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. 2016 Aug 30;113(35):9864-9.
doi: 10.1073/pnas.1604472113. Epub 2016 Aug 15.

Link of a ubiquitous human coronavirus to dromedary camels

Victor M Corman  1 , Isabella Eckerle  2 , Ziad A Memish  3 , Anne M Liljander  4 , Ronald Dijkman  5 , Hulda Jonsdottir  5 , Kisi J Z Juma Ngeiywa  6 , Esther Kamau  6 , Mario Younan  7 , Malakita Al Masri  8 , Abdullah Assiri  8 , Ilona Gluecks  9 , Bakri E Musa  10 , Benjamin Meyer  2 , Marcel A Müller  2 , Mosaad Hilali  11 , Set Bornstein  12 , Ulrich Wernery  13 , Volker Thiel  5 , Joerg Jores  14 , Jan Felix Drexler  15 , Christian Drosten  15
Affiliations

Link of a ubiquitous human coronavirus to dromedary camels

Victor M Corman et al. Proc Natl Acad Sci U S A. .

Abstract

The four human coronaviruses (HCoVs) are globally endemic respiratory pathogens. The Middle East respiratory syndrome (MERS) coronavirus (CoV) is an emerging CoV with a known zoonotic source in dromedary camels. Little is known about the origins of endemic HCoVs. Studying these viruses' evolutionary history could provide important insight into CoV emergence. In tests of MERS-CoV-infected dromedaries, we found viruses related to an HCoV, known as HCoV-229E, in 5.6% of 1,033 animals. Human- and dromedary-derived viruses are each monophyletic, suggesting ecological isolation. One gene of dromedary viruses exists in two versions in camels, full length and deleted, whereas only the deleted version exists in humans. The deletion increased in size over a succession starting from camelid viruses via old human viruses to contemporary human viruses. Live isolates of dromedary 229E viruses were obtained and studied to assess human infection risks. The viruses used the human entry receptor aminopeptidase N and replicated in human hepatoma cells, suggesting a principal ability to cause human infections. However, inefficient replication in several mucosa-derived cell lines and airway epithelial cultures suggested lack of adaptation to the human host. Dromedary viruses were as sensitive to the human type I interferon response as HCoV-229E. Antibodies in human sera neutralized dromedary-derived viruses, suggesting population immunity against dromedary viruses. Although no current epidemic risk seems to emanate from these viruses, evolutionary inference suggests that the endemic human virus HCoV-229E may constitute a descendant of camelid-associated viruses. HCoV-229E evolution provides a scenario for MERS-CoV emergence.

Keywords: coronavirus; ecology; evolution; livestock; zoonotic diseases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. S1.
Fig. S1.
Virus isolation success. Virus isolation success of the HCoV-229E–related CoVs in relation to cycle threshold (CT) values obtained by specific real-time RT-PCR. In total, a HCoV-229E–related CoV was isolated from respiratory samples originating from four individual animals. Cultured virus isolates are marked with an asterisk. Samples that were also positive for MERS-CoV RNA (samples JCN38 and JCN41) were not used in this isolation trial. #Isolation of RCN1 could not be continued due to severe fungal and bacterial contamination of the original sample.
Fig. S2.
Fig. S2.
Virus growth in cell culture. (A) Virus isolation and morphology of CPE. The CPE of dromedary-derived 229E isolate ACN4 was observed in HuH-7 cells 3 d postinfection (CPE, Right; uninfected control, Left). Rounding and detachment of cells and complete cell death occurred within 2 d after the first appearance of the CPE. (B) Growth kinetics of human and dromedary viruses. HuH-7 cells were infected at an MOI of 0.5 with HCoV-229E inf-1 and two dromedary-derived HCoV-229E–related viruses (ACN4 and JCN50). Comparable production of viral RNA [expressed as log genome equivalent (GE) copies on the left axis, referring to the lines in the diagram], but more efficient production of infectious viral particles of HCoV-229E inf-1 in contrast to the HCoV-229E–related viruses derived from dromedary camels (expressed as log plaque-forming units per milliliter on the right axis, referring to the bars in the diagram) was observed.
Fig. 1.
Fig. 1.
(A) Phylogeny of HCoV-229E–related CoVs. Nodes illustrate posterior probabilities. Sequences from this study are shown in red. GER, Germany; NED, Netherlands; USA, United States of America; KEN, Kenya; GHA, Ghana. HCoV-NL63 (branch-truncated) is an outgroup. Taif/T157a/2015 and Jeddah/N60/2014 were used as described by Sabir et al. (20). (B) Genomic organization of HCoV-229E–related CoVs. ORF1ab was truncated due to graphical reasons. Boxes illustrate the regions with major genetic differences between HCoV-229E–related viruses. Red bars indicate deletions. (C and D) Deletion patterns in ORF8 homologs of HCoV-229E–related CoVs. Red lines indicate regions with deletions (numbered I to IV). Asterisks indicate triplets that would act as in-frame stop codons. Arrows represent start codons. (E) RBD of HCoV-229E and HCoV-229E–related viruses. Black dots illustrate conserved amino acid residues compared with HCoV-229E. Variables sites are shown in red (minority) or yellow (majority).
Fig. S3.
Fig. S3.
Nucleotide alignment of the ORF8 region of HCoV-229E and related bat-229E and camelid-229E CoVs. Deletions are shaded gray. Putative start and stop codons of the ORF8 in bat and camelid viruses are shaded orange. Dots represent identical nucleotides in comparison to the most recent HCoV-229E (BN1/GER/2015). In the Kenyan virus, 12 nt are deleted (positions 27–38 from conserved start codon); in the alpaca virus, 28 nt are deleted (positions 15–42); and in human viruses, several deletions occurred in different strains: 82 nt (positions 4–85), 40 nt (positions 95–134), 44 nt (positions 94–137), 38 nt (positions 198–235, excluding inf-1), and, finally, 2 nt in the most recently circulating viruses (positions 267–268).
Fig. S4.
Fig. S4.
sgRNA of ORF8 of dromedary-associated 229E viruses. Sequence analysis of ORF8 sgRNA. The leader sequence (marked orange), putative transcription regulatory sequences (TRS, marked gray), and nucleotides in the 5′-proximal part of ORF8 (yellow box) are shown. Oligonucleotide-binding sites used for amplification and RT-PCR detection are shown as green arrows.
Fig. S5.
Fig. S5.
Nucleocapsid deletion patterns. Amino acid alignment of the translated nucleocapsid genes from camelid 229E-related CoVs and HCoV-229E. Deletions are shaded black.
Fig. 2.
Fig. 2.
(A) Receptor use of HCoV-229E–related viruses. HEK-293T-hAPN cells stably express hAPN. (B) Receptor blocking with polyclonal anti-hAPN antibodies. (C) IFN susceptibility of HCoV-229E and HCoV-229E–related viruses. GE, genome equivalent.
Fig. S6.
Fig. S6.
Comparison of mammalian APN ectodomains. The amino acid sequence alignment of human (hAPN, accession no. NM_001150), dromedary (dAPN, accession no. XM_010986749), bovine (bAPN, accession no. NP_001068612), porcine (pAPN, accession no. NP_999442), and feline APN (fAPN, accession no. NP_001009252) ectodomains is shown. Amino acid positions refer to the human APN sequence starting at amino acid position 64. The four domains of the ectodomain are indicated as described by Reguera et al. (46). The spike interacting region (positions 260–353) and the core motif (DYVEKQAS, positions 288–295) known to be essential for HCoV-229E entry are marked (47, 48). The core motif is variable among mammalian APNs (49). N-glycosylation sites (NXS/T motif) that were shown to influence the spike protein receptor interactions are indicated by gray boxes (49). Amino acid identities are highest between hAPN and dAPN.
Fig. 3.
Fig. 3.
(A) Production of virus in cell culture supernatant. A549, human lung adenocarcinoma cells; Caco, human colon carcinoma cells. Multiplicity of infection = 0.1. (B) Infection of polarized primary HAE cells at two different temperatures. (C) Nucleocapsid gene sgRNA transcription in permissive HuH-7 cells and nonpermissive A549 cells (quadruplicate data, real-time RT-PCR). (D) sgRNA in HAE cultures from two different donors (#1 and #2). ACN4, JCN50: dromedary-derived 229E CoVs; 229E: HCoV-229E strain inf-1; BN1: HCoV-229E BN1/GER/2015. CT, cycle threshold; PFU, plaque-forming units.
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