A new coronavirus associated with human respiratory disease in China — TEI-tagged view

Fan WupersName, Su ZhaopersName, Bin YupersName, Yan-Mei ChenpersName, Wen WangpersName, Zhi-Gang SongpersName, Yi HupersName, Zhao-Wu TaopersName, Jun-Hua TianpersName, Yuan-Yuan PeipersName, Ming-Li YuanpersName, Yu-Ling ZhangpersName, Fa-Hui DaipersName, Yi LiupersName, Qi-Min WangpersName, Jiao-Jiao ZhengpersName, Lin XupersName, Edward C. HolmespersName, Yong-Zhen ZhangpersName (2020date). “A new coronavirus associated with human respiratory disease in China.” Naturetitle·j.

Object of study — the aetiological agent of a severe respiratory disease outbreak in Wuhan, studied through one hospitalised seafood-market worker whose bronchoalveolar lavage fluidterm was sequenced, and the new coronavirus's genome, phylogenetic relatives, recombinationterm history and likely cell-entry receptor

Research question — what agent is associated with the Wuhan respiratory outbreak, how that agent is related to known coronaviruses, and whether it could use the human receptor that SARS-CoVterm uses

Contribution — identification and complete 29,903-nucleotide genome of a new coronavirus, WH-Human 1 (WHCVterm·def, also called 2019-nCoVterm), recovered by metagenomic RNA sequencing from a single patient's lavage fluid, placed within the subgenus Sarbecovirusterm and shown by receptor-binding-domain comparison to be a plausible user of human ACE2term

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A new coronavirus associated with human respiratory disease in China Abstract Emerging infectious diseasesterm, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health 1 – 3 . Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019date. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019date while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing 4 of a sample of bronchoalveolar lavage fluidterm from the patient identified a new RNA virus strain from the family Coronaviridae , which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoVterm’). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirusterm, subgenus Sarbecovirusterm) that had previously been found in bats in China 5 . This outbreak highlights the ongoing ability of viral spill-overterm from animals to cause severe disease in humans.

Main The patient studied was a 41-year-old man with no history of hepatitis, tuberculosis or diabetes. He was admitted to and hospitalized in the Central Hospital of Wuhan on 26 December 2019date, 6 days after the onset of disease. The patient reported fever, chest tightness, unproductive cough, pain and weakness for 1 week on presentation (Table 1 ). Physical examination of cardiovascular, abdominal and neurological characteristics was that these were normal. Mild lymphopoeniaterm·def (defined as less than 9 × 10 5 cells per ml) was observed, but white blood cell and blood platelet counts were normal in a complete blood count test. Elevated levels of C-reactive protein (41.4 mg l −1 of blood; reference range, 0–6 mg l −1 ) were observed and the levels of aspartate aminotransferase, lactic dehydrogenase and creatine kinase were slightly elevated in blood chemistry tests. The patient had mild hypoxaemia with oxygen levels of 67 mm Hg as determined by an arterial blood gas test. On the first day of admission (day 6 after the onset of disease), chest radiographs were abnormal with air-space shadowing such as ground-glass opacities, focal consolidation and patchy consolidation in both lungs (Extended Data Fig. 1 ). Computed-tomography scans of the chest revealed bilateral focal consolidation, lobar consolidation and patchy consolidation, especially in the lower lung (Extended Data Fig. 1a–d ). A chest radiograph revealed a bilateral diffuse patchy and fuzzy shadow on day 5 after admission (day 11 after the onset of disease) (Extended Data Fig. 1e ). Preliminary aetiological investigations excluded the presence of influenza virus, Chlamydia pneumoniae and Mycoplasma pneumoniae using commercial pathogen antigen-detection kits, and this was confirmed by PCR. Other common respiratory pathogens, including human adenoviruses, also tested negative by quantitative PCRterm (qPCR) (Extended Data Fig. 2 ). Although a combination of antibiotic, antiviral and glucocorticoid therapy was administered, the patient exhibited respiratory failure and was given high-flow non-invasive ventilation. The condition of the patient did not improve after 3 days of treatment and he was admitted to the intensive care unit. The patient was transferred to another hospital in Wuhan for further treatment 6 days after admission. Table 1 Clinical symptoms and patient data Characteristic Patient Age (years) 41 Sex Male Date of illness onset 20 December 2019date Date of admission 26 December 2019date Signs and symptoms Fever Yes Body temperature (°C) 38.4 Cough Yes Sputum production Yes Dizzy Yes Weakness Yes Chest tightness Yes Dyspnoea Yes Bacterial culture Negative Glucocorticoid therapy Yes Antibiotic therapy Cefoselis Antiviral therapy Oseltamivir Oxygen therapy Mechanical ventilation Epidemiological investigations by the Wuhan Center for Disease Control and Prevention revealed that the patient worked at a local indoor seafood market. Notably, in addition to fish and shellfish, a variety of live wild animals—including hedgehogs, badgers, snakes and birds (turtledoves)—were available for sale in the market before the outbreak began, as well as animal carcasses and animal meat. No bats were available for sale. While the patient might have had contact with wild animals at the market, he recalled no exposure to live poultry.

To investigate the possible aetiological agents associated with this disease, we collected bronchoalveolar lavage fluidterm (BALFterm·def) and performed deep meta-transcriptomic sequencingterm. The clinical specimen was handled in a biosafety level 3 laboratory at Shanghai Public Health Clinical Center. Total RNA was extracted from 200 μl of BALFterm·def and a meta-transcriptomic library was constructed for pair-end (150-bp reads) sequencing using an Illumina MiniSeq as previously described 4 , 6 – 8 . In total, we generated 56,565,928 sequence reads that were de novo-assembled and screened for potential aetiological agents. Of the 384,096 contigsterm assembled by Megahitterm 9 , the longest (30,474 nucleotides (nt)) had a high abundance and was closely related to a bat SARS-like coronavirus (CoV) isolate—bat SL-CoVZC45term (GenBank accession number MG772933 )—that had previously been sampled in China, with a nucleotide identityterm·var of 89.1% (Supplementary Tables 1 , 2 ). The genome sequence of this virus, as well as its termini, were determined and confirmed by reverse-transcription PCR (RT–PCR) 10 and 5′/3′ rapid amplification of cDNA ends (RACEterm), respectively. This virus strain was designated as WH-Human 1 coronavirusterm (WHCVterm·def) (and has also been referred to as ‘2019-nCoVterm’) and its whole genome sequence (29,903 nt) has been assigned GenBank accession number MN908947 . Remapping the RNA-sequencing data to the complete genome of WHCVterm·def resulted in an assembly of 123,613 reads, providing 99.99% genome coverageterm·var at a mean depth of 6.04× (range, 0.01–78.84×) (Extended Data Fig. 3 ). The viral loadterm in the BALFterm·def sample was estimated by qPCR to be 3.95 × 10 8 copies per ml (Extended Data Fig. 4 ).

The viral genome organization of WHCVterm·def was determined by sequence alignment to two representative members of the genus Betacoronavirusterm: a coronavirus associated with humans (SARS-CoVterm Tor2, GenBank accession number AY274119 ) and a coronavirus associated with bats (bat SL-CoVZC45term, GenBank accession number MG772933 ). The un-translational regions and open-reading frameterm (ORF) of WHCVterm·def were mapped on the basis of this sequence alignment and ORF prediction. The WHCVterm·def viral genome was similar to these two coronaviruses (Fig. 1 and Supplementary Table 3 ). The order of genes (5′ to 3′) was as follows: replicase ORF1ab , spike ( S ), envelope ( E ), membrane ( M ) and nucleocapsid ( N ). WHCVterm·def has 5′ and 3′ terminal sequences that are typical of betacoronavirusesterm, with 265 nt at the 5′ terminal end and 229 nt at the 3′ terminal end. The predicted replicase ORF1ab gene of WHCVterm·def is 21,291 nt in length and contained 16 predicted non-structural proteinsterm (Supplementary Table 4 ), followed by (at least) 13 downstream ORFs. Additionally, WHCVterm·def shares a highly conserved domain (LLRKNGNKG: amino acids 122–130) in nsp1 with SARS-CoVterm. The predicted S , ORF3a , E , M and N genes of WHCVterm·def are 3,822, 828, 228, 669 and 1,260 nt in length, respectively. In addition to these ORF regions, which are shared by all members of the subgenus Sarbecovirusterm, WHCVterm·def is similar to SARS-CoVterm in that it carries a predicted ORF8term gene (with a length of 366 nt) that is located between the M and N ORF genes. The functions of WHCVterm·def ORFs were predicted on the basis of those of known coronaviruses and are described in Supplementary Table 5 . In a manner similar to SARS-CoVterm Tor2, a leader transcription regulatory sequenceterm (TRSterm·def) and nine putative body TRSsterm·def could be readily identified upstream of the 5′ end of the ORF in WHCVterm·def, and the putative conserved TRSterm·def core sequence appeared in two forms—ACGAAC or CUAAAC (Supplementary Table 6 ). Fig. 1 Genome organization of SARS and SARS-like CoVs. The organization of genes for WHCVterm·def, bat SL-CoVZC45term and SARS-CoVterm Tor2.

To determine the evolutionary relationships between WHCVterm·def and previously identified coronaviruses, we estimated phylogenetic trees on the basis of the nucleotide sequences of the whole-genome sequence, the non-structural proteinterm genes ORF1a and ORF1b , and the main structural proteins encoded by the S , E , M and N genes (Fig. 2 and Extended Data Fig. 5 ). In all phylogenies, WHCVterm·def clustered with members of the subgenus Sarbecovirusterm, including the SARS-CoVterm that was responsible for the global SARS pandemic 1 , 2 of 2002–2003, as well as a number of SARS-like coronaviruses that have been obtained from bats 5 , 11 – 13 . However, WHCVterm·def changed topological position within the subgenus Sarbecovirusterm depending on which gene was used, which suggests that recombinationterm has occurred in this group of viruses in the past (Fig. 2 and Extended Data Fig. 5 ). Specifically, in the S gene tree (Extended Data Fig. 5 ), WHCVterm·def was most closely related to the bat coronavirus SL-CoVZC45 with 82.3% amino acid identityterm·var (and around 77.2% amino acid identityterm·var to SARS-CoVterm; Supplementary Table 3 ) whereas in the ORF1b phylogeny, WHCVterm·def fell in a basal position within the subgenus Sarbecovirusterm (Fig. 2 ). This topological division, which probably reflects recombinationterm among the bat sarbecovirusesterm, was also observed in the phylogenetic trees estimated for conserved domains in the replicase polyprotein pp1ab (Extended Data Fig. 6 ). Fig. 2 Maximum likelihood phylogenetic trees of nucleotide sequences of the ORF1a , ORF1b , E and M genes of WHCVterm·def and related coronaviruses. a , Phylogenetic trees of ORF1a . b , Phylogenetic trees of ORF1b . c , Phylogenetic trees of E . d , Phylogenetic trees of M . EriCoV, Erinaceus coronavirus. Numbers (>70) above or below the branches indicate percentage bootstrap values for the associated nodes. The trees were mid-point rooted for clarity only. The scale bar represents the number of substitutions per site.

To better understand the potential of WHCVterm·def to infect humans, the receptor-binding domainterm (RBDterm·def) of its spike proteinterm was compared with those of SARS-CoVsterm and bat SARS-like CoVs. The RBDterm·def sequences of WHCVterm·def were more closely related to those of SARS-CoVsterm (73.8–74.9% amino acid identityterm·var) and SARS-like CoVs, including strains Rs4874, Rs7327 and Rs4231 (75.9–76.9% amino acid identityterm·var), that are able to use the human ACE2term receptor for cell entry 11 (Supplementary Table 7 ). In addition, the RBDterm·def of the spike proteinterm from WHCVterm·def was only one amino acid longer than the RBDterm·def of the spike proteinterm from SARS-CoVterm (Extended Data Fig. 7a ). By contrast, other bat SARS-like CoVs—including the Rp3 strain that cannot bind to human ACE2term 14 —had amino acid deletions at positions 433–437 and 460–472 compared with the sequence in SARS-CoVsterm (Extended Data Fig. 7a ). The previously determined 15 crystal structure of the RBDterm·def of the spike proteinterm of SARS-CoVterm complexed with human ACE2term (Protein Data Bank (PDB) 2AJF) revealed that regions 433–437 and 460–472 directly interact with human ACE2term and hence may be important in determining species specificityterm (Extended Data Fig. 7b ). We predicted the three-dimensional protein structures of the RBDterm·def domains of the spike proteinterm of WHCVterm·def, Rs4874 and Rp3 by protein homology modellingterm using the SWISS-MODEL server and compared them to the crystal structure of RBDterm·def domain of the spike proteinterm of SARS-CoVterm (PDB 2GHV) (Extended Data Fig. 7c–f ). In accordance with the sequence alignment, the predicted protein structures of the RBDterm·def domains of WHCVterm·def and Rs4874 were closely related to that of SARS-CoVterm and different from the predicted structure of the RBDterm·def domain from Rp3. In addition, the N terminus of the spike proteinterm of WHCVterm·def is more similar to that of SARS-CoVterm than other human coronaviruses (HKU1 and OC43) (Extended Data Fig. 8 ) that can bind to sialic acid 16 . In summary, the high similarities of the amino acid sequences and predicted protein structures of the RBDterm·def domains of WHCVterm·def and SARS-CoVterm suggest that WHCVterm·def may efficiently use human ACE2term as a receptor for cellular entry, which could potentially facilitate human-to-human transmissionterm 11 , 17 , 18 .

To further characterize the putative recombinationterm events in the evolutionary history of the sarbecovirusesterm, the whole-genome sequence of WHCVterm·def and four representative coronaviruses—bat SARS-like CoV Rp3, CoVZC45, CoVZXC21 and SARS-CoVterm Tor2—were analysed using the Recombinationterm Detection Program v.4 (RDP4) 19 . Although the similarity plots suggested that possible recombinationterm events had occurred between WHCVterm·def and SARS-CoVsterm or SARS-like CoVs (Extended Data Fig. 9 ), there was no significant evidence for recombinationterm across the genome as a whole. However, some evidence for past recombinationterm was detected in the S gene of WHCVterm·def, SARS-CoVterm and bat SARS-like CoVs (WIV1 and RsSHC014) ( P < 3.147 × 10 −3 to P < 9.198 × 10 −9 ), for which the similarity plots suggested the presence of recombination breakpointsterm at nucleotides 1,029 and 1,652, which separate the S gene of WHCVterm·def into three regions (Fig. 3 ). In phylogenies of the nucleotide fragments from 1 to 1,029 and from 1,652 to the end of the sequence, WHCVterm·def was most closely related to bat SL-CoVZC45term and bat SL-CoVZXC21, whereas in the region of nucleotides 1,030 to 1,651 (the RBDterm·def region) WHCVterm·def grouped with SARS-CoVterm and bat SARS-like CoVs (WIV1 and RsSHC014) that are capable of direct human transmission 17 , 20 . Despite these recombinationterm events, which seem relatively common among sarbecovirusesterm, there is no evidence that recombinationterm has facilitated the emergence of WHCVterm·def. Fig. 3 Possible recombinationterm events in the S gene of sarbecovirusesterm. a , The sequence similarity plot reveals two putative recombination breakpointsterm (black dashed linespersName), with their locations indicated at the bottom. The plot shows similarity comparisons of the S gene of WHCVterm·def (query) compared with the sequences of SARS-CoVterm Tor2 and bat SARS-like CoVs WIV1, Rf1 and CoVZC45. b , Phylogenies of the major parental region (1–1,028 and 1,653–3,804) and minor parental region (1,029–1,652). Phylogenies were estimated using a maximum likelihood methodterm and were mid-point rooted for clarity only. Numbers above or below the branches indicate percentage bootstrap values. The scale bar represents the number of substitutions per site.

Coronaviruses are associated with a number of infectious disease outbreaks in humans, including SARS in 2002–2003 and Middle East respiratory syndrome (MERS) in 2012 1 , 21 . Four other coronaviruses—human coronaviruses HKU1, OC43, NL63 and 229E—are also associated with respiratory disease 22 – 25 . Although SARS-like coronaviruses have been widely identified in mammals including bats since 2005 in China 10 , 26 – 28 , the exact origin of human-infected coronaviruses remains unclear. Here we describe a new coronavirus—WHCVterm·def—in the BALFterm·def from a patient who experienced severe respiratory disease in Wuhan, China. Phylogenetic analysis suggests that WHCVterm·def is a member of the genus Betacoronavirusterm (subgenus Sarbecovirusterm) that has some genomic and phylogenetic similarities to SARS-CoVterm 1 , particularly in the RBDterm·def of the spike proteinterm. These genomic and clinical similarities to SARS, as well as its high abundance in clinical samples, provides evidence for an association between WHCVterm·def and the ongoing outbreak of respiratory disease in Wuhan and across the world. Although the isolation of the virus from only a single patient is not sufficient to conclude that it caused these respiratory symptoms, our findings have been independently corroborated in further patients in a separate study 29 .

The identification of multiple SARS-like CoVs in bats have led to the idea that these animals act as hosts of a natural reservoirterm of these viruses 22 , 23 . Although SARS-like viruses have been identified widely in bats in China, viruses identical to SARS-CoVterm have not yet been documented. Notably, WHCVterm·def is most closely related to bat coronaviruses, and shows 100% amino acid similarity to bat SL-CoVZC45term in the nsp7 and E proteins (Supplementary Table 3 ). Thus, these data suggest that bats are a possible host for the viral reservoir of WHCVterm·def. However, as a variety of animal species were for sale in the market when the disease was first reported, further studies are needed to determine the natural reservoirterm and any intermediate hoststerm of WHCVterm·def.

Note added in proof: Since this paper was accepted, the ICTV has designated the virus as SARS-CoV-2term 30 ; in addition, the WHO has released the official name of the disease caused by this virus, which is COVID-19term 31 .

Methods Data reporting No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment.

Patient information and collection of clinical data and sample A patient presenting with acute onset of fever (temperature over 37.5 °C), cough and chest tightness, who was admitted to the Central Hospital of Wuhan, in Wuhan, China, was considered to be a suspected caseterm. During admission, BALFterm·def was collected and stored at −80 °C until further processing. Demographic, clinical and laboratory data were retrieved from the clinical records of the patient. The study was reviewed and approved by the ethics committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Signed written informed consent was obtained from the patient.

RNA library construction and sequencing Total RNA was extracted from the BALFterm·def sample using the RNeasy Plus Universal Mini kit (Qiagen) following the manufacturer’s instructions. The quantity and quality of the RNA solution was assessed using a Qbit machine and an Agilent 2100 Bioanalyzer (Agilent Technologies) before library construction and sequencing. An RNA library was then constructed using the SMARTer Stranded Total RNA-Seq kit v.2 (TaKaRa). Ribosomal RNA depletion was performed during library construction following the manufacturer’s instructions. Paired-end (150-bp reads) sequencing of the RNA library was performed on the MiniSeq platform (Illumina). Library preparation and sequencing were carried out at the Shanghai Public Health Clinical Center, Fudan University, Shanghai, China.

Data processing and identification of the viral agent Sequencing reads were first adaptor and quality trimmed using the Trimmomatic program 32 . The remaining 56,565,928 reads were assembled de novo using both Megahitterm (v.1.1.3) 9 and Trinity (v.2.5.1) 33 with default parameter settings. Megahitterm generated a total of 384,096 assembled contigsterm·def (size range of 200–30,474 nt), whereas Trinity generated 1,329,960 contigsterm with a size range of 201–11,760 nt. All of these assembled contigsterm·def were compared (using BLASTn and Diamond BLASTx) against the entire non-redundant (nr) nucleotide and protein databases, with e values set to 1 × 10 −10 and 1 × 10 −5 , respectively. To identify possible aetiological agents present in the sequencing data, the abundance of the assembled contigsterm·def was first evaluated as the expected counts using the RSEM program 34 implemented in Trinity. Non-human reads (23,712,657 reads), generated by filtering host reads using the human genome (human release 32, GRCh38.p13, downloaded from Gencode) by Bowtie2 35 , were used for the RSEM abundance assessment.

As the longest contigsterm generated by Megahitterm (30,474 nt) and Trinity (11,760 nt) both showed high similarity to the bat SARS-like coronavirus isolate bat SL-CoVZC45term and were found at a high abundance (Supplementary Tables 1 , 2 ), the longer sequence (30,474 nt)—which covered almost the whole virus genome—was used for primer design for PCR confirmation and determination of the genome termini. Primers used for PCR, qPCR and RACEterm experiments are listed in Supplementary Table 8 . The PCR assay was conducted as previously described 10 and the complete genome termini was determined using the Takara SMARTer RACEterm 5′/3′ kit (TaKaRa) following the manufacturer’s instructions. Subsequently, the genome coverageterm·var and sequencing depth were determined by remapping all of the adaptor- and quality-trimmed reads to the whole genome of WHCVterm·def using Bowtie2 35 and Samtools 36 .

The viral loadsterm of WHCVterm·def in BALFterm·def were determined by quantitative real-time RT–PCR using the Takara One Step PrimeScript RT–PCR kit (Takara RR064A) following the manufacturer’s instructions. Real-time RT–PCR was performed using 2.5 μl RNA with 8 pmol of each primer and 4 pmol probe under the following conditions: reverse transcription at 42 °C for 10 min, 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The reactions were performed and detected by ABI 7500 Real-Time PCR Systems. The PCR product covering the Taqman primers and probe region was cloned into pLB vector using the Lethal Based Simple Fast Cloning Kit (TianGen) as standards for quantitative viral loadterm test.

Virus genome characterization and phylogenetic analysis For the newly identified virus genome, the potential ORFs were predicted and annotated using the conserved signatures of the cleavage sites recognized by coronavirus proteinases, and were processed in the Lasergene software package (v.7.1, DNAstar). The viral genes were aligned using the L-INS-i algorithm implemented in MAFFT (v.7.407) 37 .

Phylogenetic analyses were then performed using the nucleotide sequences of various CoV gene datasets: (1) whole genome, (2) ORF1a, (3) ORF1b, (4) nsp5 (3CLpro), (5) RdRp (nsp12), (6) nsp13 (Hel), (7) nsp14 (ExoN), (8) nsp15 (NendoU), (9) nsp16 (O-MT), (10) spike (S) and (11) nucleocapsid (N). Phylogenetic trees were inferred using the maximum likelihood methodterm implemented in the PhyML program (v.3.0) 38 , using the generalized time reversible substitution model and subtree pruning and regrafting branch swapping. Bootstrap support values were calculated from 1,000 pseudo-replicate trees. The best-fitting model of nucleotide substitution was determined using MEGA (v.5) 39 . Amino acid identities among sequences were calculated using the MegAlign program implemented in the Lasergene software package (v.7.1, DNAstar).

Genome recombinationterm analysis Potential recombinationterm events in the history of the sarbecovirusesterm were assessed using both the RDP4 19 and Simplot (v.3.5.1) 40 . The RDP4 analysis was conducted based on the complete genome (nucleotide) sequence, using RDP, GENECONV, BootScan, maximum chi square, Chimera, SISCAN and 3SEQ methods. Putative recombinationterm events were identified with a Bonferroni corrected P -value cut-off of 0.01. Similarity plots were inferred using Simplot to further characterize potential recombinationterm events, including the location of possible breakpoints.

Analysis of the RBDterm·def domain of the spike proteinterm of WHCVterm·def An amino acid sequence alignment of RBDterm·def sequences from WHCVterm·def, SARS-CoVsterm and bat SARS-like CoVs was performed using MUSCLE 41 . The predicted protein structures of the RBDterm·def of the spike proteinterm were estimated based on target–template alignment using ProMod3 on SWISS-MODEL server ( https://swissmodel.expasy.org/ ). The sequences of the RBDterm·def domains spike of WHCVterm·def, Rs4874 and Rp3 were searched by BLAST against the primary amino acid sequence contained in the SWISS-MODEL template library (last update, 9 January 2020; last included PDB release, 3 January 2020). Models were built based on the target–template alignment using ProMod3. The global and per-residue model quality was assessed using the QMEAN scoring function 42 . The PDB files of the predicted protein structures were displayed and compared with the crystal structures of the spike RBDterm·def of SARS-CoVterm (PDB 2GHV) 43 and the crystal of structure of the spike RBDterm·def of SARS-CoVterm complexed with human ACE2term (PDB 2AJF) 15 .

Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

Online content Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-020-2008-3.

Supplementary information This file contains Supplementary Tables 1-8.

Reporting Summary