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https://doi.org/10.1038/s41586-020-2008-3 Fan Wu1,7, Su Zhao2,7, Bin Yu3,7, Yan-Mei Chen1,7, Wen Wang4,7, Zhi-Gang Song1,7, Yi Hu2,7,
Zhao-Wu Tao2, Jun-Hua Tian3, Yuan-Yuan Pei1, Ming-Li Yuan2, Yu-Ling Zhang1, Fa-Hui Dai1,
Received: 7 January 2020
Yi Liu1, Qi-Min Wang1, Jiao-Jiao Zheng1, Lin Xu1, Edward C. Holmes1,5 & Yong-Zhen Zhang1,4,6 ✉
Accepted: 28 January 2020
The patient studied was a 41-year-old man with no history of hepa- the onset of disease) (Extended Data Fig. 1e). Preliminary aetiological
titis, tuberculosis or diabetes. He was admitted to and hospitalized investigations excluded the presence of influenza virus, Chlamydia
in the Central Hospital of Wuhan on 26 December 2019, 6 days after pneumoniae and Mycoplasma pneumoniae using commercial pathogen
the onset of disease. The patient reported fever, chest tightness, antigen-detection kits, and this was confirmed by PCR. Other common
unproductive cough, pain and weakness for 1 week on presentation respiratory pathogens, including human adenoviruses, also tested
(Table 1). Physical examination of cardiovascular, abdominal and negative by quantitative PCR (qPCR) (Extended Data Fig. 2). Although
neurological characteristics was that these were normal. Mild lym- a combination of antibiotic, antiviral and glucocorticoid therapy was
phopoenia (defined as less than 9 × 105 cells per ml) was observed, administered, the patient exhibited respiratory failure and was given
but white blood cell and blood platelet counts were normal in a com- high-flow non-invasive ventilation. The condition of the patient did not
plete blood count test. Elevated levels of C-reactive protein (41.4 mg l−1 improve after 3 days of treatment and he was admitted to the intensive
of blood; reference range, 0–6 mg l−1) were observed and the levels care unit. The patient was transferred to another hospital in Wuhan for
of aspartate aminotransferase, lactic dehydrogenase and creatine further treatment 6 days after admission.
kinase were slightly elevated in blood chemistry tests. The patient Epidemiological investigations by the Wuhan Center for Disease
had mild hypoxaemia with oxygen levels of 67 mm Hg as determined Control and Prevention revealed that the patient worked at a local
by an arterial blood gas test. On the first day of admission (day 6 after indoor seafood market. Notably, in addition to fish and shellfish, a
the onset of disease), chest radiographs were abnormal with air-space variety of live wild animals—including hedgehogs, badgers, snakes
shadowing such as ground-glass opacities, focal consolidation and and birds (turtledoves)—were available for sale in the market before
patchy consolidation in both lungs (Extended Data Fig. 1). Computed- the outbreak began, as well as animal carcasses and animal meat. No
tomography scans of the chest revealed bilateral focal consolidation, bats were available for sale. While the patient might have had contact
lobar consolidation and patchy consolidation, especially in the lower with wild animals at the market, he recalled no exposure to live poultry.
lung (Extended Data Fig. 1a–d). A chest radiograph revealed a bilateral To investigate the possible aetiological agents associated with
diffuse patchy and fuzzy shadow on day 5 after admission (day 11 after this disease, we collected bronchoalveolar lavage fluid (BALF) and
1
Shanghai Public Health Clinical Center, Fudan University, Shanghai, China. 2Department of Pulmonary and Critical Care Medicine, The Central Hospital of Wuhan, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, China. 3Wuhan Center for Disease Control and Prevention, Wuhan, China. 4Department of Zoonosis, National Institute for
Communicable Disease Control and Prevention, China Center for Disease Control and Prevention, Beijing, China. 5Marie Bashir Institute for Infectious Diseases and Biosecurity, School of Life
and Environmental Sciences and School of Medical Sciences, The University of Sydney, Sydney, New South Wales, Australia. 6School of Public Health, Fudan University, Shanghai, China.
7
These authors contributed equally: Fan Wu, Su Zhao, Bin Yu, Yan-Mei Chen, Wen Wang, Zhi-Gang Song, Yi Hu. ✉e-mail: zhangyongzhen@shphc.org.cn
10
1a S 3b M 8 N
WHCV 29,903 bp
1b 3a
E 6 7b 9b
7a 9a
10
Bat SL-CoVZC45 1a S 3b M 8 N 29,802 bp
1b 3a
E 6 7b 9b
7a 9a
8a 8b
2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000 18,000 20,000 22,000 24,000 26,000 28,000
Position
Fig. 1 | Genome organization of SARS and SARS-like CoVs. The organization of genes for WHCV, bat SL-CoVZC45 and SARS-CoV Tor2.
c d 100
HCoV-HKU1
MHV
Ro-BatCoV GCCDC1 Embecovirus
99 80 ChRCoV HKU24
BtRt-BetaCoV/GX2018 Nobecovirus HCoV-OC43
Ro-BatCoV HKU9 EriCoV
HCoV-HKU1 98
Ty-BatCoV HKU4
89 Merbecovirus
MHV Pi-BatCoV HKU5
77 Embecovirus 88
ChRCoV HKU24 MERS-CoV
HCoV-OC43 Ro-BatCoV HKU9
EriCoV 99
91
MERS-CoV BtRt-BetaCoV/GX2018 Nobecovirus
Merbecovirus Ro-BatCoV GCCDC1
70 Ty-BatCoV HKU4 91
71 Pi-BatCoV HKU5 Bat Hp-BetaCoV Hibecovirus
98
Bat Hp-BetaCoV Hibecovirus 81
WHCV
98
Bat CoV BM48-31/BGR/2008 Bat SL-CoVZC45
91 97 Bat SL-CoVZXC21
96 WHCV
89 Bat SL-CoVZC45 99 Bat CoV BM48-31/BGR/2008
Bat SL-CoVZXC21 BtCoV Cp/Yunnan2011
Bat SARS-like CoV RsSHC014 79 LYRa11
74 Bat SARS-like CoV WIV16 88
BtRs-BetaCoV/GX2013
Bat SARS-like CoV WIV1 BtCoV/279/2005
Bat SARS-like CoV Rs3367 99
Bat SARS-CoV Rm1
Bat SARS-like CoV Rs4874 Bat SARS-CoV HKU3
Bat SARS-CoV Rs672 Bat SARS-CoV Rp3
BtRf-BetaCoV/JL2012 99 BtCoV Rp/Shaanxi2011
Bat CoV JTMC15 BtRs-BetaCoV/YN2013
89 BtCoV/273/2005
BtRs-BetaCoV/YN2018C
93 Bat SARS-CoV Rf1 87 Bat SARS-like CoV Rs4231
BtRf-BetaCoV/HeB2013 BtRl-BetaCoV/SC2018
BtRf-BetaCoV/SX2013
77 Civet SARS-CoV SZ3
BtRs-BetaCoV/YN2018A SARS-CoV WH20
LYRa11 93
SARS-CoV BJ01 Sarbecovirus
82 Bat SARS-CoV Rp3 Sarbecovirus 74 SARS-CoV TOR2
92 Bat SARS-CoV HKU3 BtRs-BetaCoV/YN2018A
75 BtCoV Rp/Shaanxi2011 BtRf-BetaCoV/HeB2013
87 BtCoV/279/2005
Bat SARS-CoV Rm1 82 BtRf-BetaCoV/SX2013
BtRs-BetaCoV/YN2013 99
BtRf-BetaCoV/JL2012
BtCoV Cp/Yunnan2011 Bat CoV JTMC15
BtRs-BetaCoV/GX2013 BtCoV/273/2005
Bat SARS-like CoV Rf4092 Bat SARS-CoV Rf1
83 SARS-CoV TOR2 99 Bat SARS-like CoV YNLF 34C
SARS-CoV BJ01 Bat SARS-like CoV Rf4092
Bat SARS-like CoV YNLF 34C Bat SARS-like CoV Rs4874
SARS-CoV WH20 Bat SARS-like CoV Rs3367
Civet SARS-CoV SZ3 Bat SARS-like CoV WIV1
97
Bat SARS-like CoV Rs4231 Bat SARS-like CoV WIV16
84 BtRs-BetaCoV/YN2018B 89 BtRs-BetaCoV/YN2018D
BtRs-BetaCoV/YN2018D 75 BtRs-BetaCoV/YN2018B
BtRl-BetaCoV/SC2018 Bat SARS-like CoV RsSHC014
0.5 BtRs-BetaCoV/YN2018C 0.5
Bat SARS-CoV Rs672
Fig. 2 | Maximum likelihood phylogenetic trees of nucleotide sequences of (>70) above or below the branches indicate percentage bootstrap values for the
the ORF1a, ORF1b, E and M genes of WHCV and related coronaviruses. associated nodes. The trees were mid-point rooted for clarity only. The scale
a, Phylogenetic trees of ORF1a. b, Phylogenetic trees of ORF1b. c, Phylogenetic bar represents the number of substitutions per site.
trees of E. d, Phylogenetic trees of M. EriCoV, Erinaceus coronavirus. Numbers
ORF1b phylogeny, WHCV fell in a basal position within the subgenus with those of SARS-CoVs and bat SARS-like CoVs. The RBD sequences
Sarbecovirus (Fig. 2). This topological division, which probably reflects of WHCV were more closely related to those of SARS-CoVs (73.8–74.9%
recombination among the bat sarbecoviruses, was also observed in the amino acid identity) and SARS-like CoVs, including strains Rs4874,
phylogenetic trees estimated for conserved domains in the replicase Rs7327 and Rs4231 (75.9–76.9% amino acid identity), that are able to use
polyprotein pp1ab (Extended Data Fig. 6). the human ACE2 receptor for cell entry11 (Supplementary Table 7). In
To better understand the potential of WHCV to infect humans, the addition, the RBD of the spike protein from WHCV was only one amino
receptor-binding domain (RBD) of its spike protein was compared acid longer than the RBD of the spike protein from SARS-CoV (Extended
0.9
0.8
0.7
0.6
Similarity 0.5
0.4
0.3
Fig. 3 | Possible recombination events in the S gene of sarbecoviruses. CoVZC45. b, Phylogenies of the major parental region (1–1,028 and 1,653–
a, The sequence similarity plot reveals two putative recombination breakpoints 3,804) and minor parental region (1,029–1,652). Phylogenies were estimated
(black dashed lines), with their locations indicated at the bottom. The plot using a maximum likelihood method and were mid-point rooted for clarity
shows similarity comparisons of the S gene of WHCV (query) compared with only. Numbers above or below the branches indicate percentage bootstrap
the sequences of SARS-CoV Tor2 and bat SARS-like CoVs WIV1, Rf1 and values. The scale bar represents the number of substitutions per site.
Data Fig. 7a). By contrast, other bat SARS-like CoVs—including the Rp3 genome as a whole. However, some evidence for past recombination
strain that cannot bind to human ACE214—had amino acid deletions was detected in the S gene of WHCV, SARS-CoV and bat SARS-like CoVs
at positions 433–437 and 460–472 compared with the sequence in (WIV1 and RsSHC014) (P < 3.147 × 10−3 to P < 9.198 × 10−9), for which the
SARS-CoVs (Extended Data Fig. 7a). The previously determined15 crystal similarity plots suggested the presence of recombination breakpoints
structure of the RBD of the spike protein of SARS-CoV complexed with at nucleotides 1,029 and 1,652, which separate the S gene of WHCV into
human ACE2 (Protein Data Bank (PDB) 2AJF) revealed that regions three regions (Fig. 3). In phylogenies of the nucleotide fragments from
433–437 and 460–472 directly interact with human ACE2 and hence 1 to 1,029 and from 1,652 to the end of the sequence, WHCV was most
may be important in determining species specificity (Extended Data closely related to bat SL-CoVZC45 and bat SL-CoVZXC21, whereas in the
Fig. 7b). We predicted the three-dimensional protein structures of the region of nucleotides 1,030 to 1,651 (the RBD region) WHCV grouped
RBD domains of the spike protein of WHCV, Rs4874 and Rp3 by protein with SARS-CoV and bat SARS-like CoVs (WIV1 and RsSHC014) that are
homology modelling using the SWISS-MODEL server and compared capable of direct human transmission17,20. Despite these recombination
them to the crystal structure of RBD domain of the spike protein of events, which seem relatively common among sarbecoviruses, there is
SARS-CoV (PDB 2GHV) (Extended Data Fig. 7c–f). In accordance with no evidence that recombination has facilitated the emergence of WHCV.
the sequence alignment, the predicted protein structures of the RBD Coronaviruses are associated with a number of infectious disease
domains of WHCV and Rs4874 were closely related to that of SARS-CoV outbreaks in humans, including SARS in 2002–2003 and Middle East
and different from the predicted structure of the RBD domain from respiratory syndrome (MERS) in 20121,21. Four other coronaviruses—
Rp3. In addition, the N terminus of the spike protein of WHCV is more human coronaviruses HKU1, OC43, NL63 and 229E—are also associ-
similar to that of SARS-CoV than other human coronaviruses (HKU1 ated with respiratory disease22–25. Although SARS-like coronaviruses
and OC43) (Extended Data Fig. 8) that can bind to sialic acid16. In sum- have been widely identified in mammals including bats since 2005 in
mary, the high similarities of the amino acid sequences and predicted China10,26–28, the exact origin of human-infected coronaviruses remains
protein structures of the RBD domains of WHCV and SARS-CoV suggest unclear. Here we describe a new coronavirus—WHCV—in the BALF from
that WHCV may efficiently use human ACE2 as a receptor for cellular a patient who experienced severe respiratory disease in Wuhan, China.
entry, which could potentially facilitate human-to-human transmis- Phylogenetic analysis suggests that WHCV is a member of the genus
sion11,17,18. Betacoronavirus (subgenus Sarbecovirus) that has some genomic and
To further characterize the putative recombination events in the evo- phylogenetic similarities to SARS-CoV1, particularly in the RBD of the
lutionary history of the sarbecoviruses, the whole-genome sequence spike protein. These genomic and clinical similarities to SARS, as well as
of WHCV and four representative coronaviruses—bat SARS-like CoV its high abundance in clinical samples, provides evidence for an associa-
Rp3, CoVZC45, CoVZXC21 and SARS-CoV Tor2—were analysed using the tion between WHCV and the ongoing outbreak of respiratory disease
Recombination Detection Program v.4 (RDP4)19. Although the similar- in Wuhan and across the world. Although the isolation of the virus
ity plots suggested that possible recombination events had occurred from only a single patient is not sufficient to conclude that it caused
between WHCV and SARS-CoVs or SARS-like CoVs (Extended Data these respiratory symptoms, our findings have been independently
Fig. 9), there was no significant evidence for recombination across the corroborated in further patients in a separate study29.
32. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina
sequence data. Bioinformatics 30, 2114–2120 (2014). Acknowledgements This study was supported by the Special National Project on investigation
33. Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-seq data without a of basic resources of China (grant SQ2019FY010009) and the National Natural Science
reference genome. Nat. Biotechnol. 29, 644–652 (2011). Foundation of China (grants 81861138003 and 31930001). E.C.H. is supported by an ARC
34. Li, B., Ruotti, V., Stewart, R. M., Thomson, J. A. & Dewey, C. N. RNA-seq gene expression Australian Laureate Fellowship (FL170100022).
estimation with read mapping uncertainty. Bioinformatics 26, 493–500 (2010).
35. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods Author contributions Y.-Z.Z. conceived and designed the study. S.Z., Y.H., Z.-W.T. and M.-L.Y.
9, 357–359 (2012). performed the clinical work and sample collection. B.Y. and J.-H.T. performed the
36. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, epidemiological investigation and sample collection. F.W., Z.-G.S., L.X., Y.-Y.P., Y.-L.Z., F.-H.D.,
2078–2079 (2009). Y.L., J.-J.Z. and Q.-M.W. performed the experiments. Y.-M.C., W.W., F.W., E.C.H. and Y.-Z.Z.
37. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: analysed the data. Y.-Z.Z., E.C.H. and F.W. wrote the paper with input from all authors. Y.-Z.Z. led
improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). the study.
38. Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood
phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010). Competing interests The authors declare no competing interests.
39. Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum
likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, Additional information
2731–2739 (2011). Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
40. Lole, K. S. et al. Full-length human immunodeficiency virus type 1 genomes from subtype 2008-3.
C-infected seroconverters in India, with evidence of intersubtype recombination. J. Virol. Correspondence and requests for materials should be addressed to Y.-Z.Z.
73, 152–160 (1999). Peer review information Nature thanks Nicholas Loman and the other, anonymous, reviewer(s)
41. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high for their contribution to the peer review of this work.
throughput. Nucleic Acids Res. 32, 1792–1797 (2004). Reprints and permissions information is available at http://www.nature.com/reprints.
Extended Data Fig. 1 | Chest radiographs of the patient. a–d, Computed- and patchy consolidation were clearly observed, especially in the lower lung.
tomography scans of the chest were obtained on the day of admission (day 6 e, A chest radiograph was obtained on day 5 after admission (day 11 after the
after the onset of disease). Bilateral focal consolidation, lobar consolidation onset of disease). Bilateral diffuse patchy and fuzzy shadows were observed.
Extended Data Fig. 3 | Mapped read count plot of the WHCV genome. The histograms show the coverage depth per base of the WHCV genome. The mean
sequencing depth of the WHCV genome was 604.21 nt.
Extended Data Fig. 5 | Maximum likelihood phylogenetic trees of the percentage bootstrap values. The trees were mid-point rooted for clarity only.
nucleotide sequences of the whole genome, and S and N genes of WHCV and The scale bar represents the number of substitutions per site.
related coronaviruses. Numbers (>70) above or below the branches indicate
Extended Data Fig. 8 | Amino acid sequence comparison of the N-terminal that can bind to sialic acid and the SARS-CoVs that cannot (SZ3, WH20, BJ0 and
domain of the spike protein. Amino acid sequence comparison of the Tor2). The key residues16 for sialic acid binding on BCoV, MHV, and HCoV OC43
N-terminal domain of the spike protein of WHCV, bovine coronavirus (BCoV), and HKU1 are highlighted by orange squares.
mouse hepatitis virus (MHV) and human coronaviruses (HCoV OC43 and HKU1)
Reporting Summary
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Give P values as exact values whenever suitable.
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Our web collection on statistics for biologists contains articles on many of the points above.
The whole genome sequence obtained in this study was submitted to GenBank with the accession number MN908947.
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Replication The de novo assembly of reads was performed using two programs.
The whole genome viral sequence obtained from read assembly was confirmed by PCR assays.
The results from phylogenetic and recombination analyses were confirmed by multiple runs.
Randomization Not applicable. The goal of this study was to find out the possible aetiologic agent associated with the severe respiratory disease occurred
recently in the city of Wuhan, Hubei province, China. Since we could obtain the BALF sample from only one patient who exhibited severe
respiratory syndrome including fever, dizzy and cough, hence, randomization was not applicable to this study.
Blinding Not applicable. Only one RNA library was generated in this study and thus no group allocation was performed.
Recruitment The patient who exhibited clinic signs of respiratory disease including fever and cough was recruited.
October 2018
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