A pneumonia outbreak associated with a new coronavirus of probable bat origin

Abstract

Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats1-4. Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans5-7. Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1. Genome characterization of 2019-nCoV.

a, Metagenomics analysis of next-generation sequencing of BALF from patient ICU06. b, Genomic organization of 2019-nCoV WIV04. M, membrane. c, Similarity plot based on the full-length genome sequence of 2019-nCoV WIV04. Full-length genome sequences of SARS-CoV BJ01, bat SARSr-CoV WIV1, bat coronavirus RaTG13 and ZC45 were used as reference sequences. d, Phylogenetic tree based on nucleotide sequences of complete genomes of coronaviruses. MHV, murine hepatitis virus; PEDV, porcine epidemic diarrhoea virus; TGEV, porcine transmissible gastroenteritis virus.The scale bars represent 0.1 substitutions per nucleotide position. Descriptions of the settings and software that was used are included in the Methods.

Fig. 2. Molecular and serological investigation of patient samples.

a, Molecular detection of 2019-nCoV in seven patients. Patient information can be found in Extended Data Tables 1, 2. Detection methods are described in the Methods. AS, anal swab; OS, oral swab. b, Dynamics of 2019-nCoV antibody levels in one patient who showed signs of disease on 23 December 2019 (ICU-06). OD ratio, optical density at 450–630 nm. The right and left y axes indicate ELISA OD ratios for IgM and IgG, respectively. c, Serological test of 2019-nCoV antibodies in five patients (Extended Data Table 2). The asterisk indicates data collected from patient ICU-06 on 10 January 2020. b, c, The cut-off was to 0.2 for the IgM analysis and to 0.3 for the IgG analysis, according to the levels of healthy controls.

Fig. 3. Analysis of the receptor use of 2019-nCoV.

Determination of virus infectivity in HeLa cells that expressed or did not express (untransfected) ACE2. The expression of ACE2 plasmid with S tag was detected using mouse anti-S tag monoclonal antibody. hACE2, human ACE2; bACE2, ACE2 of Rhinolophus sinicus (bat); cACE2, civet ACE2; sACE2, swine ACE2 (pig); mACE2, mouse ACE2. Green, ACE2; red, viral protein (N); blue, DAPI (nuclei). Scale bars, 10 μm.

Extended Data Fig. 1. NGS raw reads of sample WIV04 mapping to the 2019-nCoV sequence.

The x axis indicates the genome nucleotide position and the y axis represents the read depth of the mapping.

Extended Data Fig. 2. Phylogenetic trees based on the complete S and RdRp gene sequences of coronaviruses.

a, b, Phylogenetic trees on the basis of the gene sequences of S (a) and RdRp (b) are shown. 2019-nCoV and bat CoV RaTG13 are shown in bold and in red. The trees were constructed using the maximum likelihood method using the GTR + G substitution model with bootstrap values determined by 1,000 replicates. Bootstraps values of more than 50% are shown.

Extended Data Fig. 3. Amino acid sequence alignment of the S1 protein of the 2019-nCoV to SARS-CoV and selected bat SARSr-CoVs.

The receptor-binding motif of SARS-CoV and the homologous region of other coronaviruses are indicated by the red box. The key amino acid residues involved in the interaction with human ACE2 are numbered at the top of the aligned sequences. The short insertions in the N-terminal domain of the 2019-nCoV are indicated by the blue boxes. Bat CoV RaTG13 was obtained from R. affinis, found in Yunnan province. Bat CoV ZC45 was obtained from R. sinicus, found in Zhejiang province.

Extended Data Fig. 4. Molecular detection method used to detect 2019-nCoV.

a, Standard curve for qPCR primers. The PCR product of the S gene that was serial diluted in the range of 108 to 101 (lines from left to right) was used as a template. Primer sequences and experimental conditions are described in the Methods. b, Specificity of the qPCR primers. Nucleotide samples from the indicated pathogens were used.

Extended Data Fig. 5. Amino acid sequence alignment of the nucleocapsid protein of 2019-nCoV to bat SARSr-CoV Rp3 and SARS-CoV BJ01.

Bat SARSr-CoV Rp3 was obtained from R. sinicus, which is found in Guangxi province.

Extended Data Fig. 6. Isolation and antigenic characterization of 2019-nCoV.

a, b, Vero E6 cells are shown at 24 h after infection with mock virus (a) or 2019-nCoV (b). c, d, Mock-virus-infected (c) or 2019-nCoV-infected (d) samples were stained with rabbit serum raised against recombinant SARSr-CoV Rp3 N protein (red) and DAPI (blue). The experiment was conducted twice independently with similar results. e, The ratio of the number of reads related to 2019-nCoV among the total number of virus-related reads in metagenomics analysis of supernatants from Vero E6 cell cultures. f, Virus growth in Vero E6 cells. g, Viral particles in the ultrathin sections were imaged using electron microscopy at 200 kV. The sample was from virus-infected Vero E6 cells. The inset shows the viral particles in an intra-cytosolic vacuole.

Extended Data Fig. 7. Analysis of 2019-nCoV receptor usage.

Determination of virus infectivity in HeLa cells with or without the expression of human APN and DPP4. The expression of ACE2, APN and DPP4 plasmids with S tag were detected using mouse anti-S tag monoclonal antibody. ACE2, APN and DPP4 proteins (green), viral protein (red) and nuclei (blue) are shown. Scale bars, 10 μm.