T100 system. The CM5 chip was immobilized with the anti-mouse antibody for flow cells 1 and 2 (FC1 and FC2, respectively). The mFc-fused RBD proteins were then injected and captured on FC2, while FC1 was used as a negative control. Human CD26 or human ACE2 proteins were then injected, and the binding responses were measured. The immobilized anti-mouse antibody was regenerated with 10 mM glycine (pH 1.7) (GE Healthcare).

Crystallization

The crystallization trials were performed with 1 μL of protein being mixed with 1 μL of the reservoir solution and then equilibrating against 100 μL of the reservoir solution at 4 °C by the vapor-diffusion sitting-drop method. The initial crystallization was screened using the commercially available kits. Diffractive crystals of the HKU9-RBD protein were finally obtained under 0.1 M sodium citrate tribasic dihydrate (pH 7.0) and 12% PEG 20000 with a protein concentration of 2.2 mg/mL. Derivative crystals were obtained by soaking the crystals in the reservoir solution containing 1 mM KAuBr4·2H2O for 48 h at 4 °C.

Data Collection, Integration, and Structure Determination

For data collection, all crystals were flash-cooled in liquid nitrogen after a brief soaking in the reservoir solution with the addition of 20% (v/v) glycerol. The diffraction data for the native (wavelength of 1.03906 Å) and Au derivative crystals (wavelength of 1.03906 Å) of HKU9-RBD were collected at Shanghai Synchrotron Radiation Facility (SSRF) BL17U. All data were processed with HKL2000. (49) The ice rings that form in the crystal flash cooling process were excluded from data processing, and the final overall completeness for the data set is 97.1%.
The structure of HKU9-RBD was determined by the SAD method. After location of Au sites by SHELXD (50) with the Au-SAD data, the identified positions were then refined and the phases were calculated with the SAD experimental phasing module of PHASER. (51) The real space constraints were further applied to the electron density map in DM. (52) The initial model was built with Autobuild in the PHENIX package. (53) Additional missing residues were added manually in COOT. (54) The final model was refined with phenix.refine in PHENIX (53) with energy minimization, isotropic ADP refinement, and bulk solvent modeling. The stereochemical qualities of the final model were assessed with MolProbity. (55) The Ramachandran plot distributions for the residues in the HKU9-RBD structure were 94.64, 5.36, and 0% for favored, allowed, and outlier regions, respectively. Data collection and refinement statistics are summarized in Table 1. All structural figures were generated using PyMol (http://www.pymol.org).
Table 1. Data Collection and Refinement Statistics
  HKU9-RBD (Protein Data Bank entry 5GYQ) Au derivative HKU9-RBD
Data Collection
space group P21 P1
wavelength (Å) 1.03906 1.03906
unit cell dimensions    
a, b, c (Å) 42.7, 36.0, 62.9 36.0, 46.6, 57.3
α, β, γ (deg) 90.0, 102.7, 90.0 80.4, 88.8, 88.5
resolutiona (Å) 50.00–2.10 (2.18–2.10) 50.00–2.48 (2.57–2.48)
no. of observed reflections 101588 52401
completeness (%) 97.1 (80.7) 97.7 (96.9)
redundancy 9.4 (9.4) 4.1 (3.7)
Rmergeb (%) 6.1 (15.4) 9.2 (39.0)
I/σI 34.023 (12.057) 16.960 (4.637)
CC1/2 0.998 (0.988) 0.986 (0.915)
Refinement
resolution (Å) 41.7–2.10  
no. of reflections 10811  
completeness for range (%) 97.0  
Rwork/Rfreec 0.1700/0.2006  
no. of atoms    
protein 1367  
water 128  
B factor (Å2)    
protein 28.7  
water 34.1  
root-mean-square deviation    
bond lengths (Å) 0.003  
bond angles (deg) 0.820  
Ramachandran plotd (%)    
favored 94.64  
allowed 5.36  
outliers 0.00  
a

Values for the outermost resolution shell are given in parentheses.

b

Rmerge = ∑i∑hkl|Ii – ⟨I⟩|/∑i∑hklIi, where Ii is the observed intensity and ⟨I⟩ is the average intensity from multiple measurements.

c

Rwork = ∑||Fo| – |Fc||/∑|Fo|, where Fo and Fc are the structure factor amplitudes from the data and the model, respectively. Rfree is the R factor for a subset (5%) of reflections that were selected prior to refinement calculations and were not included in the refinement.

d

Ramachandran plots were generated by using MolProbity.

Results

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HKU9-RBD Does Not Bind the SARS-CoV or MERS-CoV Receptor

We first characterized the sequence of BatCoV HKU9 S by using a series of bioinformatic methods. This 1274-residue protein exhibits typical features of coronavirus S proteins (e.g., the presence of characteristic heptad repeats 1 and 2 in the S2 subunit), though the S1/S2 cleavage site potentially processed by furin-like proteases was not detected (Figure 1A). Along the full-length protein, the amino acid sequence identity between BatCoV HKU9 S and other betaCoVSs is rather limited (e.g., 27.9% identical to MERS-CoV S, 28.0% identical to HKU4 S, and 30.4% identical to SARS-CoV S). Nevertheless, we were able to identify the RBD region based on the characteristic cysteine residues of the core subdomain (Figure 1B), which were shown, in the thus-far available RBD structures, (35,38,39,56) to form three conserved disulfide bonds stabilizing the core fold. The subsequent HKU9-RBD was allocated to the S region spanning residues 355–521 (Figure 1A). In comparison to other RBD sequences, the HKU9-RBD exhibits a comparable length in the core subdomain (Figure 1B) but is dramatically shortened in the external region (Figure 1C).

Figure 1

Figure 1. Sequence features of HKU9-RBD. (A) Schematic representation of BatCoV HKU9 S. The indicated domain elements were defined on the basis of either the pairwise sequence alignment results or the bioinformatics predictions. The signal peptide (SP), transmembrane domain (TM), and heptad repeats 1 and 2 (HR1 and HR2, respectively) were predicted with the SignalP 4.0 server, TMHMM server, and Learncoil-VMF program, respectively, while the N-terminal domain (NTD) and RBD were deduced by alignment with the N-terminal galectin-like domain of murine hepatitis virus S and MERS-RBD, respectively. The S1/S2 site potentially cleaved by furin-like proteases could not be ascertained and is therefore labeled with a question mark. (B and C) Structure-based alignment of the HKU9-, SARS-, MERS-, and HKU4-RBD sequences. The arrows and spiral lines indicate strands and helices, respectively. These secondary structure elements were labeled as illustrated in Figure 3. The conserved cysteine residues that form three disulfide bonds in the structures are marked with Arabic numerals 1–3. The core subdomain is conserved among the four RBD structures, but the external subdomain is structurally irrelevant. We therefore present the sequences separately. The two elements that anchor the external subdomain to the core subdomain are highlighted with black boxes. (B) Core subdomain sequence. (C) External subdomain sequence.

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To test if HKU9-RBD could react with either SARS-CoV receptor ACE2 (42) or MERS-CoV receptor CD26, (34) the RBD and the receptor–ectodomain proteins were individually prepared in insect cells and purified to homogeneity. The ligand–receptor interaction was then characterized via SPR BIAcore by passing ACE2 or CD26 over the immobilized RBD proteins. As expected, potent interactions were observed for both the SARS-RBD–ACE2 (KD = 0.265 μM) (Figure 2A) and MERS-RBD–CD26 (KD = 52.8 nM) (Figure 2B) binding pairs. The revealed kinetics were very similar to those reported previously, (35,39) validating the integrity of our testing system. Under the same condition, however, neither ACE2 (Figure 2C) nor CD26 (Figure 2D) interacted with HKU9-RBD. To exclude the possibility that HKU9-RBD could be nonfunctional because of immobilization or because of the absence of some important post-translational modifications on the protein, we purified the mFc-fused HKU9-RBD proteins in mammalian (293T) cells and assessed the abilities to bind CD26 or ACE2 proteins using a captured SPR method. In the same way, there was no detectable binding of mFc-fused HKU9-RBD to ACE2 or CD26 (Figure 2G), while the mFc-fused SARS-RBD protein bound to ACE2 (Figure 2E) and the mFc-fused MERS-RBD bound to CD26 (Figure 2F) well. BatCoV HKU9, therefore, could utilize neither the SARS-CoV receptor nor the MERS-CoV receptor for cell entry. Rather, it must utilize a unique cellular receptor for entry.

Figure 2

Figure 2. Characterization of HKU9-RBD by SPR assays. The indicated RBD proteins expressed by insect cells were immobilized on CM5 chips and tested for the binding with gradient concentrations of human ACE2 or CD26 using a BIAcore 3000 machine. The recorded kinetic profiles are shown: (A) human ACE2 and SARS-RBD, (B) human CD26 and MERS-RBD, (C) human ACE2 and HKU9-RBD, and (D) human CD26 and HKU9-RBD. Clearly shown is the fact that HKU9-RBD does not bind either ACE2 or CD26, in the context of which SARS-RBD and MERS-RBD bind their respective receptors. Then we purified the mFc-fused HKU9-RBD proteins in mammalian (293T) cells and assembled the abilities to bind CD26 or ACE2 proteins using a captured SPR method by a BIAcore T100 system. The anti-mouse antibodies were immobilized on CM5 chips. The mFc-fused RBD proteins were then captured (3 μg/mL for 60 s) by the antibodies and tested for binding to human ACE2 or CD26. (E) The mFc-fused SARS-RBD (SARS-RBD-mFc) did not bind to CD26 but bound to ACE2 well. (F) The mFc-fused MERS-RBD (MERS-RBD-mFc) did not bind to ACE2 but bound to CD26 well. (G) The mFc-fused HKU9-RBD (HKU9-RBD-mFc) does not bind either ACE2 or CD26.

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Crystal Structure of HKU9-RBD

We further set out to investigate the structural features of HKU9-RBD via crystallography. The protein was successfully crystallized; a 2.1 Å data set was collected (Table 1), and the structure was determined by using the single-wavelength anomalous diffraction (SAD) method. The determined structure, with an Rwork of 0.1700 and an Rfree of 0.2006, contains a single molecule in the crystallographic asymmetric unit. Clear electron densities were traced for 176 consecutive HKU9-RBD residues, extending from S355 to A520. These amino acids fold into a compact structure, which can be further divided into two subdomains as shown schematically in the other RBD structures. (35,38,39) The core subdomain comprises eight β-strands and six helices (α or 310). Five long strands (βc1−βc5) are arranged in an antiparallel manner, forming the scaffold center of the core (core-center). This core-center sheet is further wrapped by the surface helices and loops. It is notable that the six helices (H1–H6) are sporadically distributed on the two sheet faces, thereby leading to an overall globular fold for the core subdomain. On one lateral side of the core-center sheet, the external subdomain covers the core like a hat, while on the distal opposite side, three small strands (βp1−βp3) constitute a parallel peripheral sheet (core-peripheral), ensuring that the N- and C-termini of HKU9-RBD are in the proximity. As expected, the characteristic cysteine residues (Figure 1B) form three disulfide bonds in the core subdomain, further stabilizing the core structure from the interior. Of these, two (C357–C381 and C411–C517) are located in core-peripheral, contributing to the orientation of the RBD termini; one (C399/C452) resides in the core-center, linking strands βc2 and βc4 (Figure 3). Overall, the residue boundaries of the core subdomain observed in the structure are quite consistent with those deduced from the results of sequence alignment (Figure 1B).

Figure 3

Figure 3. Crystal structure of HKU9-RBD. The core and external subdomains are colored magenta and green, respectively. The core subdomain is further divided into a center region (core-center) and a peripheral region (core-peripheral), which are encircled. The core-center strands and helices are labeled βc1−βc5 and H1–H6, respectively, while the core-peripheral strands are marked βp1−βp3. The disulfide bonds and the RBD termini are labeled. The core subdomain is further presented in a surface representation in the right panel to highlight the top positioning of the external subdomain like a hat.

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The external subdomain of HKU9-RBD consists of 42 residues from L458 to V499 (Figure 1C). These amino acids extend out of strand βc4 of the core-center sheet, first orient as a loop along the core subdomain like a clamp, then fold back to form a solvent-exposed α-helix (H1′), and finally proceed into core strand βc5 (Figure 3). This observed structure differs dramatically from those of SARS-RBD and MERS-RBD, which are shown to be devoid of any helical components in the external region. (38,39) The unique external fold of HKU9-RBD could well explain its inability to bind either ACE2 or CD26.

Structural Conservation of the RBD Core Subdomain in BetaCoVs

Previously, three betaCoV RBD structures have been reported, including one lineage B structure (SARS-RBD (38)) and two lineage C structures (MERS-RBD (39) and HKU4-RBD (35)). These structures indicated an interspecies conservation in the core fold among betaCoVs. (39) BatCoV HKU9 is a representative member of betaCoV lineage D. (11) We therefore compared the currently available RBD structures with the HKU9-RBD structure determined in this study. As expected, a significant similarity was observed in the core subdomain (Figure 4A–D). Superimposition of the core structures revealed the root-mean-square deviation (rmsd) values ranging from 0.66 to 2.82 Å (Table 2), demonstrating the quite similar core folds (though with a low level of sequence identity) among the four RBDs. The most conserved part was seen in the core-center sheet. This five-stranded scaffold element as well as the single interstrand disulfide bond is invariably reserved in all the structures. In the core-peripheral region, however, a small variance in strand composition is noted. In HKU9-RBD, it contains three short β-strands, arranged in a small parallel β-sheet. Both SARS-RBD and MERS-RBD retain two of these strands, whereas HKU4-RBD is devoid of any detectable strand elements in this region. Despite the observed difference in strand formula, the core-peripherals of the RBDs exhibit a similar orientation and present the same scheme in which the domain N-terminus is in the proximity of its C-terminus. Extra common features in core-peripheral strands lie in the two disulfide bonds in the region, which are structurally and topologically conserved in the four structures (Figure 4A–D).

Figure 4

Figure 4. Structural and topological comparison of available betaCoV RBD structures. Four structures, including those of HKU9-, SARS-, MERS-, and HKU4-RBD, were oriented similarly and are presented as cartoons in parallel. The core-center, core-peripheral, and the external subdomain are encircled and highlighted in yellow. For each structure, the topological arrangement of the core-center and core-peripheral strands as well as of the external components is depicted. The core strands that flank the external subdomain are colored red and blue, respectively. Yellow lines indicate disulfide bonds. The N- and C-termini are highlighted: (A) HKU9-RBD, (B) SARS-RBD, (C) MERS-RBD, and (D) HKU4-RBD. The similarity in the topological arrangement of the external subdomain as an insertion between two core strands is illustrated.

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Table 2. Statistics of the Core Subdomain Deviations among Available BetaCoV RBD Structuresa
  HKU9-RBD MERS-RBD SARS-RBD HKU4-RBD
HKU9-RBD – 2.07 Å (104 Cα atoms) 1.92 Å (100 Cα atoms) 1.37 Å (94 Cα atoms)
MERS-RBD   – 2.82 Å (75 Cα atoms) 0.66 Å (109 Cα atoms)
SARS-RBD     – 1.95 Å (82 Cα atoms)
HKU4-RBD       –
a

The RBD core subdomain structures were superimposed onto each other by PyMol in a pairwise manner to calculate the rmsd values, which are listed in the table. The values in parentheses indicate the number of equivalent Cα atoms that were selected for rmsd calculations.

In contrast to the core conservation, the external subdomains of the four RBDs are divergent in structures. HKU9-RBD presents a single H1′ helix in the external region, whereas SARS-RBD is loop-dominated but contains two extra small β-strands. The external subdomains of MERS-RBD and HKU4-RBD, however, resemble each other and are predominantly a rigid β-sheet composed of four β-strands. Despite the structural irrelevance, the external subdomains are clearly topological equivalents in these structures, being present as an insertion between two core-center strands (Figure 4A–D).

Homologous Interaction Mode Anchoring the External Subdomain to the Core Subdomain

By superimposing the available RBD structures, we unexpectedly identified two major elements in the external subdomain that could be well-aligned (Figure 5A). The first element (element 1) spans approximately seven residues (Y464–F470 in HKU9-RBD, Y438–R444 in SARS-RBD, Y497–C503 in MERS-RBD, and Y501–C507 in HKU4-RBD) (Figures 1C and 5C) and proceeds along the core subdomain surface to be lodged between helices H2 and H6 (based on the secondary element definition of HKU9-RBD) (Figure 5A). The second element (element 2) contains eight amino acids (P471–Q478 in HKU9-RBD, K447–D454 in SARS-RBD, L517–S524 in MERS-RBD, and Y522–S529 in HKU4-RBD) (Figures 1C and 5C), extending as a curved loop covering helix H6 of the core subdomain (Figure 5A). It is interesting that these two elements are “saddled” upon the core helices, anchoring the external subdomain to the core subdomain (Figure 5A). We therefore further explored the amino acid interaction details at this core–external interface.

Figure 5

Figure 5. Homologous intersubdomain amino acid interactions anchoring the external subdomain to the core subdomain. (A) Superimposition of the betaCoV RBD (HKU9-RBD in green, SARS-RBD in yellow, MERS-RBD in blue, and HKU4-RBD in cyan) structures highlighting the external elements that can be well-aligned. These two elements, with seven (element 1) and eight (element 2) amino acids, respectively, engage mainly core subdomain helices H2 and H6 for the intersubdomain interactions. To facilitate comparison, the element residues were successively assigned a position marker (a–g for element 1 and a–h for element 2), which is highlighted. (B) Characterization of the element residues for their contributions to the intersubdomain binding. The two external elements are presented as cartoons, while the core subdomain is shown at the surface. At each position, the residue is marked sequentially with the position marker, the amino acid identity and numbering, the interacting mode/type, and the side-chain orientation. For the interaction mode, the hydrophobic or van der Waals interactions are indicated with encircled Ps, the side-chain H-bonds with encircled Ss, and main-chain H-bonds with encircled Ms. The side-chain orientations are indicated with arrows. (C) Summary of the intersubdomain interactions specified in panel B. The element sequences of the four RBDs are aligned and listed. A + indicates that a certain type of interaction is commonly observed at the position, while a +/– indicates that the interaction type is specific to some but not all of the four RBDs. The arrows mark the side-chain orientations.

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Each element residue was scrutinized for both the side-chain orientation and the intersubdomain interactions. To facilitate the analyses and comparison, the two elements were assigned a position marker for each amino acid (a–g for element 1 and a–h for element 2) (Figure 5B,C). In element 1, residue a is invariably a tyrosine in the four RBDs. This amino acid orients its bulky side chain toward the core subdomain, providing strong hydrophobic contacts. An extra side-chain H-bond is also observed at this position in HKU9-RBD. Residue b extended away from the core surface and exhibited little conservation. The residue, however, invariably contributes to the subdomain anchoring by providing a main-chain H-bond. Following residue b, the amino acids are preferably facing toward the core at position c and spreading parallel to the core surface at position d. Multiple van der Waals (vdw) contacts and conserved main-chain H-bonds are observed at these two positions, respectively. A small discrepancy is seen in SARS-RBD, which orients its c residue outward for the bulky solvent region. The remaining three element 1 amino acids at positions e–g are distant from the core subdomain and completely solvent-exposed, therefore contributing little to the core–external interactions (Figure 5B,C).
In element 2, both residues a and d are oriented parallel to the surface of the core subdomain. The configuration allows the amino acids to provide apolar vdw contacts to strengthen core–external subdomain binding. A certain extent of amino acid conservation was observed at position d where a proline is favored to facilitate the turning of the loop. Following these two positions, residues b and e insert their side chains into two surface pockets of the core subdomain. At position b, the residue is conservatively hydrophobic and has a middle-sized side chain (Val/Ile/Leu). It is accommodated in a shallow apolar pocket, creating strong stacking forces bonding the core and external subdomains. In addition, the residue also contributes a main-chain H-bond to the subdomain binding. For residue e, its accommodating pocket is deep and large, therefore allowing for amino acid variance at the position (Gly in HKU9- and HKU4-RBD, Phe in SARS-RBD, and Asn in MERS-RBD). In the four RBDs, this residue e invariably forms H-bonds with the core subdomain residue via the main-chain atom but may also provide side-chain H-bond interactions (e.g., in MERS-RBD) or multiple vdw contacts (e.g., in SARS-RBD). Extra core–external interactions in this region were further observed at position g, where the residue is oriented parallel to or toward the core subdomain and thereby contributes to the binding via hydrophobic and side-chain H-bond interactions. It is also of interest that these important interface residues of element 2 are regularly interspersed by amino acids at positions c, f, and h, which are solvent-exposed and rarely interact with the core subdomain (Figure 5B,C).
In summary, the four coronaviral RBD structures show homologous amino acid interaction patterns for intersubdomain binding. The binding relies mainly on two elements in the external subdomain, which are oriented similarly in these structures (Figure 5A). Despite the lower level of conservation in the element sequences, the side-chain orientation and the interaction modes (hydrophobic, vdw, or H-bond contacts) at each position are, in most cases, similar or homologous (Figure 5C).

Discussion

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Bats have been found to harbor the largest natural genetic pools for new coronarivuses or coronaviral genes. The origin of a majority of the betaCoVs could be traced back to bats; (57) e.g., a recent study isolated, in Chinese horseshoe bats, a live SARS-like coronavirus that can utilize the SARS-CoV receptor of ACE2 for cell entry, (22) thereby providing the strongest evidence of the bat origin of this pandemic human pathogen. In addition, two studies reported the identification of gene fragments in bats that are almost identical to those of MERS-CoV, (26,29) indicating that MERS-CoV likely also originates from bats. Noting the recent reports showing the adaptation of batCoV HKU4 for binding to human cells by recognizing CD26, (35) we believe that preparing for the unforeseeable events of potential interspecies transmission by other bat-derived betaCoVs is an urgent need. BatCoV HKU9 is an important lineage D betaCoV (11) and has been demonstrated to be widespread and circulating in different bat species. (43−46) Noting the determinative role of the coronaviral S RBD in the process of crossing species barriers (as has been structurally illustrated in other coronaviruses (35,38,39)), we characterized the structural and functional features of the homologous RBD protein of batCoV HKU9 S. The determined structure revealed a core subdomain that resembles those observed in SARS-, MERS-, and HKU4-RBD but a unique external subdomain that is composed of a single helix. Because the RBD external subdomain contains the key motifs [denoted the receptor binding motif (RBM)], interfacing with the receptor, (35,38,39) the unique external fold of HKU9-RBD therefore is in accord with our functional data showing its inability to react with either ACE2 or CD26. Which host molecule could be recognized by HKU9-RBD as a functional cell entry receptor remains to be investigated. Nevertheless, taking into account the single helical component in the external subdomain of HKU9-RBD, we expect the RBM to be located on the solvent-access side of the helix, which might facilitate future attempts to identify the receptor.
It should be noted that coronavirus RBDs are not necessarily located in the C-terminal half of the S1 subunit. Previous structural and mutagenesis data showed that the RBD of MHV S is located in the S1 N-terminal half (NTD). (41,58) Nevertheless, the current data seem to favor the notion that the CTD is prioritized over the NTD to function as the receptor binding entity, as the majority of the coronaviruses (e.g., SARS-CoV, (38) MERS-CoV, (39) batCoV HKU4, (35) human coronavirus NL63, (59) transmissible gastroenteritis virus, (60) etc.) harbor a CTD as the RBD. It is interesting that the current available betaCoV CTD/RBD structures (35,38,39) all keep the N- and C-termini on the same side opposite from the location of the external subdomain. This arrangement mode would lead the S1 N-terminal half to being sterically underneath the C-terminal half, thereby projecting the CTD distant from the viral envelope for a trans-interaction with the receptors. Our structural study demonstrated that HKU9-RBD retains the same character and therefore stands a better chance of being the authentic receptor binding entity.
In comparison to SARS-, MERS-, and HKU4-RBD whose structures are available, (35,38,39) HKU9-RBD differs in the external fold but reserves a resembled core subdomain structure. The most conserved part lies in the core-center sheet that is composed of five antiparallel strands and functions as the scaffold of the core subdomain. The sheet is sterically and structurally conserved in all the RBD structures. Additional conserved elements include the core-center helices and core-peripheral structures. Nevertheless, these elements could vary in their secondary element compositions. For example, a recent study of the structure of HKU4-RBD (35) showed that its N-terminal-most part does not fold into a characteristic helix as observed in the structures of SARS-RBD (38) and MERS-RBD. (39) For core-peripheral, the number of strands was found to vary from zero (as in HKU4-RBD) to three (as in HKU9-RBD) (Figure 4). This has added dramatic complexities to the nomenclature of the RBD secondary elements. The situation would be even worse were the external subdomains that could vary significantly in structure taken into account. We therefore suggest that the core-center strands and helices be designated as βcs (βc1−βc5) and Hs (H1, H2, etc.), respectively, and that the core-peripheral strands be designated as βps (βp1, βp2, etc.) and the external elements as H′s (H1′, H2′, etc.) or β′s (β1′, β2′, etc.). This terminological strategy should be able to facilitate the comparison of homologous RBD structures and to reflect the fact that the external subdomain is topologically an insertion between two equivalent core-center strands.
The long evolutionary history, high mutation rates, and many genetic artifices of RNA viruses often lead to conundrums in the study of the origin of viruses. It would be even more difficult to track the evolutionary traces in the viral surface proteins that are normally under great evolutionary pressure. The evolutionary records, however, are more likely to be conserved in the tertiary structures than in the amino acid sequences. In betaCoVs, the interlineage sequence identity in the S RBD is rather limited. Nevertheless, we observed several conserved features in the betaCoV RBD structures. These include (1) a conserved core-center as the scaffold of the core subdomain, (2) a similar core-peripheral where the RBD termini are clinched in the proximity, (3) a similar topological arrangement of the external subdomain as an insertion between two core strands, and (4) a homologous intersubdomain binding mode anchoring the external subdomain to the core subdomain. The features indicate a common ancestor S protein that divergently evolves into different species. During evolution, the core subdomain is structurally reserved, whereas the external subdomain folds into variant structures to engage different receptors. It is also noteworthy that the aforementioned core features have been structurally validated in betaCoV lineages B (SARS-RBD), C (MERS- and HKU4-RBD), and D (HKU9-RBD) but not yet in lineage A. Structural studies of the equivalent S RBDs of the lineage A members should be conducted in the future.

Author Information

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  • Corresponding Author
    • George F. Gao - National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing 102206, China;  CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China;  School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province 230026, China;  Laboratory of Protein Engineering and Vaccines, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China;  Research Network of Immunity and Health (RNIH), Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing 100101, China;  Email: [email protected] [email protected]
  • Authors
    • Canping Huang - National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC), Beijing 102206, China
    • Jianxun Qi - CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
    • Guangwen Lu - West China Hospital Emergency Department (WCHED), State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan 610041, China
    • Qihui Wang - CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
    • Yuan Yuan - School of Life Sciences, University of Science and Technology of China, Hefei, Anhui Province 230026, China
    • Ying Wu - CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
    • Yanfang Zhang - CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
    • Jinghua Yan - CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
  • Funding

    This work was supported by the National Natural Science Foundation of China (NSFC, Grants 81461168030 and 81290342), the China National Grand S&T Special Project (Grant 2014ZX10004-001-006), and the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDB08020100). G.L. is supported in part by NSFC (Grants 31400154, 81522026, and 31570157) and the Sichuan Outstanding Youth Science & Technology Funding (Grant 2016JQ0001). G.F.G. is a leading principle investigator of the NSFC Innovative Research Group (Grant 81321063).

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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Assistance in diffraction data collection by the staff at the Shanghai Synchrotron Radiation Facility (SSRF beamline 17U) is acknowledged. We thank Dr. Zheng Fan (Institute of Microbiology, Chinese Academy of Sciences) and Yuanyuan Chen and Zhenwei Yang (Institute of Biophysics, Chinese Academy of Sciences) for their sophisticated technical support in the SPR assay.

References

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    The existence of coronaviruses in bats was unknown until the recent discovery of bat-SARS-CoV in Chinese horseshoe bats and a novel group 1 coronavirus in other bat species. Among 309 bats of 13 species captured from 20 different locations in rural areas of Hong Kong over a 16-mo period, coronaviruses were amplified from anal swabs of 37 (12%) bats by RT-PCR. Phylogenetic anal. of RNA-dependent RNA polymerase (pol) and helicase genes revealed six novel coronaviruses from six different bat species, in addn. to the two previously described coronaviruses. Among the six novel coronaviruses, four were group 1 coronaviruses (bat-CoV HKU2 from Chinese horseshoe bat Rhinolophus sinicus, bat-CoV HKU6 from Rickett's big-footed bat Myotis ricketti, bat-CoV HKU7 from greater bent-winged bat Miniopterus magnater and bat-CoV HKU8 from lesser bent-winged bat Miniopterus pusillus) and two were group 2 coronaviruses (bat-CoV HKU4 from lesser bamboo bat Tylonycteris pachypus and bat-CoV HKU5 from Japanese pipistrelle Pipistrellus abramus). An astonishing diversity of coronaviruses was obsd. in bats.
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  11. 11
    Woo, P. C., Wang, M., Lau, S. K., Xu, H., Poon, R. W., Guo, R., Wong, B. H., Gao, K., Tsoi, H. W., Huang, Y., Li, K. S., Lam, C. S., Chan, K. H., Zheng, B. J., and Yuen, K. Y. (2007) Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features. J. Virol. 81, 1574– 1585,  DOI: 10.1128/JVI.02182-06
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    11
    Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features
    Woo, Patrick C. Y.; Wang, Ming; Lau, Susanna K. P.; Xu, Huifang; Poon, Rosana W. S.; Guo, Rongtong; Wong, Beatrice H. L.; Gao, Kai; Tsoi, Hoi-wah; Huang, Yi; Li, Kenneth S. M.; Lam, Carol S. F.; Chan, Kwok-hung; Zheng, Bo-jian; Yuen, Kwok-yung
    Journal of Virology (2007), 81 (4), 1574-1585CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
    Twelve complete genomes of three novel coronaviruses, i.e., bat coronavirus HKU4 (bat-CoV HKU4), bat-CoV HKU5 (putative group 2c), and bat-CoV HKU9 (putative group 2d), were sequenced. Comparative genome anal. showed that the various open reading frames (ORFs) of the genomes of the three coronaviruses had significantly higher amino acid identities to those of other group 2 coronaviruses than group 1 and 3 coronaviruses. Phylogenetic trees constructed using chymotrypsin-like protease, RNA-dependent RNA polymerase, helicase, spike, and nucleocapsid all showed that the group 2a and 2b and putative group 2c and 2d coronaviruses are more closely related to each other than to group 1 and 3 coronaviruses. Unique genomic features distinguishing between these four subgroups, including the no. of papain-like proteases, the presence or absence of hemagglutinin esterase, small ORFs between the membrane and nucleocapsid genes and ORFs (NS7a and NS7b), bulged stem-loop and pseudoknot structures downstream of the nucleocapsid gene, transcription regulatory sequence, and ribosomal recognition signal for the envelope gene, were also obsd. This is the first time that NS7a and NS7b downstream of the nucleocapsid gene have been found in a group 2 coronavirus. The high Ka/Ks ratio of NS7a and NS7b in bat-CoV HKU9 implies that these two group 2d-specific genes are under high selective pressure and hence are rapidly evolving. The four subgroups of group 2 coronaviruses probably originated from a common ancestor. Further mol. epidemiol. studies on coronaviruses in the bats of other countries, as well as in other animals, and complete genome sequencing will shed more light on coronavirus diversity and their evolutionary histories.
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  12. 12
    Fouchier, R. A., Kuiken, T., Schutten, M., van Amerongen, G., van Doornum, G. J., van den Hoogen, B. G., Peiris, M., Lim, W., Stohr, K., and Osterhaus, A. D. (2003) Aetiology: Koch’s postulates fulfilled for SARS virus. Nature 423, 240,  DOI: 10.1038/423240a
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    Aetiology: Koch's postulates fulfilled for SARS virus
    Fouchier, Ron A. M.; Kuiken, Thijs; Schutten, Martin; van Amerongen, Geert; van Doornum, Gerard J. J.; van den Hoogen, Bernadette G.; Peiris, Malik; Lim, Wilina; Stoehr, Klaus; Osterhaus, Albert D. M. E.
    Nature (London, United Kingdom) (2003), 423 (6937), 240CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    There is no expanded citation for this reference.
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  13. 13
    Ksiazek, T. G., Erdman, D., Goldsmith, C. S., Zaki, S. R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J. A., Lim, W., Rollin, P. E., Dowell, S. F., Ling, A. E., Humphrey, C. D., Shieh, W. J., Guarner, J., Paddock, C. D., Rota, P., Fields, B., DeRisi, J., Yang, J. Y., Cox, N., Hughes, J. M., LeDuc, J. W., Bellini, W. J., Anderson, L. J., and SARS Working Group (2003) A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1953– 1966,  DOI: 10.1056/NEJMoa030781
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    13
    A novel coronavirus associated with severe acute respiratory syndrome
    Ksiazek, Thomas G.; Erdman, Dean; Goldsmith, Cynthia S.; Zaki, Sherif R.; Peret, Teresa; Emery, Shannon; Tong, Suxiang; Urbani, Carlo; Comer, James A.; Lim, Wilina; Rollin, Pierre E.; Dowell, Scott F.; Ling, Ai-Ee; Humphrey, Charles D.; Shieh, Wan-Ju; Guarner, Jeannette; Paddock, Christopher D.; Rota, Paul; Fields, Barry; DeRisi, Joseph; Yang, Jyh-Yuan; Cox, Nancy; Hughes, James M.; LeDuc, James W.; Bellini, William J.; Anderson, Larry J.; Cannon, A. D. L.; Curtis, M.; Farrar, B.; Morgan, L.; Pezzanite, L.; Sanchez, A. J.; Slaughter, K. A.; Stevens, T. L.; Stockton, P. C.; Wagoner, K. D.; Sanchez, A.; Nichol, S.; Vincent, M.; Osborne, J.; Honig, J.; Brickson, B. R.; Holloway, B.; McCaustland, K.; Lingappa, J.; Lowe, L.; Scott, S.; Lu, X.; Villamarzo, Y.; Cook, B.; Chen, Q.; Birge, C.; Shu, B.; Pallansch, M.; Tatti, K. M.; Morken, T.; Smith, C.; Greer, P.; White, E.; McGlothen, T.; Bhatnagar, J.; Patel, M.; Bartlett, J.; Montague, J.; Lee, W.; Packard, M.; Thompson, H. A.; Moen, A.; Fukuda, K.; Uyeki, T.; Harper, S.; Klimov, A.; Lindstrom, S.; Benson, R.; Carlone, G.; Facklam, R.; Fields, P.; Levett, P.; Mayer, L.; Talkington, D.; Thacker, W. L.; Tondella, M. L. C.; Whitney, C.; Robertson, B.; Warnock, D.; Brooks, T.; Schrag, S.; Rosenstein, N.; Arthur, R.; Ganem, D.; Poutanen, S. M.; Chen, T.-J.; Hsiao, C.-H.; Wai-Fu, N. G.; Ho, M.; Keung, T.-K.; Nghiem, K. H.; Nguyen, H. K. L.; Le, M. Q.; Nguyen, H. H. T.; Hoang, L. T.; Vu, T. H.; Vu, H. Q.; Chunsuttiwat, S.
    New England Journal of Medicine (2003), 348 (20), 1953-1966CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
    A worldwide outbreak of severe acute respiratory syndrome (SARS) was assocd. with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiol. agent of this outbreak. We received clin. specimens from patients in 7 countries and tested them, using virus-isolation techniques, electron-microscopical and histol. studies, and mol. and serol. assays, in an attempt to identify a wide range of potential pathogens. None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathol. features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examn. revealed ultrastructural features characteristic of coronaviruses. Immunohistochem. and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers the authors identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate were used to demonstrate a virus-specific serol. response. This virus may never before have circulated in the U.S. population. Conclusions: A novel coronavirus is assocd. with this outbreak, and the evidence indicates that this virus has an etiol. role in SARS. Because of the death of Dr. Carlo Urbani, the authors propose that this first isolate be named the Urbani strain of SARS-assocd. coronavirus.
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  14. 14
    Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R. A., Berger, A., Burguiere, A. M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J. C., Muller, S., Rickerts, V., Sturmer, M., Vieth, S., Klenk, H. D., Osterhaus, A. D., Schmitz, H., and Doerr, H. W. (2003) Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1967– 1976,  DOI: 10.1056/NEJMoa030747
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    14
    Identification of a novel coronavirus in patients with severe acute respiratory syndrome
    Drosten, Christian; Guenther, Stephan; Preiser, Wolfgang; van der Werf, Sylvie; Brodt, Hans-Reinhard; Becker, Stephan; Rabenau, Holger; Panning, Marcus; Kolesnikova, Larissa; Fouchier, Ron A. M.; Berger, Annemarie; Burguiere, Ana-Maria; Cinatl, Jindrich; Eickmann, Markus; Escriou, Nicolas; Grywna, Klaus; Kramme, Stefanie; Manuguerra, Jean-Claude; Mueller, Stefanie; Rickerts, Volker; Stuermer, Martin; Vieth, Simon; Klenk, Hans-Dieter; Osterhaus, Albert D. M. E.; Schmitz, Herbert; Doerr, Hans Wilheim
    New England Journal of Medicine (2003), 348 (20), 1967-1976CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
    The severe acute respiratory syndrome (SARS) has recently been identified as a new clin. entity. SARS is thought to be caused by an unknown infectious agent. Clin. specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and mol. techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a PCR (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60% of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clin. specimens from patients with SARS but not in controls. High concns. of viral RNA of up to 100 million mols. per mL were found in sputum. Viral RNA was also detected at extremely low concns. in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS.
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  15. 15
    Rota, P. A., Oberste, M. S., Monroe, S. S., Nix, W. A., Campagnoli, R., Icenogle, J. P., Penaranda, S., Bankamp, B., Maher, K., Chen, M. H., Tong, S., Tamin, A., Lowe, L., Frace, M., DeRisi, J. L., Chen, Q., Wang, D., Erdman, D. D., Peret, T. C., Burns, C., Ksiazek, T. G., Rollin, P. E., Sanchez, A., Liffick, S., Holloway, B., Limor, J., McCaustland, K., Olsen-Rasmussen, M., Fouchier, R., Gunther, S., Osterhaus, A. D., Drosten, C., Pallansch, M. A., Anderson, L. J., and Bellini, W. J. (2003) Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394– 1399,  DOI: 10.1126/science.1085952
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    15
    Characterization of a novel coronavirus associated with severe acute respiratory syndrome
    Rota, Paul A.; Oberste, M. Steven; Monroe, Stephan S.; Nix, W. Allan; Campagnoli, Ray; Icenogle, Joseph P.; Penaranda, Silvia; Bankamp, Bettina; Maher, Kaija; Chen, Min-hsin; Tong, Suxiong; Tamin, Azaibi; Lowe, Luis; Frace, Michael; DeRisi, Joseph L.; Chen, Qi; Wang, David; Erdman, Dean D.; Peret, Teresa C. T.; Burns, Cara; Ksiazek, Thomas G.; Rollin, Pierre E.; Sanchez, Anthony; Liffick, Stephanie; Holloway, Brian; Limor, Josef; McCaustland, Karen; Olsen-Rasmussen, Melissa; Fouchier, Ron; Guenther, Stephan; Osterhaus, Albert D. M. E.; Drosten, Christian; Pallansch, Mark A.; Anderson, Larry J.; Bellini, William J.
    Science (Washington, DC, United States) (2003), 300 (5624), 1394-1399CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    In Mar. 2003, a novel coronavirus (SARS-CoV) was discovered in assocn. with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was detd., and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
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  16. 16
    de Groot, R. J., Baker, S. C., Baric, R. S., Brown, C. S., Drosten, C., Enjuanes, L., Fouchier, R. A., Galiano, M., Gorbalenya, A. E., Memish, Z. A., Perlman, S., Poon, L. L., Snijder, E. J., Stephens, G. M., Woo, P. C., Zaki, A. M., Zambon, M., and Ziebuhr, J. (2013) Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J. Virol. 87, 7790– 7792,  DOI: 10.1128/JVI.01244-13
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    16
    Middle east respiratory syndrome coronavirus (MERS-CoV): announcement of the coronavirus study group
    de Groot, Raoul J.; Baker, Susan C.; Baric, Ralph S.; Brown, Caroline S.; Drosten, Christian; Enjuanes, Luis; Fouchier, Ron A. M.; Galiano, Monica; Gorbalenya, Alexander E.; Memish, Ziad A.; Perlman, Stanley; Poon, Leo L. M.; Snijder, Eric J.; Stephens, Gwen M.; Woo, Patrick C. Y.; Zaki, Ali M.; Zambon, Maria; Ziebuhr, John
    Journal of Virology (2013), 87 (14), 7790-7792CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
    A review. A brief review including epidemiol., description, and consensus name for Middle east respiratory syndrome coronavirus (MERS-CoV).
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    Zaki, A. M., van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D., and Fouchier, R. A. (2012) Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 367, 1814– 1820,  DOI: 10.1056/NEJMoa1211721
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    Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia
    Zaki, Ali Moh; van Boheemen, Sander; Bestebroer, Theo M.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.
    New England Journal of Medicine (2012), 367 (19), 1814-1820CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
    A previously unknown coronavirus was isolated from the sputum of a 60-yr-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clin. data, virus isolation, and mol. identification are presented. The clin. picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
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    Bermingham, A., Chand, M. A., Brown, C. S., Aarons, E., Tong, C., Langrish, C., Hoschler, K., Brown, K., Galiano, M., Myers, R., Pebody, R. G., Green, H. K., Boddington, N. L., Gopal, R., Price, N., Newsholme, W., Drosten, C., Fouchier, R. A., and Zambon, M. (2012) Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012. Euro Surveill 17, 20290
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    Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012
    Bermingham A; Chand M A; Brown C S; Aarons E; Tong C; Langrish C; Hoschler K; Brown K; Galiano M; Myers R; Pebody R G; Green H K; Boddington N L; Gopal R; Price N; Newsholme W; Drosten C; Fouchier R A; Zambon M
    Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin (2012), 17 (40), 20290 ISSN:.
    Coronaviruses have the potential to cause severe transmissible human disease, as demonstrated by the severe acute respiratory syndrome (SARS) outbreak of 2003. We describe here the clinical and virological features of a novel coronavirus infection causing severe respiratory illness in a patient transferred to London, United Kingdom, from the Gulf region of the Middle East.
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    Woo, P. C., Lau, S. K., Chu, C. M., Chan, K. H., Tsoi, H. W., Huang, Y., Wong, B. H., Poon, R. W., Cai, J. J., Luk, W. K., Poon, L. L., Wong, S. S., Guan, Y., Peiris, J. S., and Yuen, K. Y. (2005) Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 79, 884– 895,  DOI: 10.1128/JVI.79.2.884-895.2005
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    19
    Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia
    Woo, Patrick C. Y.; Lau, Susanna K. P.; Chu, Chung-ming; Chan, Kwok-hung; Tsoi, Hoi-wah; Huang, Yi; Wong, Beatrice H. L.; Poon, Rosana W. S.; Cai, James J.; Luk, Wei-kwang; Poon, Leo L. M.; Wong, Samson S. Y.; Guan, Yi; Peiris, J. S. Malik; Yuen, Kwok-yung
    Journal of Virology (2005), 79 (2), 884-895CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
    Despite extensive lab. investigations in patients with respiratory tract infections, no microbiol. cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here the authors report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-yr-old man with pneumonia who had just returned from Shenzhen, China. Quant. reverse transcription-PCR showed that the amt. of CoV-HKU1 RNA was 8.5 to 9.6×106 copies per mL in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with IgM titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, resp. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic anal. reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, neg. for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an addnl. specimen, with a viral load of 1.13×106 copies per mL, from a 35-yr-old woman with pneumonia. The data support the existence of a novel group 2 coronavirus assocd. with pneumonia in humans.
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    Lu, G. and Liu, D. (2012) SARS-like virus in the Middle East: a truly bat-related coronavirus causing human diseases. Protein Cell 3, 803– 805,  DOI: 10.1007/s13238-012-2811-1
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    SARS-like virus in the Middle East: a truly bat-related coronavirus causing human diseases
    Lu Guangwen; Liu Di
    Protein & cell (2012), 3 (11), 803-5 ISSN:.
    There is no expanded citation for this reference.
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    Lu, G., Wang, Q., and Gao, G. F. (2015) Bat-to-human: spike features determining ’host jump’ of coronaviruses SARS-CoV, MERS-CoV, and beyond. Trends Microbiol. 23, 468– 478,  DOI: 10.1016/j.tim.2015.06.003
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    21
    Bat-to-human: spike features determining 'host jump' of coronaviruses SARS-CoV, MERS-CoV, and beyond
    Lu, Guangwen; Wang, Qihui; Gao, George F.
    Trends in Microbiology (2015), 23 (8), 468-478CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
    Both severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic pathogens that crossed the species barriers to infect humans. The mechanism of viral interspecies transmission is an important scientific question to be addressed. These coronaviruses contain a surface-located spike (S) protein that initiates infection by mediating receptor-recognition and membrane fusion and is therefore a key factor in host specificity. In addn., the S protein needs to be cleaved by host proteases before executing fusion, making these proteases a second determinant of coronavirus interspecies infection. Here, we summarize the progress made in the past decade in understanding the cross-species transmission of SARS-CoV and MERS-CoV by focusing on the features of the S protein, its receptor-binding characteristics, and the cleavage process involved in priming.
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    Ge, X. Y., Li, J. L., Yang, X. L., Chmura, A. A., Zhu, G., Epstein, J. H., Mazet, J. K., Hu, B., Zhang, W., Peng, C., Zhang, Y. J., Luo, C. M., Tan, B., Wang, N., Zhu, Y., Crameri, G., Zhang, S. Y., Wang, L. F., Daszak, P., and Shi, Z. L. (2013) Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535– 538,  DOI: 10.1038/nature12711
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    Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor
    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
    Nature (London, United Kingdom) (2013), 503 (7477), 535-538CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    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 mol., 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 morphol., 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.
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  23. 23
    Lau, S. K., Woo, P. C., Li, K. S., Huang, Y., Tsoi, H. W., Wong, B. H., Wong, S. S., Leung, S. Y., Chan, K. H., and Yuen, K. Y. (2005) Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. U. S. A. 102, 14040– 14045,  DOI: 10.1073/pnas.0506735102
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    23
    Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats
    Lau, Susanna K. P.; Woo, Patrick C. Y.; Li, Kenneth S. M.; Huang, Yi; Tsoi, Hoi-Wah; Wong, Beatrice H. L.; Wong, Samson S. Y.; Leung, Suet-Yi; Chan, Kwok-Hung; Yuen, Kwok-Yung
    Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (39), 14040-14045CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and anal. of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic anal. showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were obsd. in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were obsd. between human and civet SARS-CoV genomes. In addn., the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals.
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  24. 24
    Song, H. D., Tu, C. C., Zhang, G. W., Wang, S. Y., Zheng, K., Lei, L. C., Chen, Q. X., Gao, Y. W., Zhou, H. Q., Xiang, H., Zheng, H. J., Chern, S. W., Cheng, F., Pan, C. M., Xuan, H., Chen, S. J., Luo, H. M., Zhou, D. H., Liu, Y. F., He, J. F., Qin, P. Z., Li, L. H., Ren, Y. Q., Liang, W. J., Yu, Y. D., Anderson, L., Wang, M., Xu, R. H., Wu, X. W., Zheng, H. Y., Chen, J. D., Liang, G., Gao, Y., Liao, M., Fang, L., Jiang, L. Y., Li, H., Chen, F., Di, B., He, L. J., Lin, J. Y., Tong, S., Kong, X., Du, L., Hao, P., Tang, H., Bernini, A., Yu, X. J., Spiga, O., Guo, Z. M., Pan, H. Y., He, W. Z., Manuguerra, J. C., Fontanet, A., Danchin, A., Niccolai, N., Li, Y. X., Wu, C. I., and Zhao, G. P. (2005) Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human. Proc. Natl. Acad. Sci. U. S. A. 102, 2430– 2435,  DOI: 10.1073/pnas.0409608102
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    24
    Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human
    Song, Huai-Dong; Tu, Chang-Chun; Zhang, Guo-Wei; Wang, Sheng-Yue; Zheng, Kui; Lei, Lian-Cheng; Chen, Qiu-Xia; Gao, Yu-Wei; Zhou, Hui-Qiong; Xiang, Hua; Zheng, Hua-Jun; Chern, Shur-Wern Wang; Cheng, Feng; Pan, Chun-Ming; Xuan, Hua; Chen, Sai-Juan; Luo, Hui-Ming; Zhou, Duan-Hua; Liu, Yu-Fei; He, Jian-Feng; Qin, Peng-Zhe; Li, Ling-Hui; Ren, Yu-Qi; Liang, Wen-Jia; Yu, Ye-Dong; Anderson, Larry; Wang, Ming; Xu, Rui-Heng; Wu, Xin-Wei; Zheng, Huan-Ying; Chen, Jin-Ding; Liang, Guodong; Gao, Yang; Liao, Ming; Fang, Ling; Jiang, Li-Yun; Li, Hui; Chen, Fang; Di, Biao; He, Li-Juan; Lin, Jin-Yan; Tong, Suxiang; Kong, Xiangang; Du, Lin; Hao, Pei; Tang, Hua; Bernini, Andrea; Yu, Xiao-Jing; Spiga, Ottavia; Guo, Zong-Ming; Pan, Hai-Yan; He, Wei-Zhong; Manuguerra, Jean-Claude; Fontanet, Arnaud; Danchin, Antoine; Niccolai, Neri; Li, Yi-Xue; Wu, Chung-I.; Zhao, Guo-Ping
    Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (7), 2430-2435CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic anal. suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geog. locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002-2003 epidemic. Major genetic variations in some crit. genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003.
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  25. 25
    Lau, S. K., Li, K. S., Tsang, A. K., Lam, C. S., Ahmed, S., Chen, H., Chan, K. H., Woo, P. C., and Yuen, K. Y. (2013) 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. J. Virol. 87, 8638– 8650,  DOI: 10.1128/JVI.01055-13
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    25
    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
    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
    Journal of Virology (2013), 87 (15), 8638-8650CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
    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 examd. the mol. epidemiol. 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), resp. 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 pos. selected sites than that of Ty-BatCoV HKU4, suggesting that Pi-BatCoV HKU5 may generate variants to occupy new ecol. niches along with its host in diverse habitats. Mol. clock anal. 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.
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  26. 26
    Ithete, N. L., Stoffberg, S., Corman, V. M., Cottontail, V. M., Richards, L. R., Schoeman, M. C., Drosten, C., Drexler, J. F., and Preiser, W. (2013) Close relative of human Middle East respiratory syndrome coronavirus in bat, South Africa. Emerg. Infect. Dis. 19, 1697– 1699,  DOI: 10.3201/eid1910.130946
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    Close relative of human Middle East respiratory syndrome coronavirus in bat, South Africa
    Ithete Ndapewa Laudika; Stoffberg Samantha; Corman Victor Max; Cottontail Veronika M; Richards Leigh Rosanne; Schoeman M Corrie; Drosten Christian; Drexler Jan Felix; Preiser Wolfgang
    Emerging infectious diseases (2013), 19 (10), 1697-9 ISSN:.
    There is no expanded citation for this reference.
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    Corman, V. M., Ithete, N. L., Richards, L. R., Schoeman, M. C., Preiser, W., Drosten, C., and Drexler, J. F. (2014) Rooting the phylogenetic tree of middle East respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat. J. Virol. 88, 11297– 11303,  DOI: 10.1128/JVI.01498-14
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    27
    Rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an African bat
    Corman, Victor Max; Ithete, Ndapewa Laudika; Richards, Leigh Rosanne; Schoeman, M. Corrie; Preiser, Wolfgang; Drosten, Christian; Drexler, Jan Felix
    Journal of Virology (2014), 88 (19), 11297-11303, 8 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)
    The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes lethal respiratory infections mainly on the Arabian Peninsula. The evolutionary origins of MERS-CoV are unknown. We detd. the full genome sequence of a CoV directly from fecal material obtained from a South African Neoromicia capensis bat (NeoCoV). NeoCoV shared essential details of genome architecture with MERS-CoV. Eighty-five percent of the NeoCoV genome was identical to MERS-CoV at the nucleotide level. Based on taxonomic criteria, NeoCoV and MERS-CoV belonged to one viral species. The presence of a genetically divergent S1 subunit within the NeoCoV spike gene indicated that intraspike recombination events may have been involved in the emergence of MERS-CoV. NeoCoV constitutes a sister taxon of MERS-CoV, placing the MERS-CoV root between a recently described virus from African camels and all other viruses. This suggests a higher level of viral diversity in camels than in humans. Together with serol. evidence for widespread MERS-CoV infection in camelids sampled up to 20 years ago in Africa and the Arabian Peninsula, the genetic data indicate that camels act as sources of virus for humans rather than vice versa. The majority of camels on the Arabian Peninsula is imported from the Greater Horn of Africa, where several Neoromicia species occur. The acquisition of MERS-CoV by camels from bats might have taken place in sub-Saharan Africa. Camelids may represent mixing vessels for MERS-CoV and other mammalian CoVs.
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    Yang, L., Wu, Z., Ren, X., Yang, F., Zhang, J., He, G., Dong, J., Sun, L., Zhu, Y., Zhang, S., and Jin, Q. (2014) MERS-related betacoronavirus in Vespertilio superans bats, China. Emerg. Infect. Dis. 20, 1260– 1262,  DOI: 10.3201/eid.2006.140318
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    MERS-related betacoronavirus in Vespertilio superans bats, China
    Yang Li; Wu Zhiqiang; Ren Xianwen; Yang Fan; Zhang Junpeng; He Guimei; Dong Jie; Sun Lilian; Zhu Yafang; Zhang Shuyi; Jin Qi
    Emerging infectious diseases (2014), 20 (7), 1260-2 ISSN:.
    There is no expanded citation for this reference.
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  29. 29
    Memish, Z. A., Mishra, N., Olival, K. J., Fagbo, S. F., Kapoor, V., Epstein, J. H., Alhakeem, R., Durosinloun, A., Al Asmari, M., Islam, A., Kapoor, A., Briese, T., Daszak, P., Al Rabeeah, A. A., and Lipkin, W. I. (2013) Middle East respiratory syndrome coronavirus in bats, Saudi Arabia. Emerg. Infect. Dis. 19, 1819– 1823,  DOI: 10.3201/eid1911.131172
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    Middle east respiratory syndrome coronavirus in bats, Saudi Arabia
    Memish, Ziad A.; Mishra, Nischay; Olival, Kevin J.; Fagbo, Shamsudeen F.; Kapoor, Vishal; Epstein, Jonathan H.; Al Hakeem, Rafat; Al Asmari, Mushabab; Islam, Ariful; Kapoor, Amit; Briese, Thomas; Daszak, Peter; Al Rabeeah, Abdullah A.; Lipkin, W. Ian
    Emerging Infectious Diseases (2013), 19 (11), 1819-1823CODEN: EIDIFA; ISSN:1080-6040. (Centers for Disease Control and Prevention)
    The source of human infection with Middle East respiratory syndrome coronavirus remains unknown. Mol. investigation indicated that bats in Saudi Arabia are infected with several alphacoronaviruses and betacoronaviruses. Virus from 1 bat showed 100% nucleotide identity to virus from the human index case-patient. Bats might play a role in human infection.
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    De Benedictis, P., Marciano, S., Scaravelli, D., Priori, P., Zecchin, B., Capua, I., Monne, I., and Cattoli, G. (2014) Alpha and lineage C betaCoV infections in Italian bats. Virus Genes 48, 366– 371,  DOI: 10.1007/s11262-013-1008-x
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    Alpha and lineage C betaCoV infections in Italian bats
    De Benedictis, Paola; Marciano, Sabrina; Scaravelli, Dino; Priori, Pamela; Zecchin, Barbara; Capua, Ilaria; Monne, Isabella; Cattoli, Giovanni
    Virus Genes (2014), 48 (2), 366-371CODEN: VIGEET; ISSN:0920-8569. (Springer)
    AlphaCoV and lineage C betaCoV, genetically similar to those identified in Spanish related bat species, have been detected in Italian Myotis blythii and Eptesicus serotinus, resp., out of 75 anal swabs collected from Vespertilionidae between 2009 and 2012. Sequence anal. of the 816-bp obtained RdRp sequence fragment indicates a 96.9% amino acid identity of the Italian lineage C betaCoV with the recent Middle East Respiratory Syndrome Coronavirus (MERS-CoV, Genbank accession no. KF192507). This is the first documented occurrence of a lineage C betaCoV in the Italian bat population, notably in E. serotinus.
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  31. 31
    Annan, A., Baldwin, H. J., Corman, V. M., Klose, S. M., Owusu, M., Nkrumah, E. E., Badu, E. K., Anti, P., Agbenyega, O., Meyer, B., Oppong, S., Sarkodie, Y. A., Kalko, E. K., Lina, P. H., Godlevska, E. V., Reusken, C., Seebens, A., Gloza-Rausch, F., Vallo, P., Tschapka, M., Drosten, C., and Drexler, J. F. (2013) Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe. Emerg. Infect. Dis. 19, 456– 459,  DOI: 10.3201/eid1903.121503
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    Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe
    Annan Augustina; Baldwin Heather J; Corman Victor Max; Klose Stefan M; Owusu Michael; Nkrumah Evans Ewald; Badu Ebenezer Kofi; Anti Priscilla; Agbenyega Olivia; Meyer Benjamin; Oppong Samuel; Sarkodie Yaw Adu; Kalko Elisabeth K V; Lina Peter H C; Godlevska Elena V; Reusken Chantal; Seebens Antje; Gloza-Rausch Florian; Vallo Peter; Tschapka Marco; Drosten Christian; Drexler Jan Felix
    Emerging infectious diseases (2013), 19 (3), 456-9 ISSN:.
    We screened fecal specimens of 4,758 bats from Ghana and 272 bats from 4 European countries for betacoronaviruses. Viruses related to the novel human betacoronavirus EMC/2012 were detected in 46 (24.9%) of 185 Nycteris bats and 40 (14.7%) of 272 Pipistrellus bats. Their genetic relatedness indicated EMC/2012 originated from bats.
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  32. 32
    Azhar, E. I., El-Kafrawy, S. A., Farraj, S. A., Hassan, A. M., Al-Saeed, M. S., Hashem, A. M., and Madani, T. A. (2014) Evidence for camel-to-human transmission of MERS coronavirus. N. Engl. J. Med. 370, 2499– 2505,  DOI: 10.1056/NEJMoa1401505
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    Evidence for camel-to-human transmission of MERS coronavirus
    Azhar, Esam I.; El-Kafrawy, Sherif A.; Farraj, Suha A.; Hassan, Ahmed M.; Al-Saeed, Muneera S.; Hashem, Anwar M.; Madani, Tariq A.
    New England Journal of Medicine (2014), 370 (26), 2499-2505, 7 pp.CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
    We describe the isolation and sequencing of Middle East respiratory syndrome coronavirus (MERS-CoV) obtained from a dromedary camel and from a patient who died of lab.-confirmed MERS-CoV infection after close contact with camels that had rhinorrhea. Nasal swabs collected from the patient and from one of his nine camels were pos. for MERS-CoV RNA. In addn., MERS-CoV was isolated from the patient and the camel. The full genome sequences of the two isolates were identical. Serol. data indicated that MERS-CoV was circulating in the camels but not in the patient before the human infection occurred. These data suggest that this fatal case of human MERS-CoV infection was transmitted through close contact with an infected camel.
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  33. 33
    Reusken, C. B., Haagmans, B. L., Muller, M. A., Gutierrez, C., Godeke, G. J., Meyer, B., Muth, D., Raj, V. S., Smits-De Vries, L., Corman, V. M., Drexler, J. F., Smits, S. L., El Tahir, Y. E., De Sousa, R., van Beek, J., Nowotny, N., van Maanen, K., Hidalgo-Hermoso, E., Bosch, B. J., Rottier, P., Osterhaus, A., Gortazar-Schmidt, C., Drosten, C., and Koopmans, M. P. (2013) Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect. Dis. 13, 859– 866,  DOI: 10.1016/S1473-3099(13)70164-6
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    33
    Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study
    Reusken, Chantal BEM; Haagmans, Bart L.; Mueller, Marcel A.; Gutierrez, Carlos; Godeke, Gert-Jan; Meyer, Benjamin; Muth, Doreen; Raj, V. Stalin; Vries, Laura Smits-De; Corman, Victor M.; Drexler, Jan-Felix; Smits, Saskia L.; El Tahir, Yasmin E.; De Sousa, Rita; van Beek, Janko; Nowotny, Norbert; van Maanen, Kees; Hidalgo-Hermoso, Ezequiel; Bosch, Berend-Jan; Rottier, Peter; Osterhaus, Albert; Gortazar-Schmidt, Christian; Drosten, Christian; Koopmans, Marion PG
    Lancet Infectious Diseases (2013), 13 (10), 859-866CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)
    Background: A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, mol. clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. Methods: We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. Findings: 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralizing antibody titers varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralization by bovine coronavirus antibodies. Interpretation: MERS-CoV or a related virus has infected camel populations. Both titers and seroprevalences in sera from different locations in Oman suggest widespread infection.
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    Raj, V. S., Mou, H., Smits, S. L., Dekkers, D. H., Muller, M. A., Dijkman, R., Muth, D., Demmers, J. A., Zaki, A., Fouchier, R. A., Thiel, V., Drosten, C., Rottier, P. J., Osterhaus, A. D., Bosch, B. J., and Haagmans, B. L. (2013) Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495, 251– 254,  DOI: 10.1038/nature12005
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    Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC
    Raj, V. Stalin; Mou, Huihui; Smits, Saskia L.; Dekkers, Dick H. W.; Mueller, Marcel A.; Dijkman, Ronald; Muth, Doreen; Demmers, Jeroen A. A.; Zaki, Ali; Fouchier, Ron A. M.; Thiel, Volker; Drosten, Christian; Rottier, Peter J. M.; Osterhaus, Albert D. M. E.; Bosch, Berend Jan; Haagmans, Bart L.
    Nature (London, United Kingdom) (2013), 495 (7440), 251-254CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Most human coronaviruses cause mild upper respiratory tract disease but may be assocd. with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome anal. revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addn., it will contribute critically to our understanding of the pathogenesis and epidemiol. of this emerging human coronavirus, and may facilitate the development of intervention strategies.
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  35. 35
    Wang, Q., Qi, J., Yuan, Y., Xuan, Y., Han, P., Wan, Y., Ji, W., Li, Y., Wu, Y., Wang, J., Iwamoto, A., Woo, P. C., Yuen, K. Y., Yan, J., Lu, G., and Gao, G. F. (2014) Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26. Cell Host Microbe 16, 328– 337,  DOI: 10.1016/j.chom.2014.08.009
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    Bat Origins of MERS-CoV Supported by Bat Coronavirus HKU4 Usage of Human Receptor CD26
    Wang, Qihui; Qi, Jianxun; Yuan, Yuan; Xuan, Yifang; Han, Pengcheng; Wan, Yuhua; Ji, Wei; Li, Yan; Wu, Ying; Wang, Jianwei; Iwamoto, Aikichi; Woo, Patrick C. Y.; Yuen, Kwok-Yung; Yan, Jinghua; Lu, Guangwen; Gao, George F.
    Cell Host & Microbe (2014), 16 (3), 328-337CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)
    The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) is phylogenetically closely related to the bat coronaviruses (BatCoVs) HKU4 and HKU5. However, the evolutionary pathway of MERS-CoV is still unclear. A receptor binding domain (RBD) in the MERS-CoV envelope-embedded spike protein specifically engages human CD26 (hCD26) to initiate viral entry. The high sequence identity in the viral spike protein prompted us to investigate if HKU4 and HKU5 can recognize hCD26 for cell entry. We found that HKU4-RBD, but not HKU5-RBD, binds to hCD26, and pseudotyped viruses embedding HKU4 spike can infect cells via hCD26 recognition. The structure of the HKU4-RBD/hCD26 complex revealed a hCD26-binding mode similar overall to that obsd. for MERS-RBD. HKU4-RBD, however, is less adapted to hCD26 than MERS-RBD, explaining its lower affinity for receptor binding. Our findings support a bat origin for MERS-CoV and indicate the need for surveillance of HKU4-related viruses in bats.
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    Yang, Y., Du, L., Liu, C., Wang, L., Ma, C., Tang, J., Baric, R. S., Jiang, S., and Li, F. (2014) Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc. Natl. Acad. Sci. U. S. A. 111, 12516– 12521,  DOI: 10.1073/pnas.1405889111
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    Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus
    Yang, Yang; Du, Lanying; Liu, Chang; Wang, Lili; Ma, Cuiqing; Tang, Jian; Baric, Ralph S.; Jiang, Shibo; Li, Fang
    Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (34), 12516-12521CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.
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    Wang, Q., Wong, G., Lu, G., Yan, J., and Gao, G. F. (2016) MERS-CoV spike protein: Targets for vaccines and therapeutics. Antiviral Res. 133, 165– 177,  DOI: 10.1016/j.antiviral.2016.07.015
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    MERS-CoV spike protein: Targets for vaccines and therapeutics
    Wang, Qihui; Wong, Gary; Lu, Guangwen; Yan, Jinghua; Gao, George F.
    Antiviral Research (2016), 133 (), 165-177CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
    The disease outbreak caused by Middle East respiratory syndrome coronavirus (MERS-CoV) is still ongoing in the Middle East. Over 1700 people have been infected since it was first reported in Sept. 2012. Despite great efforts, licensed vaccines or therapeutics against MERS-CoV remain unavailable. The MERS-CoV spike (S) protein is an important viral antigen known to mediate host-receptor binding and virus entry, as well as induce robust humoral and cell-mediated responses in humans during infection. In this review, we highlight the importance of the S protein in the MERS-CoV life cycle, summarize recent advances in the development of vaccines and therapeutics based on the S protein, and discuss strategies that can be explored to develop new medical countermeasures against MERS-CoV.
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    Li, F., Li, W., Farzan, M., and Harrison, S. C. (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864– 1868,  DOI: 10.1126/science.1116480
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    Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor
    Li, Fang; Li, Wenhui; Farzan, Michael; Harrison, Stephen C.
    Science (Washington, DC, United States) (2005), 309 (5742), 1864-1868CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    The spike protein (S) of SARS coronavirus (SARS-CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). A defined receptor-binding domain (RBD) on S mediates this interaction. The crystal structure at 2.9 angstrom resoln. of the RBD bound with the peptidase domain of human ACE2 shows that the RBD presents a gently concave surface, which cradles the N-terminal lobe of the peptidase. The at. details at the interface between the two proteins clarify the importance of residue changes that facilitate efficient cross-species infection and human-to-human transmission. The structure of the RBD suggests ways to make truncated disulfide-stabilized RBD variants for use in the design of coronavirus vaccines.
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    Lu, G., Hu, Y., Wang, Q., Qi, J., Gao, F., Li, Y., Zhang, Y., Zhang, W., Yuan, Y., Bao, J., Zhang, B., Shi, Y., Yan, J., and Gao, G. F. (2013) Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature 500, 227– 231,  DOI: 10.1038/nature12328
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    Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26
    Lu, Guangwen; Hu, Yawei; Wang, Qihui; Qi, Jianxun; Gao, Feng; Li, Yan; Zhang, Yanfang; Zhang, Wei; Yuan, Yuan; Bao, Jinku; Zhang, Buchang; Shi, Yi; Yan, Jinghua; Gao, George F.
    Nature (London, United Kingdom) (2013), 500 (7461), 227-231CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV. CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. Here we delineate the mol. basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissocn. const. of 16.7nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 β-propeller. The at. details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.
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    Wang, N., Shi, X., Jiang, L., Zhang, S., Wang, D., Tong, P., Guo, D., Fu, L., Cui, Y., Liu, X., Arledge, K. C., Chen, Y. H., Zhang, L., and Wang, X. (2013) Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell Res. 23, 986– 993,  DOI: 10.1038/cr.2013.92
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    Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4
    Wang, Nianshuang; Shi, Xuanling; Jiang, Liwei; Zhang, Senyan; Wang, Dongli; Tong, Pei; Guo, Dongxing; Fu, Lili; Cui, Ye; Liu, Xi; Arledge, Kelly C.; Chen, Ying-Hua; Zhang, Linqi; Wang, Xinquan
    Cell Research (2013), 23 (8), 986-993CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
    The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor, dipeptidyl peptidase 4 (DPP4). Sequence comparison and modeling anal. have revealed a putative receptor-binding domain (RBD) on the viral spike, which mediates this interaction. We report the 3.0 Å-resoln. crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 β-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor-binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are crit. for viral binding to DPP4 and entry into the target cell. The at. details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.
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    Peng, G., Sun, D., Rajashankar, K. R., Qian, Z., Holmes, K. V., and Li, F. (2011) Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor. Proc. Natl. Acad. Sci. U. S. A. 108, 10696– 10701,  DOI: 10.1073/pnas.1104306108
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    Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor
    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang
    Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (26), 10696-10701, S10696/1-S10696/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    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 mol. 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same β-sandwich fold as human galectins (S-lectins) and addnl. 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. Crit. 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 anal. and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the β-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.
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    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXovFenu7g%253D&md5=fecc9ff68277af2e239eaaf122c27300
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    Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., Choe, H., and Farzan, M. (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450– 454,  DOI: 10.1038/nature02145
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    Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus
    Li, Wenhui; Moore, Michael J.; Vasilieva, Natalya; Sui, Jianhua; Wong, Swee Kee; Berne, Michael A.; Somasundaran, Mohan; Sullivan, John L.; Luzuriaga, Katherine; Greenough, Thomas C.; Choe, Hyeryun; Farzan, Michael
    Nature (London, United Kingdom) (2003), 426 (6965), 450-454CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), assoc. with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a sol. form of ACE2, but not of the related enzyme ACE1, blocked assocn. of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.
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    Ge, X., Li, Y., Yang, X., Zhang, H., Zhou, P., Zhang, Y., and Shi, Z. (2012) Metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in China. J. Virol. 86, 4620– 4630,  DOI: 10.1128/JVI.06671-11
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    Metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in China
    Ge, Xingyi; Li, Yan; Yang, Xinglou; Zhang, Huajun; Zhou, Peng; Zhang, Yunzhi; Shi, Zhengli
    Journal of Virology (2012), 86 (8), 4620-4630CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
    Increasing data indicate that bats harbor diverse viruses, some of which cause severe human diseases. In this study, sequence-independent amplification and high-throughput sequencing (Solexa) were applied to the metagenomic anal. of viruses in bat fecal samples collected from 6 locations in China. A total of 8746,417 reads with a length of 306,124,595 bp were obtained. Among these reads, 13,541 (0.15%) had similarity to phage sequences and 9170 (0.1%) had similarity to eukaryotic virus sequences. A total of 129 assembled contigs (>100 nucleotides) were constructed and compared with GenBank: 32 contigs were related to phages, and 97 were related to eukaryotic viruses. The most frequent reads and contigs related to eukaryotic viruses were homologous to densoviruses, dicistroviruses, coronaviruses, parvoviruses, and tobamoviruses, a range that includes viruses from invertebrates, vertebrates, and plants. Most of the contigs had low identities to known viral genomic or protein sequences, suggesting that a large no. of novel and genetically diverse insect viruses as well as putative mammalian viruses are transmitted by bats in China. This study provides the first preliminary understanding of the virome of some bat populations in China, which may guide the discovery and isolation of novel viruses in the future.
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    Lau, S. K., Poon, R. W., Wong, B. H., Wang, M., Huang, Y., Xu, H., Guo, R., Li, K. S., Gao, K., Chan, K. H., Zheng, B. J., Woo, P. C., and Yuen, K. Y. (2010) Coexistence of different genotypes in the same bat and serological characterization of Rousettus bat coronavirus HKU9 belonging to a novel Betacoronavirus subgroup. J. Virol. 84, 11385– 11394,  DOI: 10.1128/JVI.01121-10
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    Coexistence of different genotypes in the same bat and serological characterization of Rousettus bat coronavirus HKU9 belonging to a novel Betacoronavirus subgroup
    Lau, Susanna K. P.; Poon, Rosana W. S.; Wong, Beatrice H. L.; Wang, Ming; Huang, Yi; Xu, Huifang; Guo, Rongtong; Li, Kenneth S. M.; Gao, Kai; Chan, Kwok-Hung; Zheng, Bo-Jian; Woo, Patrick C. Y.; Yuen, Kwok-Yung
    Journal of Virology (2010), 84 (21), 11385-11394CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
    Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D (Nobecovirus), from Leschenault's rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, resp., suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination anal. using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as sep. subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault's rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, resp. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault's rousette.
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    Tao, Y., Tang, K., Shi, M., Conrardy, C., Li, K. S., Lau, S. K., Anderson, L. J., and Tong, S. (2012) Genomic characterization of seven distinct bat coronaviruses in Kenya. Virus Res. 167, 67– 73,  DOI: 10.1016/j.virusres.2012.04.007
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    Genomic characterization of seven distinct bat coronaviruses in Kenya
    Tao, Ying; Tang, Kevin; Shi, Mang; Conrardy, Christina; Li, Kenneth S. M.; Lau, Susanna K. P.; Anderson, Larry J.; Tong, Suxiang
    Virus Research (2012), 167 (1), 67-73CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
    To better understand the genetic diversity and genomic features of 41 coronaviruses (CoVs) identified from Kenya bats in 2006, seven CoVs as representatives of seven different phylogenetic groups identified from partial polymerase gene sequences, were subjected to extensive genomic sequencing. As a result, 15-16 kb nucleotide sequences encoding complete RNA dependent RNA polymerase, spike, envelope, membrane, and nucleocapsid proteins plus other open reading frames (ORFs) were generated. Sequences anal. confirmed that the CoVs from Kenya bats are divergent members of Alphacoronavirus and Betacoronavirus genera. Furthermore, the CoVs BtKY22, BtKY41, and BtKY43 in Alphacoronavirus genus and BtKY24 in Betacoronavirus genus are likely representatives of 4 novel CoV species. BtKY27 and BtKY33 are members of the established bat CoV species in Alphacoronavirus genus and BtKY06 is a member of the established bat CoV species in Betacoronavirus genus. The genome organization of these seven CoVs is similar to other known CoVs from the same groups except for differences in the no. of putative ORFs following the N gene. The present results confirm a significant diversity of CoVs circulating in Kenya bats. These Kenya bat CoVs are phylogenetically distant from any previously described human and animal CoVs. However, because of the examples of host switching among CoVs after relatively minor sequence changes in S1 domain of spike protein, a further surveillance in animal reservoirs and understanding the interface between host susceptibility is crit. for predicting and preventing the potential threat of bat CoVs to public health.
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    Tong, S., Conrardy, C., Ruone, S., Kuzmin, I. V., Guo, X., Tao, Y., Niezgoda, M., Haynes, L., Agwanda, B., Breiman, R. F., Anderson, L. J., and Rupprecht, C. E. (2009) Detection of novel SARS-like and other coronaviruses in bats from Kenya. Emerg. Infect. Dis. 15, 482– 485,  DOI: 10.3201/eid1503.081013
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    Detection of novel SARS-like and other coronaviruses in bats from Kenya
    Tong Suxiang; Conrardy Christina; Ruone Susan; Kuzmin Ivan V; Guo Xiling; Tao Ying; Niezgoda Michael; Haynes Lia; Agwanda Bernard; Breiman Robert F; Anderson Larry J; Rupprecht Charles E
    Emerging infectious diseases (2009), 15 (3), 482-5 ISSN:.
    Diverse coronaviruses have been identified in bats from several continents but not from Africa. We identified group 1 and 2 coronaviruses in bats in Kenya, including SARS-related coronaviruses. The sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans.
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    Zhang, W., Qi, J., Shi, Y., Li, Q., Gao, F., Sun, Y., Lu, X., Lu, Q., Vavricka, C. J., Liu, D., Yan, J., and Gao, G. F. (2010) Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus. Protein Cell 1, 459– 467,  DOI: 10.1007/s13238-010-0059-1
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    Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus
    Zhang, Wei; Qi, Jianxun; Shi, Yi; Li, Qing; Gao, Feng; Sun, Yeping; Lu, Xishan; Lu, Qiong; Vavricka, Christopher J.; Liu, Di; Yan, Jinghua; Gao, George F.
    Protein & Cell (2010), 1 (5), 459-467CODEN: PCREFB; ISSN:1674-800X. (Higher Education Press)
    Influenza virus is the causative agent of the seasonal and occasional pandemic flu. The current H1N1 influenza pandemic, announced by the WHO in June 2009, is highly contagious and responsible for global economic losses and fatalities. Although the H1N1 gene segments have three origins in terms of host species, the virus has been named swine-origin influenza virus (S-OIV) due to a predominant swine origin. 2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects. Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection. Primary sequence anal. of the 2009 S-OIV hemagglutinin (HA) reveals its closest relationship to that of the 1918 pandemic influenza virus, however, anal. at the structural level is necessary to critically assess the functional significance. In this report, we report the crystal structure of sol. hemagglutinin H1 (09H1) at 2.9 Å, illustrating that the 09H1 is very similar to the 1918 pandemic HA (18H1) in overall structure and the structural modules, including the five defined antiboby (Ab)-binding epitopes. Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV, and people born around the 1918 are resistant to the current pandemic, yet younger generations are more susceptible to the 2009 pandemic.
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    The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and detd. its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.
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    To localize the epitopes recognized by monoclonal antibodies (MAbs) specific for the S1 subunit of the murine coronavirus JHMV spike protein, the authors have expressed S1 proteins with different deletions from the C-terminus of S1. S1utt is composed of the entire 769-amino-acid (aa) S1 protein; S1NM, S1N, S1N(330), and S1N(220) are deletion mutants with 594, 453, 330, and 220 aa from the N terminus of the S1 protein. The expressed S1 deletion mutant proteins were examd. for reactivities to a panel of MAbs. All MAbs classified in groups A and B, those reactive to most mouse hepatitis virus (MHV) strains and those specific for isolate JHMV, resp., recognized S1N(330) and the larger S1 deletion mutants but failed to react with S1N(220). MAbs in group C, specific for the larger S protein of JHMV, reacted only with the S1utt protein without any deletion. These results indicated that the domain composed of the N-terminal 330 aa comprised the cluster of conformational epitopes recognized by MAbs in groups A and B. It was also shown that the epitopes of MAbs in group C were not restricted to the region missing in the smaller S protein. These results together with the fact that all MAbs in group B retained high neutralizing activity suggested the possibility that the N-terminal 330 aa are responsible for binding to the MHV-specific receptors. To investigate this possibility, the authors expressed the receptor protein and examd. the binding of each S1 deletion mutant to the receptor. It was demonstrated that the S1N(330) protein as well as other S1 deletion mutants larger than S1N(330) bound to the receptor. These results indicated that a domain composed of 330 aa at the N terminus of the S1 protein is responsible for binding to the MHV-specific receptor.
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    Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang
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    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). How different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors, was investigated. The crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2 was detd. NL63-CoV RBD has a novel β-sandwich core structure consisting of 2 layers of β-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homol. 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.
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    Reguera, J., Santiago, C., Mudgal, G., Ordono, D., Enjuanes, L., and Casasnovas, J. M. (2012) Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies. PLoS Pathog. 8, e1002859,  DOI: 10.1371/journal.ppat.1002859
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    Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies
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    PLoS Pathogens (2012), 8 (8), e1002859CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
    The coronaviruses (CoVs) are enveloped viruses of animals and humans assocd. 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 dets. virus-binding specificity for recessed glycan-contg. 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 dets. 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.
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  • Abstract

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    Figure 1

    Figure 1. Sequence features of HKU9-RBD. (A) Schematic representation of BatCoV HKU9 S. The indicated domain elements were defined on the basis of either the pairwise sequence alignment results or the bioinformatics predictions. The signal peptide (SP), transmembrane domain (TM), and heptad repeats 1 and 2 (HR1 and HR2, respectively) were predicted with the SignalP 4.0 server, TMHMM server, and Learncoil-VMF program, respectively, while the N-terminal domain (NTD) and RBD were deduced by alignment with the N-terminal galectin-like domain of murine hepatitis virus S and MERS-RBD, respectively. The S1/S2 site potentially cleaved by furin-like proteases could not be ascertained and is therefore labeled with a question mark. (B and C) Structure-based alignment of the HKU9-, SARS-, MERS-, and HKU4-RBD sequences. The arrows and spiral lines indicate strands and helices, respectively. These secondary structure elements were labeled as illustrated in Figure 3. The conserved cysteine residues that form three disulfide bonds in the structures are marked with Arabic numerals 1–3. The core subdomain is conserved among the four RBD structures, but the external subdomain is structurally irrelevant. We therefore present the sequences separately. The two elements that anchor the external subdomain to the core subdomain are highlighted with black boxes. (B) Core subdomain sequence. (C) External subdomain sequence.

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    Figure 2

    Figure 2. Characterization of HKU9-RBD by SPR assays. The indicated RBD proteins expressed by insect cells were immobilized on CM5 chips and tested for the binding with gradient concentrations of human ACE2 or CD26 using a BIAcore 3000 machine. The recorded kinetic profiles are shown: (A) human ACE2 and SARS-RBD, (B) human CD26 and MERS-RBD, (C) human ACE2 and HKU9-RBD, and (D) human CD26 and HKU9-RBD. Clearly shown is the fact that HKU9-RBD does not bind either ACE2 or CD26, in the context of which SARS-RBD and MERS-RBD bind their respective receptors. Then we purified the mFc-fused HKU9-RBD proteins in mammalian (293T) cells and assembled the abilities to bind CD26 or ACE2 proteins using a captured SPR method by a BIAcore T100 system. The anti-mouse antibodies were immobilized on CM5 chips. The mFc-fused RBD proteins were then captured (3 μg/mL for 60 s) by the antibodies and tested for binding to human ACE2 or CD26. (E) The mFc-fused SARS-RBD (SARS-RBD-mFc) did not bind to CD26 but bound to ACE2 well. (F) The mFc-fused MERS-RBD (MERS-RBD-mFc) did not bind to ACE2 but bound to CD26 well. (G) The mFc-fused HKU9-RBD (HKU9-RBD-mFc) does not bind either ACE2 or CD26.

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    Figure 3

    Figure 3. Crystal structure of HKU9-RBD. The core and external subdomains are colored magenta and green, respectively. The core subdomain is further divided into a center region (core-center) and a peripheral region (core-peripheral), which are encircled. The core-center strands and helices are labeled βc1−βc5 and H1–H6, respectively, while the core-peripheral strands are marked βp1−βp3. The disulfide bonds and the RBD termini are labeled. The core subdomain is further presented in a surface representation in the right panel to highlight the top positioning of the external subdomain like a hat.

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    Figure 4

    Figure 4. Structural and topological comparison of available betaCoV RBD structures. Four structures, including those of HKU9-, SARS-, MERS-, and HKU4-RBD, were oriented similarly and are presented as cartoons in parallel. The core-center, core-peripheral, and the external subdomain are encircled and highlighted in yellow. For each structure, the topological arrangement of the core-center and core-peripheral strands as well as of the external components is depicted. The core strands that flank the external subdomain are colored red and blue, respectively. Yellow lines indicate disulfide bonds. The N- and C-termini are highlighted: (A) HKU9-RBD, (B) SARS-RBD, (C) MERS-RBD, and (D) HKU4-RBD. The similarity in the topological arrangement of the external subdomain as an insertion between two core strands is illustrated.

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    Figure 5

    Figure 5. Homologous intersubdomain amino acid interactions anchoring the external subdomain to the core subdomain. (A) Superimposition of the betaCoV RBD (HKU9-RBD in green, SARS-RBD in yellow, MERS-RBD in blue, and HKU4-RBD in cyan) structures highlighting the external elements that can be well-aligned. These two elements, with seven (element 1) and eight (element 2) amino acids, respectively, engage mainly core subdomain helices H2 and H6 for the intersubdomain interactions. To facilitate comparison, the element residues were successively assigned a position marker (a–g for element 1 and a–h for element 2), which is highlighted. (B) Characterization of the element residues for their contributions to the intersubdomain binding. The two external elements are presented as cartoons, while the core subdomain is shown at the surface. At each position, the residue is marked sequentially with the position marker, the amino acid identity and numbering, the interacting mode/type, and the side-chain orientation. For the interaction mode, the hydrophobic or van der Waals interactions are indicated with encircled Ps, the side-chain H-bonds with encircled Ss, and main-chain H-bonds with encircled Ms. The side-chain orientations are indicated with arrows. (C) Summary of the intersubdomain interactions specified in panel B. The element sequences of the four RBDs are aligned and listed. A + indicates that a certain type of interaction is commonly observed at the position, while a +/– indicates that the interaction type is specific to some but not all of the four RBDs. The arrows mark the side-chain orientations.

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      Ksiazek, Thomas G.; Erdman, Dean; Goldsmith, Cynthia S.; Zaki, Sherif R.; Peret, Teresa; Emery, Shannon; Tong, Suxiang; Urbani, Carlo; Comer, James A.; Lim, Wilina; Rollin, Pierre E.; Dowell, Scott F.; Ling, Ai-Ee; Humphrey, Charles D.; Shieh, Wan-Ju; Guarner, Jeannette; Paddock, Christopher D.; Rota, Paul; Fields, Barry; DeRisi, Joseph; Yang, Jyh-Yuan; Cox, Nancy; Hughes, James M.; LeDuc, James W.; Bellini, William J.; Anderson, Larry J.; Cannon, A. D. L.; Curtis, M.; Farrar, B.; Morgan, L.; Pezzanite, L.; Sanchez, A. J.; Slaughter, K. A.; Stevens, T. L.; Stockton, P. C.; Wagoner, K. D.; Sanchez, A.; Nichol, S.; Vincent, M.; Osborne, J.; Honig, J.; Brickson, B. R.; Holloway, B.; McCaustland, K.; Lingappa, J.; Lowe, L.; Scott, S.; Lu, X.; Villamarzo, Y.; Cook, B.; Chen, Q.; Birge, C.; Shu, B.; Pallansch, M.; Tatti, K. M.; Morken, T.; Smith, C.; Greer, P.; White, E.; McGlothen, T.; Bhatnagar, J.; Patel, M.; Bartlett, J.; Montague, J.; Lee, W.; Packard, M.; Thompson, H. A.; Moen, A.; Fukuda, K.; Uyeki, T.; Harper, S.; Klimov, A.; Lindstrom, S.; Benson, R.; Carlone, G.; Facklam, R.; Fields, P.; Levett, P.; Mayer, L.; Talkington, D.; Thacker, W. L.; Tondella, M. L. C.; Whitney, C.; Robertson, B.; Warnock, D.; Brooks, T.; Schrag, S.; Rosenstein, N.; Arthur, R.; Ganem, D.; Poutanen, S. M.; Chen, T.-J.; Hsiao, C.-H.; Wai-Fu, N. G.; Ho, M.; Keung, T.-K.; Nghiem, K. H.; Nguyen, H. K. L.; Le, M. Q.; Nguyen, H. H. T.; Hoang, L. T.; Vu, T. H.; Vu, H. Q.; Chunsuttiwat, S.
      New England Journal of Medicine (2003), 348 (20), 1953-1966CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
      A worldwide outbreak of severe acute respiratory syndrome (SARS) was assocd. with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiol. agent of this outbreak. We received clin. specimens from patients in 7 countries and tested them, using virus-isolation techniques, electron-microscopical and histol. studies, and mol. and serol. assays, in an attempt to identify a wide range of potential pathogens. None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathol. features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examn. revealed ultrastructural features characteristic of coronaviruses. Immunohistochem. and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers the authors identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate were used to demonstrate a virus-specific serol. response. This virus may never before have circulated in the U.S. population. Conclusions: A novel coronavirus is assocd. with this outbreak, and the evidence indicates that this virus has an etiol. role in SARS. Because of the death of Dr. Carlo Urbani, the authors propose that this first isolate be named the Urbani strain of SARS-assocd. coronavirus.
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    14. 14
      Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R. A., Berger, A., Burguiere, A. M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J. C., Muller, S., Rickerts, V., Sturmer, M., Vieth, S., Klenk, H. D., Osterhaus, A. D., Schmitz, H., and Doerr, H. W. (2003) Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348, 1967– 1976,  DOI: 10.1056/NEJMoa030747
      14
      Identification of a novel coronavirus in patients with severe acute respiratory syndrome
      Drosten, Christian; Guenther, Stephan; Preiser, Wolfgang; van der Werf, Sylvie; Brodt, Hans-Reinhard; Becker, Stephan; Rabenau, Holger; Panning, Marcus; Kolesnikova, Larissa; Fouchier, Ron A. M.; Berger, Annemarie; Burguiere, Ana-Maria; Cinatl, Jindrich; Eickmann, Markus; Escriou, Nicolas; Grywna, Klaus; Kramme, Stefanie; Manuguerra, Jean-Claude; Mueller, Stefanie; Rickerts, Volker; Stuermer, Martin; Vieth, Simon; Klenk, Hans-Dieter; Osterhaus, Albert D. M. E.; Schmitz, Herbert; Doerr, Hans Wilheim
      New England Journal of Medicine (2003), 348 (20), 1967-1976CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
      The severe acute respiratory syndrome (SARS) has recently been identified as a new clin. entity. SARS is thought to be caused by an unknown infectious agent. Clin. specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and mol. techniques. A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a PCR (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60% of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clin. specimens from patients with SARS but not in controls. High concns. of viral RNA of up to 100 million mols. per mL were found in sputum. Viral RNA was also detected at extremely low concns. in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. The novel coronavirus might have a role in causing SARS.
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    15. 15
      Rota, P. A., Oberste, M. S., Monroe, S. S., Nix, W. A., Campagnoli, R., Icenogle, J. P., Penaranda, S., Bankamp, B., Maher, K., Chen, M. H., Tong, S., Tamin, A., Lowe, L., Frace, M., DeRisi, J. L., Chen, Q., Wang, D., Erdman, D. D., Peret, T. C., Burns, C., Ksiazek, T. G., Rollin, P. E., Sanchez, A., Liffick, S., Holloway, B., Limor, J., McCaustland, K., Olsen-Rasmussen, M., Fouchier, R., Gunther, S., Osterhaus, A. D., Drosten, C., Pallansch, M. A., Anderson, L. J., and Bellini, W. J. (2003) Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394– 1399,  DOI: 10.1126/science.1085952
      15
      Characterization of a novel coronavirus associated with severe acute respiratory syndrome
      Rota, Paul A.; Oberste, M. Steven; Monroe, Stephan S.; Nix, W. Allan; Campagnoli, Ray; Icenogle, Joseph P.; Penaranda, Silvia; Bankamp, Bettina; Maher, Kaija; Chen, Min-hsin; Tong, Suxiong; Tamin, Azaibi; Lowe, Luis; Frace, Michael; DeRisi, Joseph L.; Chen, Qi; Wang, David; Erdman, Dean D.; Peret, Teresa C. T.; Burns, Cara; Ksiazek, Thomas G.; Rollin, Pierre E.; Sanchez, Anthony; Liffick, Stephanie; Holloway, Brian; Limor, Josef; McCaustland, Karen; Olsen-Rasmussen, Melissa; Fouchier, Ron; Guenther, Stephan; Osterhaus, Albert D. M. E.; Drosten, Christian; Pallansch, Mark A.; Anderson, Larry J.; Bellini, William J.
      Science (Washington, DC, United States) (2003), 300 (5624), 1394-1399CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      In Mar. 2003, a novel coronavirus (SARS-CoV) was discovered in assocn. with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was detd., and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
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    16. 16
      de Groot, R. J., Baker, S. C., Baric, R. S., Brown, C. S., Drosten, C., Enjuanes, L., Fouchier, R. A., Galiano, M., Gorbalenya, A. E., Memish, Z. A., Perlman, S., Poon, L. L., Snijder, E. J., Stephens, G. M., Woo, P. C., Zaki, A. M., Zambon, M., and Ziebuhr, J. (2013) Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group. J. Virol. 87, 7790– 7792,  DOI: 10.1128/JVI.01244-13
      16
      Middle east respiratory syndrome coronavirus (MERS-CoV): announcement of the coronavirus study group
      de Groot, Raoul J.; Baker, Susan C.; Baric, Ralph S.; Brown, Caroline S.; Drosten, Christian; Enjuanes, Luis; Fouchier, Ron A. M.; Galiano, Monica; Gorbalenya, Alexander E.; Memish, Ziad A.; Perlman, Stanley; Poon, Leo L. M.; Snijder, Eric J.; Stephens, Gwen M.; Woo, Patrick C. Y.; Zaki, Ali M.; Zambon, Maria; Ziebuhr, John
      Journal of Virology (2013), 87 (14), 7790-7792CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
      A review. A brief review including epidemiol., description, and consensus name for Middle east respiratory syndrome coronavirus (MERS-CoV).
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    17. 17
      Zaki, A. M., van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D., and Fouchier, R. A. (2012) Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 367, 1814– 1820,  DOI: 10.1056/NEJMoa1211721
      17
      Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia
      Zaki, Ali Moh; van Boheemen, Sander; Bestebroer, Theo M.; Osterhaus, Albert D. M. E.; Fouchier, Ron A. M.
      New England Journal of Medicine (2012), 367 (19), 1814-1820CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
      A previously unknown coronavirus was isolated from the sputum of a 60-yr-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clin. data, virus isolation, and mol. identification are presented. The clin. picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
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    18. 18
      Bermingham, A., Chand, M. A., Brown, C. S., Aarons, E., Tong, C., Langrish, C., Hoschler, K., Brown, K., Galiano, M., Myers, R., Pebody, R. G., Green, H. K., Boddington, N. L., Gopal, R., Price, N., Newsholme, W., Drosten, C., Fouchier, R. A., and Zambon, M. (2012) Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012. Euro Surveill 17, 20290
      18
      Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012
      Bermingham A; Chand M A; Brown C S; Aarons E; Tong C; Langrish C; Hoschler K; Brown K; Galiano M; Myers R; Pebody R G; Green H K; Boddington N L; Gopal R; Price N; Newsholme W; Drosten C; Fouchier R A; Zambon M
      Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin (2012), 17 (40), 20290 ISSN:.
      Coronaviruses have the potential to cause severe transmissible human disease, as demonstrated by the severe acute respiratory syndrome (SARS) outbreak of 2003. We describe here the clinical and virological features of a novel coronavirus infection causing severe respiratory illness in a patient transferred to London, United Kingdom, from the Gulf region of the Middle East.
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    19. 19
      Woo, P. C., Lau, S. K., Chu, C. M., Chan, K. H., Tsoi, H. W., Huang, Y., Wong, B. H., Poon, R. W., Cai, J. J., Luk, W. K., Poon, L. L., Wong, S. S., Guan, Y., Peiris, J. S., and Yuen, K. Y. (2005) Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 79, 884– 895,  DOI: 10.1128/JVI.79.2.884-895.2005
      19
      Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia
      Woo, Patrick C. Y.; Lau, Susanna K. P.; Chu, Chung-ming; Chan, Kwok-hung; Tsoi, Hoi-wah; Huang, Yi; Wong, Beatrice H. L.; Poon, Rosana W. S.; Cai, James J.; Luk, Wei-kwang; Poon, Leo L. M.; Wong, Samson S. Y.; Guan, Yi; Peiris, J. S. Malik; Yuen, Kwok-yung
      Journal of Virology (2005), 79 (2), 884-895CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
      Despite extensive lab. investigations in patients with respiratory tract infections, no microbiol. cause can be identified in a significant proportion of patients. In the past 3 years, several novel respiratory viruses, including human metapneumovirus, severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and human coronavirus NL63, were discovered. Here the authors report the discovery of another novel coronavirus, coronavirus HKU1 (CoV-HKU1), from a 71-yr-old man with pneumonia who had just returned from Shenzhen, China. Quant. reverse transcription-PCR showed that the amt. of CoV-HKU1 RNA was 8.5 to 9.6×106 copies per mL in his nasopharyngeal aspirates (NPAs) during the first week of the illness and dropped progressively to undetectable levels in subsequent weeks. He developed increasing serum levels of specific antibodies against the recombinant nucleocapsid protein of CoV-HKU1, with IgM titers of 1:20, 1:40, and 1:80 and IgG titers of <1:1,000, 1:2,000, and 1:8,000 in the first, second and fourth weeks of the illness, resp. Isolation of the virus by using various cell lines, mixed neuron-glia culture, and intracerebral inoculation of suckling mice was unsuccessful. The complete genome sequence of CoV-HKU1 is a 29,926-nucleotide, polyadenylated RNA, with G+C content of 32%, the lowest among all known coronaviruses with available genome sequence. Phylogenetic anal. reveals that CoV-HKU1 is a new group 2 coronavirus. Screening of 400 NPAs, neg. for SARS-CoV, from patients with respiratory illness during the SARS period identified the presence of CoV-HKU1 RNA in an addnl. specimen, with a viral load of 1.13×106 copies per mL, from a 35-yr-old woman with pneumonia. The data support the existence of a novel group 2 coronavirus assocd. with pneumonia in humans.
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    20. 20
      Lu, G. and Liu, D. (2012) SARS-like virus in the Middle East: a truly bat-related coronavirus causing human diseases. Protein Cell 3, 803– 805,  DOI: 10.1007/s13238-012-2811-1
      20
      SARS-like virus in the Middle East: a truly bat-related coronavirus causing human diseases
      Lu Guangwen; Liu Di
      Protein & cell (2012), 3 (11), 803-5 ISSN:.
      There is no expanded citation for this reference.
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    21. 21
      Lu, G., Wang, Q., and Gao, G. F. (2015) Bat-to-human: spike features determining ’host jump’ of coronaviruses SARS-CoV, MERS-CoV, and beyond. Trends Microbiol. 23, 468– 478,  DOI: 10.1016/j.tim.2015.06.003
      21
      Bat-to-human: spike features determining 'host jump' of coronaviruses SARS-CoV, MERS-CoV, and beyond
      Lu, Guangwen; Wang, Qihui; Gao, George F.
      Trends in Microbiology (2015), 23 (8), 468-478CODEN: TRMIEA; ISSN:0966-842X. (Elsevier Ltd.)
      Both severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic pathogens that crossed the species barriers to infect humans. The mechanism of viral interspecies transmission is an important scientific question to be addressed. These coronaviruses contain a surface-located spike (S) protein that initiates infection by mediating receptor-recognition and membrane fusion and is therefore a key factor in host specificity. In addn., the S protein needs to be cleaved by host proteases before executing fusion, making these proteases a second determinant of coronavirus interspecies infection. Here, we summarize the progress made in the past decade in understanding the cross-species transmission of SARS-CoV and MERS-CoV by focusing on the features of the S protein, its receptor-binding characteristics, and the cleavage process involved in priming.
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    22. 22
      Ge, X. Y., Li, J. L., Yang, X. L., Chmura, A. A., Zhu, G., Epstein, J. H., Mazet, J. K., Hu, B., Zhang, W., Peng, C., Zhang, Y. J., Luo, C. M., Tan, B., Wang, N., Zhu, Y., Crameri, G., Zhang, S. Y., Wang, L. F., Daszak, P., and Shi, Z. L. (2013) Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503, 535– 538,  DOI: 10.1038/nature12711
      22
      Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor
      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
      Nature (London, United Kingdom) (2013), 503 (7477), 535-538CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      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 mol., 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 morphol., 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.
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    23. 23
      Lau, S. K., Woo, P. C., Li, K. S., Huang, Y., Tsoi, H. W., Wong, B. H., Wong, S. S., Leung, S. Y., Chan, K. H., and Yuen, K. Y. (2005) Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. U. S. A. 102, 14040– 14045,  DOI: 10.1073/pnas.0506735102
      23
      Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats
      Lau, Susanna K. P.; Woo, Patrick C. Y.; Li, Kenneth S. M.; Huang, Yi; Tsoi, Hoi-Wah; Wong, Beatrice H. L.; Wong, Samson S. Y.; Leung, Suet-Yi; Chan, Kwok-Hung; Yuen, Kwok-Yung
      Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (39), 14040-14045CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Although the finding of severe acute respiratory syndrome coronavirus (SARS-CoV) in caged palm civets from live animal markets in China has provided evidence for interspecies transmission in the genesis of the SARS epidemic, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV. In a surveillance study for CoV in noncaged animals from the wild areas of the Hong Kong Special Administration Region, we identified a CoV closely related to SARS-CoV (bat-SARS-CoV) from 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (Rhinolophus sinicus) by using RT-PCR. Sequencing and anal. of three bat-SARS-CoV genomes from samples collected at different dates showed that bat-SARS-CoV is closely related to SARS-CoV from humans and civets. Phylogenetic anal. showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b CoV, distantly related to known group 2 CoV. Most differences between the bat-SARS-CoV and SARS-CoV genomes were obsd. in the spike genes, ORF 3 and ORF 8, which are the regions where most variations also were obsd. between human and civet SARS-CoV genomes. In addn., the presence of a 29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats by using an enzyme immunoassay. Neutralizing antibody to human SARS-CoV also was detected in bats with lower viral loads. Precautions should be exercised in the handling of these animals.
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    24. 24
      Song, H. D., Tu, C. C., Zhang, G. W., Wang, S. Y., Zheng, K., Lei, L. C., Chen, Q. X., Gao, Y. W., Zhou, H. Q., Xiang, H., Zheng, H. J., Chern, S. W., Cheng, F., Pan, C. M., Xuan, H., Chen, S. J., Luo, H. M., Zhou, D. H., Liu, Y. F., He, J. F., Qin, P. Z., Li, L. H., Ren, Y. Q., Liang, W. J., Yu, Y. D., Anderson, L., Wang, M., Xu, R. H., Wu, X. W., Zheng, H. Y., Chen, J. D., Liang, G., Gao, Y., Liao, M., Fang, L., Jiang, L. Y., Li, H., Chen, F., Di, B., He, L. J., Lin, J. Y., Tong, S., Kong, X., Du, L., Hao, P., Tang, H., Bernini, A., Yu, X. J., Spiga, O., Guo, Z. M., Pan, H. Y., He, W. Z., Manuguerra, J. C., Fontanet, A., Danchin, A., Niccolai, N., Li, Y. X., Wu, C. I., and Zhao, G. P. (2005) Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human. Proc. Natl. Acad. Sci. U. S. A. 102, 2430– 2435,  DOI: 10.1073/pnas.0409608102
      24
      Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human
      Song, Huai-Dong; Tu, Chang-Chun; Zhang, Guo-Wei; Wang, Sheng-Yue; Zheng, Kui; Lei, Lian-Cheng; Chen, Qiu-Xia; Gao, Yu-Wei; Zhou, Hui-Qiong; Xiang, Hua; Zheng, Hua-Jun; Chern, Shur-Wern Wang; Cheng, Feng; Pan, Chun-Ming; Xuan, Hua; Chen, Sai-Juan; Luo, Hui-Ming; Zhou, Duan-Hua; Liu, Yu-Fei; He, Jian-Feng; Qin, Peng-Zhe; Li, Ling-Hui; Ren, Yu-Qi; Liang, Wen-Jia; Yu, Ye-Dong; Anderson, Larry; Wang, Ming; Xu, Rui-Heng; Wu, Xin-Wei; Zheng, Huan-Ying; Chen, Jin-Ding; Liang, Guodong; Gao, Yang; Liao, Ming; Fang, Ling; Jiang, Li-Yun; Li, Hui; Chen, Fang; Di, Biao; He, Li-Juan; Lin, Jin-Yan; Tong, Suxiang; Kong, Xiangang; Du, Lin; Hao, Pei; Tang, Hua; Bernini, Andrea; Yu, Xiao-Jing; Spiga, Ottavia; Guo, Zong-Ming; Pan, Hai-Yan; He, Wei-Zhong; Manuguerra, Jean-Claude; Fontanet, Arnaud; Danchin, Antoine; Niccolai, Neri; Li, Yi-Xue; Wu, Chung-I.; Zhao, Guo-Ping
      Proceedings of the National Academy of Sciences of the United States of America (2005), 102 (7), 2430-2435CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      The genomic sequences of severe acute respiratory syndrome coronaviruses from human and palm civet of the 2003/2004 outbreak in the city of Guangzhou, China, were nearly identical. Phylogenetic anal. suggested an independent viral invasion from animal to human in this new episode. Combining all existing data but excluding singletons, we identified 202 single-nucleotide variations. Among them, 17 are polymorphic in palm civets only. The ratio of nonsynonymous/synonymous nucleotide substitution in palm civets collected 1 yr apart from different geog. locations is very high, suggesting a rapid evolving process of viral proteins in civet as well, much like their adaptation in the human host in the early 2002-2003 epidemic. Major genetic variations in some crit. genes, particularly the Spike gene, seemed essential for the transition from animal-to-human transmission to human-to-human transmission, which eventually caused the first severe acute respiratory syndrome outbreak of 2002/2003.
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    25. 25
      Lau, S. K., Li, K. S., Tsang, A. K., Lam, C. S., Ahmed, S., Chen, H., Chan, K. H., Woo, P. C., and Yuen, K. Y. (2013) 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. J. Virol. 87, 8638– 8650,  DOI: 10.1128/JVI.01055-13
      25
      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
      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
      Journal of Virology (2013), 87 (15), 8638-8650CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
      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 examd. the mol. epidemiol. 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), resp. 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 pos. selected sites than that of Ty-BatCoV HKU4, suggesting that Pi-BatCoV HKU5 may generate variants to occupy new ecol. niches along with its host in diverse habitats. Mol. clock anal. 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.
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    26. 26
      Ithete, N. L., Stoffberg, S., Corman, V. M., Cottontail, V. M., Richards, L. R., Schoeman, M. C., Drosten, C., Drexler, J. F., and Preiser, W. (2013) Close relative of human Middle East respiratory syndrome coronavirus in bat, South Africa. Emerg. Infect. Dis. 19, 1697– 1699,  DOI: 10.3201/eid1910.130946
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      Close relative of human Middle East respiratory syndrome coronavirus in bat, South Africa
      Ithete Ndapewa Laudika; Stoffberg Samantha; Corman Victor Max; Cottontail Veronika M; Richards Leigh Rosanne; Schoeman M Corrie; Drosten Christian; Drexler Jan Felix; Preiser Wolfgang
      Emerging infectious diseases (2013), 19 (10), 1697-9 ISSN:.
      There is no expanded citation for this reference.
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    27. 27
      Corman, V. M., Ithete, N. L., Richards, L. R., Schoeman, M. C., Preiser, W., Drosten, C., and Drexler, J. F. (2014) Rooting the phylogenetic tree of middle East respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat. J. Virol. 88, 11297– 11303,  DOI: 10.1128/JVI.01498-14
      27
      Rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an African bat
      Corman, Victor Max; Ithete, Ndapewa Laudika; Richards, Leigh Rosanne; Schoeman, M. Corrie; Preiser, Wolfgang; Drosten, Christian; Drexler, Jan Felix
      Journal of Virology (2014), 88 (19), 11297-11303, 8 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)
      The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes lethal respiratory infections mainly on the Arabian Peninsula. The evolutionary origins of MERS-CoV are unknown. We detd. the full genome sequence of a CoV directly from fecal material obtained from a South African Neoromicia capensis bat (NeoCoV). NeoCoV shared essential details of genome architecture with MERS-CoV. Eighty-five percent of the NeoCoV genome was identical to MERS-CoV at the nucleotide level. Based on taxonomic criteria, NeoCoV and MERS-CoV belonged to one viral species. The presence of a genetically divergent S1 subunit within the NeoCoV spike gene indicated that intraspike recombination events may have been involved in the emergence of MERS-CoV. NeoCoV constitutes a sister taxon of MERS-CoV, placing the MERS-CoV root between a recently described virus from African camels and all other viruses. This suggests a higher level of viral diversity in camels than in humans. Together with serol. evidence for widespread MERS-CoV infection in camelids sampled up to 20 years ago in Africa and the Arabian Peninsula, the genetic data indicate that camels act as sources of virus for humans rather than vice versa. The majority of camels on the Arabian Peninsula is imported from the Greater Horn of Africa, where several Neoromicia species occur. The acquisition of MERS-CoV by camels from bats might have taken place in sub-Saharan Africa. Camelids may represent mixing vessels for MERS-CoV and other mammalian CoVs.
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    28. 28
      Yang, L., Wu, Z., Ren, X., Yang, F., Zhang, J., He, G., Dong, J., Sun, L., Zhu, Y., Zhang, S., and Jin, Q. (2014) MERS-related betacoronavirus in Vespertilio superans bats, China. Emerg. Infect. Dis. 20, 1260– 1262,  DOI: 10.3201/eid.2006.140318
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      MERS-related betacoronavirus in Vespertilio superans bats, China
      Yang Li; Wu Zhiqiang; Ren Xianwen; Yang Fan; Zhang Junpeng; He Guimei; Dong Jie; Sun Lilian; Zhu Yafang; Zhang Shuyi; Jin Qi
      Emerging infectious diseases (2014), 20 (7), 1260-2 ISSN:.
      There is no expanded citation for this reference.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cfmsVOjug%253D%253D&md5=23bdcbeae6698d440c64f1a4190df947
    29. 29
      Memish, Z. A., Mishra, N., Olival, K. J., Fagbo, S. F., Kapoor, V., Epstein, J. H., Alhakeem, R., Durosinloun, A., Al Asmari, M., Islam, A., Kapoor, A., Briese, T., Daszak, P., Al Rabeeah, A. A., and Lipkin, W. I. (2013) Middle East respiratory syndrome coronavirus in bats, Saudi Arabia. Emerg. Infect. Dis. 19, 1819– 1823,  DOI: 10.3201/eid1911.131172
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      Middle east respiratory syndrome coronavirus in bats, Saudi Arabia
      Memish, Ziad A.; Mishra, Nischay; Olival, Kevin J.; Fagbo, Shamsudeen F.; Kapoor, Vishal; Epstein, Jonathan H.; Al Hakeem, Rafat; Al Asmari, Mushabab; Islam, Ariful; Kapoor, Amit; Briese, Thomas; Daszak, Peter; Al Rabeeah, Abdullah A.; Lipkin, W. Ian
      Emerging Infectious Diseases (2013), 19 (11), 1819-1823CODEN: EIDIFA; ISSN:1080-6040. (Centers for Disease Control and Prevention)
      The source of human infection with Middle East respiratory syndrome coronavirus remains unknown. Mol. investigation indicated that bats in Saudi Arabia are infected with several alphacoronaviruses and betacoronaviruses. Virus from 1 bat showed 100% nucleotide identity to virus from the human index case-patient. Bats might play a role in human infection.
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    30. 30
      De Benedictis, P., Marciano, S., Scaravelli, D., Priori, P., Zecchin, B., Capua, I., Monne, I., and Cattoli, G. (2014) Alpha and lineage C betaCoV infections in Italian bats. Virus Genes 48, 366– 371,  DOI: 10.1007/s11262-013-1008-x
      30
      Alpha and lineage C betaCoV infections in Italian bats
      De Benedictis, Paola; Marciano, Sabrina; Scaravelli, Dino; Priori, Pamela; Zecchin, Barbara; Capua, Ilaria; Monne, Isabella; Cattoli, Giovanni
      Virus Genes (2014), 48 (2), 366-371CODEN: VIGEET; ISSN:0920-8569. (Springer)
      AlphaCoV and lineage C betaCoV, genetically similar to those identified in Spanish related bat species, have been detected in Italian Myotis blythii and Eptesicus serotinus, resp., out of 75 anal swabs collected from Vespertilionidae between 2009 and 2012. Sequence anal. of the 816-bp obtained RdRp sequence fragment indicates a 96.9% amino acid identity of the Italian lineage C betaCoV with the recent Middle East Respiratory Syndrome Coronavirus (MERS-CoV, Genbank accession no. KF192507). This is the first documented occurrence of a lineage C betaCoV in the Italian bat population, notably in E. serotinus.
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    31. 31
      Annan, A., Baldwin, H. J., Corman, V. M., Klose, S. M., Owusu, M., Nkrumah, E. E., Badu, E. K., Anti, P., Agbenyega, O., Meyer, B., Oppong, S., Sarkodie, Y. A., Kalko, E. K., Lina, P. H., Godlevska, E. V., Reusken, C., Seebens, A., Gloza-Rausch, F., Vallo, P., Tschapka, M., Drosten, C., and Drexler, J. F. (2013) Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe. Emerg. Infect. Dis. 19, 456– 459,  DOI: 10.3201/eid1903.121503
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      Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe
      Annan Augustina; Baldwin Heather J; Corman Victor Max; Klose Stefan M; Owusu Michael; Nkrumah Evans Ewald; Badu Ebenezer Kofi; Anti Priscilla; Agbenyega Olivia; Meyer Benjamin; Oppong Samuel; Sarkodie Yaw Adu; Kalko Elisabeth K V; Lina Peter H C; Godlevska Elena V; Reusken Chantal; Seebens Antje; Gloza-Rausch Florian; Vallo Peter; Tschapka Marco; Drosten Christian; Drexler Jan Felix
      Emerging infectious diseases (2013), 19 (3), 456-9 ISSN:.
      We screened fecal specimens of 4,758 bats from Ghana and 272 bats from 4 European countries for betacoronaviruses. Viruses related to the novel human betacoronavirus EMC/2012 were detected in 46 (24.9%) of 185 Nycteris bats and 40 (14.7%) of 272 Pipistrellus bats. Their genetic relatedness indicated EMC/2012 originated from bats.
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    32. 32
      Azhar, E. I., El-Kafrawy, S. A., Farraj, S. A., Hassan, A. M., Al-Saeed, M. S., Hashem, A. M., and Madani, T. A. (2014) Evidence for camel-to-human transmission of MERS coronavirus. N. Engl. J. Med. 370, 2499– 2505,  DOI: 10.1056/NEJMoa1401505
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      Evidence for camel-to-human transmission of MERS coronavirus
      Azhar, Esam I.; El-Kafrawy, Sherif A.; Farraj, Suha A.; Hassan, Ahmed M.; Al-Saeed, Muneera S.; Hashem, Anwar M.; Madani, Tariq A.
      New England Journal of Medicine (2014), 370 (26), 2499-2505, 7 pp.CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      We describe the isolation and sequencing of Middle East respiratory syndrome coronavirus (MERS-CoV) obtained from a dromedary camel and from a patient who died of lab.-confirmed MERS-CoV infection after close contact with camels that had rhinorrhea. Nasal swabs collected from the patient and from one of his nine camels were pos. for MERS-CoV RNA. In addn., MERS-CoV was isolated from the patient and the camel. The full genome sequences of the two isolates were identical. Serol. data indicated that MERS-CoV was circulating in the camels but not in the patient before the human infection occurred. These data suggest that this fatal case of human MERS-CoV infection was transmitted through close contact with an infected camel.
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    33. 33
      Reusken, C. B., Haagmans, B. L., Muller, M. A., Gutierrez, C., Godeke, G. J., Meyer, B., Muth, D., Raj, V. S., Smits-De Vries, L., Corman, V. M., Drexler, J. F., Smits, S. L., El Tahir, Y. E., De Sousa, R., van Beek, J., Nowotny, N., van Maanen, K., Hidalgo-Hermoso, E., Bosch, B. J., Rottier, P., Osterhaus, A., Gortazar-Schmidt, C., Drosten, C., and Koopmans, M. P. (2013) Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect. Dis. 13, 859– 866,  DOI: 10.1016/S1473-3099(13)70164-6
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      Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study
      Reusken, Chantal BEM; Haagmans, Bart L.; Mueller, Marcel A.; Gutierrez, Carlos; Godeke, Gert-Jan; Meyer, Benjamin; Muth, Doreen; Raj, V. Stalin; Vries, Laura Smits-De; Corman, Victor M.; Drexler, Jan-Felix; Smits, Saskia L.; El Tahir, Yasmin E.; De Sousa, Rita; van Beek, Janko; Nowotny, Norbert; van Maanen, Kees; Hidalgo-Hermoso, Ezequiel; Bosch, Berend-Jan; Rottier, Peter; Osterhaus, Albert; Gortazar-Schmidt, Christian; Drosten, Christian; Koopmans, Marion PG
      Lancet Infectious Diseases (2013), 13 (10), 859-866CODEN: LIDABP; ISSN:1473-3099. (Elsevier Ltd.)
      Background: A new betacoronavirus-Middle East respiratory syndrome coronavirus (MERS-CoV)-has been identified in patients with severe acute respiratory infection. Although related viruses infect bats, mol. clock analyses have been unable to identify direct ancestors of MERS-CoV. Anecdotal exposure histories suggest that patients had been in contact with dromedary camels or goats. We investigated possible animal reservoirs of MERS-CoV by assessing specific serum antibodies in livestock. Methods: We took sera from animals in the Middle East (Oman) and from elsewhere (Spain, Netherlands, Chile). Cattle (n=80), sheep (n=40), goats (n=40), dromedary camels (n=155), and various other camelid species (n=34) were tested for specific serum IgG by protein microarray using the receptor-binding S1 subunits of spike proteins of MERS-CoV, severe acute respiratory syndrome coronavirus, and human coronavirus OC43. Results were confirmed by virus neutralisation tests for MERS-CoV and bovine coronavirus. Findings: 50 of 50 (100%) sera from Omani camels and 15 of 105 (14%) from Spanish camels had protein-specific antibodies against MERS-CoV spike. Sera from European sheep, goats, cattle, and other camelids had no such antibodies. MERS-CoV neutralizing antibody titers varied between 1/320 and 1/2560 for the Omani camel sera and between 1/20 and 1/320 for the Spanish camel sera. There was no evidence for cross-neutralization by bovine coronavirus antibodies. Interpretation: MERS-CoV or a related virus has infected camel populations. Both titers and seroprevalences in sera from different locations in Oman suggest widespread infection.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1Ggt7nM&md5=7f399ed0b22c6f2620781c36e154ace5
    34. 34
      Raj, V. S., Mou, H., Smits, S. L., Dekkers, D. H., Muller, M. A., Dijkman, R., Muth, D., Demmers, J. A., Zaki, A., Fouchier, R. A., Thiel, V., Drosten, C., Rottier, P. J., Osterhaus, A. D., Bosch, B. J., and Haagmans, B. L. (2013) Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature 495, 251– 254,  DOI: 10.1038/nature12005
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      Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC
      Raj, V. Stalin; Mou, Huihui; Smits, Saskia L.; Dekkers, Dick H. W.; Mueller, Marcel A.; Dijkman, Ronald; Muth, Doreen; Demmers, Jeroen A. A.; Zaki, Ali; Fouchier, Ron A. M.; Thiel, Volker; Drosten, Christian; Rottier, Peter J. M.; Osterhaus, Albert D. M. E.; Bosch, Berend Jan; Haagmans, Bart L.
      Nature (London, United Kingdom) (2013), 495 (7440), 251-254CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Most human coronaviruses cause mild upper respiratory tract disease but may be assocd. with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome anal. revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addn., it will contribute critically to our understanding of the pathogenesis and epidemiol. of this emerging human coronavirus, and may facilitate the development of intervention strategies.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjvFensb8%253D&md5=7b80b018010e90aaf64842d9f54c032c
    35. 35
      Wang, Q., Qi, J., Yuan, Y., Xuan, Y., Han, P., Wan, Y., Ji, W., Li, Y., Wu, Y., Wang, J., Iwamoto, A., Woo, P. C., Yuen, K. Y., Yan, J., Lu, G., and Gao, G. F. (2014) Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26. Cell Host Microbe 16, 328– 337,  DOI: 10.1016/j.chom.2014.08.009
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      Bat Origins of MERS-CoV Supported by Bat Coronavirus HKU4 Usage of Human Receptor CD26
      Wang, Qihui; Qi, Jianxun; Yuan, Yuan; Xuan, Yifang; Han, Pengcheng; Wan, Yuhua; Ji, Wei; Li, Yan; Wu, Ying; Wang, Jianwei; Iwamoto, Aikichi; Woo, Patrick C. Y.; Yuen, Kwok-Yung; Yan, Jinghua; Lu, Guangwen; Gao, George F.
      Cell Host & Microbe (2014), 16 (3), 328-337CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)
      The recently reported Middle East respiratory syndrome coronavirus (MERS-CoV) is phylogenetically closely related to the bat coronaviruses (BatCoVs) HKU4 and HKU5. However, the evolutionary pathway of MERS-CoV is still unclear. A receptor binding domain (RBD) in the MERS-CoV envelope-embedded spike protein specifically engages human CD26 (hCD26) to initiate viral entry. The high sequence identity in the viral spike protein prompted us to investigate if HKU4 and HKU5 can recognize hCD26 for cell entry. We found that HKU4-RBD, but not HKU5-RBD, binds to hCD26, and pseudotyped viruses embedding HKU4 spike can infect cells via hCD26 recognition. The structure of the HKU4-RBD/hCD26 complex revealed a hCD26-binding mode similar overall to that obsd. for MERS-RBD. HKU4-RBD, however, is less adapted to hCD26 than MERS-RBD, explaining its lower affinity for receptor binding. Our findings support a bat origin for MERS-CoV and indicate the need for surveillance of HKU4-related viruses in bats.
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    36. 36
      Yang, Y., Du, L., Liu, C., Wang, L., Ma, C., Tang, J., Baric, R. S., Jiang, S., and Li, F. (2014) Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc. Natl. Acad. Sci. U. S. A. 111, 12516– 12521,  DOI: 10.1073/pnas.1405889111
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      Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus
      Yang, Yang; Du, Lanying; Liu, Chang; Wang, Lili; Ma, Cuiqing; Tang, Jian; Baric, Ralph S.; Jiang, Shibo; Li, Fang
      Proceedings of the National Academy of Sciences of the United States of America (2014), 111 (34), 12516-12521CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.
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    37. 37
      Wang, Q., Wong, G., Lu, G., Yan, J., and Gao, G. F. (2016) MERS-CoV spike protein: Targets for vaccines and therapeutics. Antiviral Res. 133, 165– 177,  DOI: 10.1016/j.antiviral.2016.07.015
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      MERS-CoV spike protein: Targets for vaccines and therapeutics
      Wang, Qihui; Wong, Gary; Lu, Guangwen; Yan, Jinghua; Gao, George F.
      Antiviral Research (2016), 133 (), 165-177CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)
      The disease outbreak caused by Middle East respiratory syndrome coronavirus (MERS-CoV) is still ongoing in the Middle East. Over 1700 people have been infected since it was first reported in Sept. 2012. Despite great efforts, licensed vaccines or therapeutics against MERS-CoV remain unavailable. The MERS-CoV spike (S) protein is an important viral antigen known to mediate host-receptor binding and virus entry, as well as induce robust humoral and cell-mediated responses in humans during infection. In this review, we highlight the importance of the S protein in the MERS-CoV life cycle, summarize recent advances in the development of vaccines and therapeutics based on the S protein, and discuss strategies that can be explored to develop new medical countermeasures against MERS-CoV.
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    38. 38
      Li, F., Li, W., Farzan, M., and Harrison, S. C. (2005) Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864– 1868,  DOI: 10.1126/science.1116480
      38
      Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor
      Li, Fang; Li, Wenhui; Farzan, Michael; Harrison, Stephen C.
      Science (Washington, DC, United States) (2005), 309 (5742), 1864-1868CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The spike protein (S) of SARS coronavirus (SARS-CoV) attaches the virus to its cellular receptor, angiotensin-converting enzyme 2 (ACE2). A defined receptor-binding domain (RBD) on S mediates this interaction. The crystal structure at 2.9 angstrom resoln. of the RBD bound with the peptidase domain of human ACE2 shows that the RBD presents a gently concave surface, which cradles the N-terminal lobe of the peptidase. The at. details at the interface between the two proteins clarify the importance of residue changes that facilitate efficient cross-species infection and human-to-human transmission. The structure of the RBD suggests ways to make truncated disulfide-stabilized RBD variants for use in the design of coronavirus vaccines.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXpvFCisLw%253D&md5=0a7ac2fb7a76c97979d859c0489c682f
    39. 39
      Lu, G., Hu, Y., Wang, Q., Qi, J., Gao, F., Li, Y., Zhang, Y., Zhang, W., Yuan, Y., Bao, J., Zhang, B., Shi, Y., Yan, J., and Gao, G. F. (2013) Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature 500, 227– 231,  DOI: 10.1038/nature12328
      39
      Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26
      Lu, Guangwen; Hu, Yawei; Wang, Qihui; Qi, Jianxun; Gao, Feng; Li, Yan; Zhang, Yanfang; Zhang, Wei; Yuan, Yuan; Bao, Jinku; Zhang, Buchang; Shi, Yi; Yan, Jinghua; Gao, George F.
      Nature (London, United Kingdom) (2013), 500 (7461), 227-231CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV. CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. Here we delineate the mol. basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissocn. const. of 16.7nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 β-propeller. The at. details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhtVKhtbfK&md5=37bd04e399f46ba56e21f97bbd5d8b0d
    40. 40
      Wang, N., Shi, X., Jiang, L., Zhang, S., Wang, D., Tong, P., Guo, D., Fu, L., Cui, Y., Liu, X., Arledge, K. C., Chen, Y. H., Zhang, L., and Wang, X. (2013) Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell Res. 23, 986– 993,  DOI: 10.1038/cr.2013.92
      40
      Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4
      Wang, Nianshuang; Shi, Xuanling; Jiang, Liwei; Zhang, Senyan; Wang, Dongli; Tong, Pei; Guo, Dongxing; Fu, Lili; Cui, Ye; Liu, Xi; Arledge, Kelly C.; Chen, Ying-Hua; Zhang, Linqi; Wang, Xinquan
      Cell Research (2013), 23 (8), 986-993CODEN: CREEB6; ISSN:1001-0602. (NPG Nature Asia-Pacific)
      The spike glycoprotein (S) of recently identified Middle East respiratory syndrome coronavirus (MERS-CoV) targets the cellular receptor, dipeptidyl peptidase 4 (DPP4). Sequence comparison and modeling anal. have revealed a putative receptor-binding domain (RBD) on the viral spike, which mediates this interaction. We report the 3.0 Å-resoln. crystal structure of MERS-CoV RBD bound to the extracellular domain of human DPP4. Our results show that MERS-CoV RBD consists of a core and a receptor-binding subdomain. The receptor-binding subdomain interacts with DPP4 β-propeller but not its intrinsic hydrolase domain. MERS-CoV RBD and related SARS-CoV RBD share a high degree of structural similarity in their core subdomains, but are notably divergent in the receptor-binding subdomain. Mutagenesis studies have identified several key residues in the receptor-binding subdomain that are crit. for viral binding to DPP4 and entry into the target cell. The at. details at the interface between MERS-CoV RBD and DPP4 provide structural understanding of the virus and receptor interaction, which can guide development of therapeutics and vaccines against MERS-CoV infection.
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    41. 41
      Peng, G., Sun, D., Rajashankar, K. R., Qian, Z., Holmes, K. V., and Li, F. (2011) Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor. Proc. Natl. Acad. Sci. U. S. A. 108, 10696– 10701,  DOI: 10.1073/pnas.1104306108
      41
      Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor
      Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (26), 10696-10701, S10696/1-S10696/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      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 mol. 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same β-sandwich fold as human galectins (S-lectins) and addnl. 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. Crit. 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 anal. and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the β-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.
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    42. 42
      Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., Choe, H., and Farzan, M. (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450– 454,  DOI: 10.1038/nature02145
      42
      Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus
      Li, Wenhui; Moore, Michael J.; Vasilieva, Natalya; Sui, Jianhua; Wong, Swee Kee; Berne, Michael A.; Somasundaran, Mohan; Sullivan, John L.; Luzuriaga, Katherine; Greenough, Thomas C.; Choe, Hyeryun; Farzan, Michael
      Nature (London, United Kingdom) (2003), 426 (6965), 450-454CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Spike (S) proteins of coronaviruses, including the coronavirus that causes severe acute respiratory syndrome (SARS), assoc. with cellular receptors to mediate infection of their target cells. Here we identify a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), isolated from SARS coronavirus (SARS-CoV)-permissive Vero E6 cells, that efficiently binds the S1 domain of the SARS-CoV S protein. We found that a sol. form of ACE2, but not of the related enzyme ACE1, blocked assocn. of the S1 domain with Vero E6 cells. 293T cells transfected with ACE2, but not those transfected with human immunodeficiency virus-1 receptors, formed multinucleated syncytia with cells expressing S protein. Furthermore, SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Finally, anti-ACE2 but not anti-ACE1 antibody blocked viral replication on Vero E6 cells. Together our data indicate that ACE2 is a functional receptor for SARS-CoV.
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    43. 43
      Ge, X., Li, Y., Yang, X., Zhang, H., Zhou, P., Zhang, Y., and Shi, Z. (2012) Metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in China. J. Virol. 86, 4620– 4630,  DOI: 10.1128/JVI.06671-11
      43
      Metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in China
      Ge, Xingyi; Li, Yan; Yang, Xinglou; Zhang, Huajun; Zhou, Peng; Zhang, Yunzhi; Shi, Zhengli
      Journal of Virology (2012), 86 (8), 4620-4630CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
      Increasing data indicate that bats harbor diverse viruses, some of which cause severe human diseases. In this study, sequence-independent amplification and high-throughput sequencing (Solexa) were applied to the metagenomic anal. of viruses in bat fecal samples collected from 6 locations in China. A total of 8746,417 reads with a length of 306,124,595 bp were obtained. Among these reads, 13,541 (0.15%) had similarity to phage sequences and 9170 (0.1%) had similarity to eukaryotic virus sequences. A total of 129 assembled contigs (>100 nucleotides) were constructed and compared with GenBank: 32 contigs were related to phages, and 97 were related to eukaryotic viruses. The most frequent reads and contigs related to eukaryotic viruses were homologous to densoviruses, dicistroviruses, coronaviruses, parvoviruses, and tobamoviruses, a range that includes viruses from invertebrates, vertebrates, and plants. Most of the contigs had low identities to known viral genomic or protein sequences, suggesting that a large no. of novel and genetically diverse insect viruses as well as putative mammalian viruses are transmitted by bats in China. This study provides the first preliminary understanding of the virome of some bat populations in China, which may guide the discovery and isolation of novel viruses in the future.
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    44. 44
      Lau, S. K., Poon, R. W., Wong, B. H., Wang, M., Huang, Y., Xu, H., Guo, R., Li, K. S., Gao, K., Chan, K. H., Zheng, B. J., Woo, P. C., and Yuen, K. Y. (2010) Coexistence of different genotypes in the same bat and serological characterization of Rousettus bat coronavirus HKU9 belonging to a novel Betacoronavirus subgroup. J. Virol. 84, 11385– 11394,  DOI: 10.1128/JVI.01121-10
      44
      Coexistence of different genotypes in the same bat and serological characterization of Rousettus bat coronavirus HKU9 belonging to a novel Betacoronavirus subgroup
      Lau, Susanna K. P.; Poon, Rosana W. S.; Wong, Beatrice H. L.; Wang, Ming; Huang, Yi; Xu, Huifang; Guo, Rongtong; Li, Kenneth S. M.; Gao, Kai; Chan, Kwok-Hung; Zheng, Bo-Jian; Woo, Patrick C. Y.; Yuen, Kwok-Yung
      Journal of Virology (2010), 84 (21), 11385-11394CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)
      Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9), a recently identified coronavirus of novel Betacoronavirus subgroup D (Nobecovirus), from Leschenault's rousette, was previously found to display marked sequence polymorphism among genomes of four strains. Among 10 bats with complete RNA-dependent RNA polymerase (RdRp), spike (S), and nucleocapsid (N) genes sequenced, three and two sequence clades for all three genes were codetected in two and five bats, resp., suggesting the coexistence of two or three distinct genotypes of Ro-BatCoV HKU9 in the same bat. Complete genome sequencing of the distinct genotypes from two bats, using degenerate/genome-specific primers with overlapping sequences confirmed by specific PCR, supported the coexistence of at least two distinct genomes in each bat. Recombination anal. using eight Ro-BatCoV HKU9 genomes showed possible recombination events between strains from different bat individuals, which may have allowed for the generation of different genotypes. Western blot assays using recombinant N proteins of Ro-BatCoV HKU9, Betacoronavirus subgroup A (HCoV-HKU1), subgroup B (SARSr-Rh-BatCoV), and subgroup C (Ty-BatCoV HKU4 and Pi-BatCoV HKU5) coronaviruses were subgroup specific, supporting their classification as sep. subgroups under Betacoronavirus. Antibodies were detected in 75 (43%) of 175 and 224 (64%) of 350 tested serum samples from Leschenault's rousette bats by Ro-BatCoV HKU9 N-protein-based Western blot and enzyme immunoassays, resp. This is the first report describing coinfection of different coronavirus genotypes in bats and coronavirus genotypes of diverse nucleotide variation in the same host. Such unique phenomena, and the unusual instability of ORF7a, are likely due to recombination which may have been facilitated by the dense roosting behavior and long foraging range of Leschenault's rousette.
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    45. 45
      Tao, Y., Tang, K., Shi, M., Conrardy, C., Li, K. S., Lau, S. K., Anderson, L. J., and Tong, S. (2012) Genomic characterization of seven distinct bat coronaviruses in Kenya. Virus Res. 167, 67– 73,  DOI: 10.1016/j.virusres.2012.04.007
      45
      Genomic characterization of seven distinct bat coronaviruses in Kenya
      Tao, Ying; Tang, Kevin; Shi, Mang; Conrardy, Christina; Li, Kenneth S. M.; Lau, Susanna K. P.; Anderson, Larry J.; Tong, Suxiang
      Virus Research (2012), 167 (1), 67-73CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)
      To better understand the genetic diversity and genomic features of 41 coronaviruses (CoVs) identified from Kenya bats in 2006, seven CoVs as representatives of seven different phylogenetic groups identified from partial polymerase gene sequences, were subjected to extensive genomic sequencing. As a result, 15-16 kb nucleotide sequences encoding complete RNA dependent RNA polymerase, spike, envelope, membrane, and nucleocapsid proteins plus other open reading frames (ORFs) were generated. Sequences anal. confirmed that the CoVs from Kenya bats are divergent members of Alphacoronavirus and Betacoronavirus genera. Furthermore, the CoVs BtKY22, BtKY41, and BtKY43 in Alphacoronavirus genus and BtKY24 in Betacoronavirus genus are likely representatives of 4 novel CoV species. BtKY27 and BtKY33 are members of the established bat CoV species in Alphacoronavirus genus and BtKY06 is a member of the established bat CoV species in Betacoronavirus genus. The genome organization of these seven CoVs is similar to other known CoVs from the same groups except for differences in the no. of putative ORFs following the N gene. The present results confirm a significant diversity of CoVs circulating in Kenya bats. These Kenya bat CoVs are phylogenetically distant from any previously described human and animal CoVs. However, because of the examples of host switching among CoVs after relatively minor sequence changes in S1 domain of spike protein, a further surveillance in animal reservoirs and understanding the interface between host susceptibility is crit. for predicting and preventing the potential threat of bat CoVs to public health.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmvFKns7w%253D&md5=1e24cdd424f377176f745aad8b693175
    46. 46
      Tong, S., Conrardy, C., Ruone, S., Kuzmin, I. V., Guo, X., Tao, Y., Niezgoda, M., Haynes, L., Agwanda, B., Breiman, R. F., Anderson, L. J., and Rupprecht, C. E. (2009) Detection of novel SARS-like and other coronaviruses in bats from Kenya. Emerg. Infect. Dis. 15, 482– 485,  DOI: 10.3201/eid1503.081013
      46
      Detection of novel SARS-like and other coronaviruses in bats from Kenya
      Tong Suxiang; Conrardy Christina; Ruone Susan; Kuzmin Ivan V; Guo Xiling; Tao Ying; Niezgoda Michael; Haynes Lia; Agwanda Bernard; Breiman Robert F; Anderson Larry J; Rupprecht Charles E
      Emerging infectious diseases (2009), 15 (3), 482-5 ISSN:.
      Diverse coronaviruses have been identified in bats from several continents but not from Africa. We identified group 1 and 2 coronaviruses in bats in Kenya, including SARS-related coronaviruses. The sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans.
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    47. 47
      Zhang, W., Qi, J., Shi, Y., Li, Q., Gao, F., Sun, Y., Lu, X., Lu, Q., Vavricka, C. J., Liu, D., Yan, J., and Gao, G. F. (2010) Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus. Protein Cell 1, 459– 467,  DOI: 10.1007/s13238-010-0059-1
      47
      Crystal structure of the swine-origin A (H1N1)-2009 influenza A virus hemagglutinin (HA) reveals similar antigenicity to that of the 1918 pandemic virus
      Zhang, Wei; Qi, Jianxun; Shi, Yi; Li, Qing; Gao, Feng; Sun, Yeping; Lu, Xishan; Lu, Qiong; Vavricka, Christopher J.; Liu, Di; Yan, Jinghua; Gao, George F.
      Protein & Cell (2010), 1 (5), 459-467CODEN: PCREFB; ISSN:1674-800X. (Higher Education Press)
      Influenza virus is the causative agent of the seasonal and occasional pandemic flu. The current H1N1 influenza pandemic, announced by the WHO in June 2009, is highly contagious and responsible for global economic losses and fatalities. Although the H1N1 gene segments have three origins in terms of host species, the virus has been named swine-origin influenza virus (S-OIV) due to a predominant swine origin. 2009 S-OIV has been shown to highly resemble the 1918 pandemic virus in many aspects. Hemagglutinin is responsible for the host range and receptor binding of the virus and is therefore a primary indicator for the potential of infection. Primary sequence anal. of the 2009 S-OIV hemagglutinin (HA) reveals its closest relationship to that of the 1918 pandemic influenza virus, however, anal. at the structural level is necessary to critically assess the functional significance. In this report, we report the crystal structure of sol. hemagglutinin H1 (09H1) at 2.9 Å, illustrating that the 09H1 is very similar to the 1918 pandemic HA (18H1) in overall structure and the structural modules, including the five defined antiboby (Ab)-binding epitopes. Our results provide an explanation as to why sera from the survivors of the 1918 pandemics can neutralize the 2009 S-OIV, and people born around the 1918 are resistant to the current pandemic, yet younger generations are more susceptible to the 2009 pandemic.
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    48. 48
      Song, H., Qi, J., Khedri, Z., Diaz, S., Yu, H., Chen, X., Varki, A., Shi, Y., and Gao, G. F. (2016) An open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly-identified influenza D virus: basis for its broad cell tropism. PLoS Pathog. 12, e1005411,  DOI: 10.1371/journal.ppat.1005411
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      An open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly-identified influenza D virus: basis for its broad cell tropism
      Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F.
      PLoS Pathogens (2016), 12 (1), e1005411/1-e1005411/24CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
      Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. We reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we detd. the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFGgtL%252FP&md5=908fdafa477e150c619f4039d67b3953
    49. 49
      Otwinowski, Z. and Minor, W. (1997) Processing of X-ray diffraction data. Methods Enzymol. 276, 307– 326,  DOI: 10.1016/S0076-6879(97)76066-X
      49
      Processing of x-ray diffraction data collected in oscillation mode
      Otwinowski, Zbyszek; Minor, Wladek
      Methods in Enzymology (1997), 276 (Macromolecular Crystallography, Part A), 307-326CODEN: MENZAU; ISSN:0076-6879. (Academic)
      Macromol. crystallog. is an iterative process. Rarely do the first crystals provide all the necessary data to solve the biol. problem being studied. Each step benefits from experience learned in previous steps. To monitor the progress, the HKL package provides 2 tools: (1) statistics, both weighted (χ2) and unweighted (R-merge), are provided, and the Bayesian reasoning and multicomponent error model facilitates obtaining the proper error ests. and (2) visualization of the process plays a double role by helping the operator to confirm that the process of data redn., including the resulting statistics, is correct, and allowing one to evaluate problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and redn. has been reached. At that point, the effort should be directed to solving the structure. The methods presented here have been applied to solve a large variety of problems, from inorg. mols. with 5 Å unit cell to rotavirus of 700 Å diam. crystd. in 700 × 1000 × 1400 Å cell. Overall quality of the method was tested by many researchers by successful application of the programs to MAD structure detns.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXivFehsbw%253D&md5=c9536971d4e32cc35352c40fb9368131
    50. 50
      Uson, I. and Sheldrick, G. M. (1999) Advances in direct methods for protein crystallography. Curr. Opin. Struct. Biol. 9, 643– 648,  DOI: 10.1016/S0959-440X(99)00020-2
      50
      Advances in direct methods for protein crystallography
      Uson, Isabel; Sheldrick, George M.
      Current Opinion in Structural Biology (1999), 9 (5), 643-648CODEN: COSBEF; ISSN:0959-440X. (Current Biology Publications)
      A review with 43 refs. Recent advances in ab initio direct methods have enabled the soln. of crystal structures of small proteins from native x-ray data alone, i.e., without the use of fragments of known structure or the need to prep. heavy-atom or selenomethionine derivs., provided that the data are available to at. resoln. These methods are also proving to be useful for locating the selenium atoms or other anomalous scatterers in the multiple wavelength anomalous diffraction phasing of larger proteins at lower resoln.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXmslajt7g%253D&md5=07a077eda53963e2c4441a2d79a95037
    51. 51
      Read, R. J. (2001) Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr., Sect. D: Biol. Crystallogr. 57, 1373– 1382,  DOI: 10.1107/S0907444901012471
      51
      Pushing the boundaries of molecular replacement with maximum likelihood
      Read, Randy J.
      Acta Crystallographica, Section D: Biological Crystallography (2001), D57 (10), 1373-1382CODEN: ABCRE6; ISSN:0907-4449. (Munksgaard International Publishers Ltd.)
      The mol.-replacement method works well with good models and simple unit cells, but often fails with more difficult problems. Experience with likelihood in other areas of crystallog. suggests that it would improve performance significantly. For mol. replacement, the form of the required likelihood function depends on whether there is ambiguity in the relative phases of the contributions from symmetry-related mols. (e.g. rotation vs. translation searches). Likelihood functions used in structure refinement are appropriate only for translation (or six-dimensional) searches, where the correct translation will place all of the atoms in the model approx. correctly. A new likelihood function that allows for unknown relative phases is suitable for rotation searches. It is shown that correlations between sequence identity and coordinate error can be used to calibrate parameters for model quality in the likelihood functions. Multiple models of a mol. can be combined in a statistically valid way by setting up the joint probability distribution of the true and model structure factors as a multivariate complex normal distribution, from which the conditional distribution of the true structure factor given the models can be derived. Tests in a new mol.-replacement program, Beast, show that the likelihood-based targets are more sensitive and more accurate than previous targets. The new multiple-model likelihood function has a dramatic impact on success.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXmvFGltrc%253D&md5=b342c58edd40265324e0241ff75cec67
    52. 52
      Cowtan, K. D. and Zhang, K. Y. (1999) Density modification for macromolecular phase improvement. Prog. Biophys. Mol. Biol. 72, 245– 270,  DOI: 10.1016/S0079-6107(99)00008-5
      52
      Density modification for macromolecular phase improvement
      Cowtan K D; Zhang K Y
      Progress in biophysics and molecular biology (1999), 72 (3), 245-70 ISSN:0079-6107.
      Density modification provides a simple and largely automatic tool for improving phase estimates for observed structure factors. The phase information arises from a combination of the known structure factor magnitudes, the current phase estimates, and stereochemical information. The magnitudes, the current phase estimates, and stereochemical information. The addition of these phase information derived from theoretical sources renders new structures amenable to solution, and reduces the effort required to solve other structures. A diverse array of techniques which have been applied to the phase improvement problem are reviewed.
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    53. 53
      Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 213– 221,  DOI: 10.1107/S0907444909052925
      53
      PHENIX: a comprehensive Python-based system for macromolecular structure solution
      Adams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
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    54. 54
      Emsley, P. and Cowtan, K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60, 2126– 2132,  DOI: 10.1107/S0907444904019158
      54
      Coot: model-building tools for molecular graphics
      Emsley, Paul; Cowtan, Kevin
      Acta Crystallographica, Section D: Biological Crystallography (2004), D60 (12, Pt. 1), 2126-2132CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)
      CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for x-ray structure soln., structure comparison and anal., and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVars73P&md5=1be390f3bb6fd584468499ad0921161e
    55. 55
      Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 12– 21,  DOI: 10.1107/S0907444909042073
      55
      MolProbity: all-atom structure validation for macromolecular crystallography
      Chen, Vincent B.; Arendall, W. Bryan, III; Headd, Jeffrey J.; Keedy, Daniel A.; Immormino, Robert M.; Kapral, Gary J.; Murray, Laura W.; Richardson, Jane S.; Richardson, David C.
      Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 12-21CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)
      MolProbity is a structure-validation web service that provides broad-spectrum solidly based evaluation of model quality at both the global and local levels for both proteins and nucleic acids. It relies heavily on the power and sensitivity provided by optimized hydrogen placement and all-atom contact anal., complemented by updated versions of covalent-geometry and torsion-angle criteria. Some of the local corrections can be performed automatically in MolProbity and all of the diagnostics are presented in chart and graphical forms that help guide manual rebuilding. X-ray crystallog. provides a wealth of biol. important mol. data in the form of at. three-dimensional structures of proteins, nucleic acids and increasingly large complexes in multiple forms and states. Advances in automation, in everything from crystn. to data collection to phasing to model building to refinement, have made solving a structure using crystallog. easier than ever. However, despite these improvements, local errors that can affect biol. interpretation are widespread at low resoln. and even high-resoln. structures nearly all contain at least a few local errors such as Ramachandran outliers, flipped branched protein side chains and incorrect sugar puckers. It is crit. both for the crystallographer and for the end user that there are easy and reliable methods to diagnose and correct these sorts of errors in structures. MolProbity is the authors' contribution to helping solve this problem and this article reviews its general capabilities, reports on recent enhancements and usage, and presents evidence that the resulting improvements are now beneficially affecting the global database.
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    56. 56
      Chen, Y., Rajashankar, K. R., Yang, Y., Agnihothram, S. S., Liu, C., Lin, Y. L., Baric, R. S., and Li, F. (2013) Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus. J. Virol. 87, 10777– 10783,  DOI: 10.1128/JVI.01756-13
      56
      Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus
      Chen, Yaoqing; Rajashankar, Kanagalaghatta R.; Yang, Yang; Agnihothram, Sudhakar S.; Liu, Chang; Lin, Yi-Lun; Baric, Ralph S.; Li, Fang
      Journal of Virology (2013), 87 (19), 10777-10783CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)
      The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and detd. its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.
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    57. 57
      Shi, Z. (2010) Bat and virus. Protein Cell 1, 109– 114,  DOI: 10.1007/s13238-010-0029-7
      57
      Bat and virus
      Shi, Zhengli
      Protein & Cell (2010), 1 (2), 109-114CODEN: PCREFB; ISSN:1674-800X. (Higher Education Press)
      Bat, the only flying mammal and count more than 20% of the extant mammals on earth, were recently identified as a natural reservoir of emerging and reemerging infectious pathogens. Astonishing amt. (more than 70) and genetic diversity of viruses isolated from the bat have been identified in different populations throughout the world. Many studies focus on bat viruses that caused severe domestic and human diseases. However, many viruses were found in apparently healthy bats, suggesting that bats may have a specific immune system or antiviral activity against virus infections. Therefore, basic researches for bat immunol. and virus-host interactions are important for understanding bat-derived infectious diseases.
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    58. 58
      Kubo, H., Yamada, Y. K., and Taguchi, F. (1994) Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein. J. Virol. 68, 5403– 5410
      58
      Localization of neutralizing epitopes and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein
      Kubo, Hideyuki; Yamada, Yasuko K.; Taguchi, Fumihiro
      Journal of Virology (1994), 68 (9), 5403-10CODEN: JOVIAM; ISSN:0022-538X.
      To localize the epitopes recognized by monoclonal antibodies (MAbs) specific for the S1 subunit of the murine coronavirus JHMV spike protein, the authors have expressed S1 proteins with different deletions from the C-terminus of S1. S1utt is composed of the entire 769-amino-acid (aa) S1 protein; S1NM, S1N, S1N(330), and S1N(220) are deletion mutants with 594, 453, 330, and 220 aa from the N terminus of the S1 protein. The expressed S1 deletion mutant proteins were examd. for reactivities to a panel of MAbs. All MAbs classified in groups A and B, those reactive to most mouse hepatitis virus (MHV) strains and those specific for isolate JHMV, resp., recognized S1N(330) and the larger S1 deletion mutants but failed to react with S1N(220). MAbs in group C, specific for the larger S protein of JHMV, reacted only with the S1utt protein without any deletion. These results indicated that the domain composed of the N-terminal 330 aa comprised the cluster of conformational epitopes recognized by MAbs in groups A and B. It was also shown that the epitopes of MAbs in group C were not restricted to the region missing in the smaller S protein. These results together with the fact that all MAbs in group B retained high neutralizing activity suggested the possibility that the N-terminal 330 aa are responsible for binding to the MHV-specific receptors. To investigate this possibility, the authors expressed the receptor protein and examd. the binding of each S1 deletion mutant to the receptor. It was demonstrated that the S1N(330) protein as well as other S1 deletion mutants larger than S1N(330) bound to the receptor. These results indicated that a domain composed of 330 aa at the N terminus of the S1 protein is responsible for binding to the MHV-specific receptor.
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    59. 59
      Wu, K., Li, W., Peng, G., and Li, F. (2009) Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Proc. Natl. Acad. Sci. U. S. A. 106, 19970– 19974,  DOI: 10.1073/pnas.0908837106
      59
      Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor
      Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang
      Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (47), 19970-19974, S19970/1-S19970/6CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      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). How different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors, was investigated. The crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2 was detd. NL63-CoV RBD has a novel β-sandwich core structure consisting of 2 layers of β-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homol. 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.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhsFGjtLrP&md5=f60e9892de280375cc632fc56af4e09e
    60. 60
      Reguera, J., Santiago, C., Mudgal, G., Ordono, D., Enjuanes, L., and Casasnovas, J. M. (2012) Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies. PLoS Pathog. 8, e1002859,  DOI: 10.1371/journal.ppat.1002859
      60
      Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies
      Reguera, Juan; Santiago, Cesar; Mudgal, Gaurav; Ordono, Desiderio; Enjuanes, Luis; Casasnovas, Jose M.
      PLoS Pathogens (2012), 8 (8), e1002859CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)
      The coronaviruses (CoVs) are enveloped viruses of animals and humans assocd. 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 dets. virus-binding specificity for recessed glycan-contg. 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 dets. 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.
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