Supporting InformationCorman et al. 10.1073/pnas.1604472113SI MethodsExtended Ethics Statement. All animals were handled accordingto national and European legislation, namely, European UnionCouncil Directive 86/609/EEC for the protection of animals. Fur-thermore, all protocols of this study were designed and performed instrict accordance with the Kenyan legislation for animal experi-mentation and were approved by the Institutional Animal Careand Use Committee (reference no. 2014.05.15) at the InternationalLivestock Research Institute (ILRI). Work at the ILRI complieswith the United Kingdom’s Animals (Scientific Procedures) Act of1986, which contains guidelines and codes of practice for thehousing and care of animals used in scientific procedures. Licensesfor sampling and sample exportation and importation permits wereobtained from the respective countries and authorities [Kenya:National Council of Science and Technology approval no. ILRI-IREC2013-12, including approval of use of stored dromedary serataken during previous routine veterinary diagnostics in Egypt,Sudan, and Somalia; KSA: Material Transfer agreement from theMinistry of Health; Germany (import permits): MKULNV-VI-5-2501/2.2-Uni Bonn-01/14, MKULNV-VI-5-2501/2.2-Uni Bonn-02/14, MKULNV-VI-5-2501/2.2-Uni Bonn-01/15, MKULNV-VI-5-2501/2.2-Uni Bonn-02/15, and LANUV NRW-8.87-03.04.06-111].No research work permits were necessary in the United ArabEmirates because of an unrestricted research clearance into animaldiseases to the governmental Central Veterinary Research Labora-tory (represented by author U.W.). Sampling in KSA was done aspart of an outbreak response solicited by the Saudi Ministry ofHealth. The contemporary human isolate of HCoV-229E was takenfrom and by coauthor I.E. during an episode of acute rhinitis. Noanimals were killed as part of this study. All animal handling andsampling was done by trained personnel, with animal safety andcomfort as the first priority during minimally invasive sampling usingnasal swabs and peripheral venous puncture.

Whole-Genome Sequencing. Full- CoV genome sequences weregenerated directly from clinical materials. Amplification and se-quencing were done using sets of nested RT-PCR assays (primersare available upon request), followed by next-generation sequencingon an Illumina MiSeq instrument and confirmation by Sanger se-quencing. All 229E-related camel CoV sequences determined in thisstudy were submitted to the GenBank under accession numbersKT253324-27 and KU291448-49.

Serology. Sera were tested by a recombinant HCoV-229E spikeprotein IFA as described previously (38, 39). Serum samples wereused at a dilution of 1:80. Detection was based on a goat anti-llamaIgG fluorescein isothiocyanate-conjugated antibody previouslyshown to be suitable for dromedary samples (17–19).

Phylogeny. Phylogenetic analyses were done using MrBayes ver-sion 3.1 (40) utilizing a GTR+G+I nucleotide substitution model,2 million generations sampled every 100 steps, and HCoV-NL63as an outgroup. Trees were annotated using TreeAnnotator V1.5and visualized using FigTree V1.4 from the BEAST package(41). Recombination was tested using RDP V4.0 (42).

Virus Isolation and Virus Strains. Cell lines HuH-7 (human hepa-toma), VeroE6 (monkey kidney), Caco-2 (human colon carcinoma),Caki-3 (dromedary kidney), HEF (primary esophageal human fi-broblasts), and LGK-1-R.B (alpaca kidney) were used for virusisolation. Cells were cultured as described previously (22, 43).Respiratory samples were incubated onto cells either unfiltrated and

supplemented with Pen/Strep (Life Technologies) and Amphoter-icin B (GE Healthcare) or filtrated through a sterile filter (0.45-μLpore size; Millipore). Upon detection of a CPE, supernatants weretested by specific real-time RT-PCR. Two live-virus isolates ofthe dromedary-derived 229E-related viruses, termed ACN4 andJCN50, were used for the in vitro experiments. For comparison,strain HCoV-229E inf-1 (44) and a low-passage (passage 3) wild-type (wt) isolate of HCoV-229E were used. Quantification of allvirus stocks was done by plaque titration on HuH-7 cells as de-scribed previously for MERS-CoV (22).

Virus Growth Kinetics.For growth kinetics, HuH-7 cells were infectedin triplicate with a multiplicity of infection (MOI) of 0.5 plaque-forming units for each virus. After incubation for 1 h at 37 °C, cellswere washed twice with PBS and growth medium containing 10%(vol/vol) FCS was added. Supernatants were quantified by titrationon HuH-7 cells. For assessment of human mucosa-derived celllines, A549 and Caco cells were used.

IFN Susceptibility Assay. HuH-7 cells were incubated with growthmedium containing type I IFN at a concentration of 100 IU/mL,1,000 IU/mL, or no IFN (untreated) 16 h prior to infection intriplicate with HCoV-229E, ACN4, and JCN50 at an MOI of0.1 pfu/mL. After a 1-h incubation, the supernatant was removedand cells were washed twice with PBS. Replication was assessedby a real-time RT-PCR and titration.

Receptor Use Experiments. The human embryonic kidney cell lineHEK-293T was transformed by lentiviral transfer of the hAPNgene and grown under puromycin selection (cells subsequentlydesignated as HEK-293T-hAPN). Untransformed HEK-293Tcells served as controls. Cells were infected with HCoV-229Einf-1, HCoV-229E wt, ACN4, and JCN50 in triplicate with an MOIof 0.5 plaque-forming units per cell. For receptor-blocking experi-ments, HEK-293T-hAPN cells were preincubated with 10 μg/mLpolyclonal goat anti-hAPN antibody (ab93897; Abcam) for 1 h at37 °C. Half of the antibody solution was stored, and cells weresubsequently infected in triplicate by adding viruses at an MOI of0.001 for 30 min at 4 °C. The supernatant was discarded, andmedium with antibody solution was added. At time points 0, 24,48, and 72 hpi, samples from supernatants were taken for quanti-fication by real-time RT-PCR.

HAE Cultures. Primary HAE cultures from human donors wereprepared as described previously (45). Once polarized, they wereinfected with 50,000, 5,000, and 500 plaque-forming units of eachvirus and incubated at 33 °C and 37 °C. Supernatants were har-vested 24, 48, 72, and 96 hpi and quantified by real-time RT-PCR.

Identification of sgRNA Transcripts. Testing for sgRNA of N and thecamel ORF8 protein used a leader-specific primer (CoV229E-sgRNA-rtF), as well as primers and probes targeting sequencesdownstream of the start codons of the respective ORFs. In additionto fluorescent probe readouts, RT-PCR products for both sgRNAswere analyzed on agarose gel and sequenced. Oligonucleotidesequences were as follows: CoV229E-sgRNA-rtF, CTTAAGTRYCTT ATC TAT CTA YAG ATA G; CoV229E_sgRNA_N_rtR, TTG CCC TTT CTA GTT CTG AAA CG; CoV229E_sgRNA_ORF8_rtR, CCA ACT WAA AAM CAC AYA CAAACC A; CoV229E_sgRNA_N_rtP, 6-carboxyfluorescein-ATACCTCGTAATTTGGTACCCAYCAAC-6-carboxy-N,N,N,N′-tetramethylrhodamine; and CoV229E_sgRNA_ORF8_rtP,6-carboxyfluorescein-TYR TTC AAT TGT TAG TTG YTG

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CTA AYT CAG-carboxy-N,N,N,N′-tetramethylrhodamine. As-says used the SSIII RT-PCR kit (Life Technologies) with 400 nMconcentrations of each of the primers, as well as 200 nM probe.Thermal cycling involved 10 min at 55 °C for reverse transcription,followed by 3 min at 95 °C and 45 cycles of 10 s at 95 °C, 10 s at56 °C, and 20 s at 72 °C.

Microneutralization Assay. HuH-7 cells were seeded in a 96-wellplate 1 d before infection. One hundred plaque-forming units ofeach virus were preincubated in parallel with diluted serum for 1 hat 37 °C. Infection was performed in duplicate for 1 h at 37 °C.After 72 h under fresh medium, cells were fixed with formalin,stained, and assessed for the presence of CPEs.

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-timeRT-PCR. In total, a HCoV-229E–related CoV was isolated from respiratory samples originating from four individual animals. Cultured virus isolates are markedwith 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 couldnot be continued due to severe fungal and bacterial contamination of the original sample.

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 cells3 d postinfection (CPE, Right; uninfected control, Left). Rounding and detachment of cells and complete cell death occurred within 2 d after the first ap-pearance 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 twodromedary-derived HCoV-229E–related viruses (ACN4 and JCN50). Comparable production of viral RNA [expressed as log genome equivalent (GE) copies on theleft 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) wasobserved.

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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 andstop 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 inhuman 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).

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Fig.S

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Fig. S5. Nucleocapsid deletion patterns. Amino acid alignment of the translated nucleocapsid genes from camelid 229E-related CoVs and HCoV-229E. De-letions are shaded black.

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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 theectodomain 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/Tmotif) that were shown to influence the spike protein receptor interactions are indicated by gray boxes (49). Amino acid identities are highest between hAPNand dAPN.

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Table S1. Virus isolates and titers in paired serum samples

ID

RNA copies per milliliterrespiratory swab

suspension

Spike-rIFT-229Etiters

in serum

AC04/KSA/2014 5.1E+08 1:3,200JC50/KSA/2014 7.2E+08 1:800JC49/KSA/2014 4.8E+06 1:3,200JC52/KSA/2014 1.8E+07 NegativeHCoV-229E/BN1/GER/2015 4.2E+09 1:3,200

Table S2. Virus neutralization by human sera

Serum code

Challenge virus

Serum typeHCoV-229E ACN4 JCN50

CSS-52 1:20 1:40 1:20 Convalescent serumWHO-B 1:20 1:10 1:20 Blood donor serumWHO-D 1:20 1:10 1:20 Blood donor serumBSS-88 1:20 − − Blood donor serumBSS-89 1:20 − − Blood donor serumCSS-41 1:20 − − Convalescent serumCSS-48 1:20 − − Convalescent serumCSS-49 1:10 − − Convalescent serum

−, no neutralization at dilution 1:10.

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