A new coronavirus associated with human respiratory disease in China

Abstract

Emerging infectious diseases, 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 2019. 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 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing⁴ of a sample of bronchoalveolar lavage fluid 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-nCoV'). 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 Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China⁵. This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

Fig. 1. Genome organization of SARS and SARS-like CoVs.

The organization of genes for WHCV, bat SL-CoVZC45 and SARS-CoV Tor2.

Fig. 2. Maximum likelihood phylogenetic trees of nucleotide sequences of the ORF1a, ORF1b, E and M genes of WHCV 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.

Fig. 3. Possible recombination events in the S gene of sarbecoviruses.

a, The sequence similarity plot reveals two putative recombination breakpoints (black dashed lines), with their locations indicated at the bottom. The plot shows similarity comparisons of the S gene of WHCV (query) compared with the sequences of SARS-CoV 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 method 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.

Extended Data Fig. 1. Chest radiographs of the patient.

a–d, Computed-tomography scans of the chest were obtained on the day of admission (day 6 after the onset of disease). Bilateral focal consolidation, lobar consolidation and patchy consolidation were clearly observed, especially in the lower lung. e, A chest radiograph was obtained on day 5 after admission (day 11 after the onset of disease). Bilateral diffuse patchy and fuzzy shadows were observed.

Extended Data Fig. 2. Other respiratory pathogens were not detected in the BALF sample by real-time RT–PCR.

a–e, The BALF sample was tested for the presence of influenza A virus (a), the Victoria lineage of influenza B viruses (b), the Yamagata lineage of influenza B viruses (c), human adenovirus (d) and Chlamydia pneumoniae (e). Sample 1 was the BALF sample of the patient, water was used as a negative (NEG) control and positive (POS) control samples included plasmids covering the Taqman primers and probe regions of influenza A, the Victoria and Yamagata lineages of influenza B viruses, human adenovirus and Chlamydia pneumoniae.

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. 4. Quantification of WHCV in clinical samples by real-time RT–PCR.

a, Specificity evaluation of the WHCV primers. Test samples comprised clinical samples that were positive for at least one of the following viruses: influenza A virus (09H1N1 and H3N2), influenza B virus, human adenovirus, respiratory syncytial virus, rhinovirus, parainfluenza virus type 1–4, human bocavirus, human metapneumovirus, coronavirus OC43, coronavirus NL63, coronavirus 229E and coronavirus HKU1. Only the standard plasmid of WHCV (WHCV 15,704–16,846 bp in a pLB vector) led to positive amplification (brown curve). b, Amplification curve of the DNA standard for WHCV. From left to right, the DNA concentrations were 1.8 × 10⁸, 1.8 × 10⁷, 1.8 × 10⁶, 1.8 × 10⁵, 1.8 × 10⁴ and 1.8 × 10³. c, Linear fitted curve of C_t values to concentrations of the WHCV DNA standard. d, Quantification of WHCV in the BALF sample by real-time RT–PCR. The WHCV DNA standard was used as positive control (POS), water (NEG) and blank were used as negative controls. The amplification curve of the BALF sample is shown in green.

Extended Data Fig. 5. Maximum likelihood phylogenetic trees of the nucleotide sequences of the whole genome, and S and N genes of WHCV and related coronaviruses.

Numbers (>70) above or below the branches indicate percentage bootstrap values. The trees were mid-point rooted for clarity only. The scale bar represents the number of substitutions per site.

Extended Data Fig. 6. Maximum likelihood phylogenetic trees of the nucleotide sequences of the 3CL, RdRp, Hel, ExoN, NendoU and O-MT genes of WHCV and related coronaviruses.

Numbers (>70) above or below the branches indicate percentage bootstrap values. The trees were mid-point rooted for clarity only. The scale bar represents the number of substitutions per site.

Extended Data Fig. 7. Analysis of RBD of the spike protein of WHCV coronavirus.

a, Amino acid sequence alignments of RBD sequences of SARS-like CoVs. Three bat SARS-like CoVs—which could efficiently use the human ACE2 as receptor—had an RBD sequence of similar size to SARS-CoV. WHCV contains a single Val470 insertion. The key amino acid residues involved in the interaction with human ACE2 are marked by orange squares. By contrast, five bat SARS-like CoVs, including Rp3, which has previously been found not to bind to ACE2—had amino acid deletions in two motifs (amino acids 433–437 and 460–472, highlighted by red boxes) compared with those of SARS-CoV.11 b, The two motifs (amino acids 433–437 and 460–472) are shown in red for the crystal structure of the RBD of the spike protein of SARS-CoV in complex with the human ACE2 receptor (PDB 2AJF). Human ACE2 is shown in blue and the RBD of the spike protein of SARS-CoV is shown in green. Important residues in human ACE2 that interact with the RBD of the spike protein of SARS-CoV are marked. c, Predicted protein structure of the RBD of the spike protein of WHCV based on target–template alignment using ProMod3 on the SWISS-MODEL server. d, Predicted structure of the RBD of the spike protein of SARS-like CoV Rs4874. e, Predicted structure of the RBD of the spike protein of SARS-like CoV Rp3. f, Crystal structure of the RBD of the spike protein of SARS-CoV (green) (PDB 2GHV). Motifs that resemble amino acids 433–437 and 460–472 of the spike protein of SARS-CoV are shown in red.

Extended Data Fig. 8. Amino acid sequence comparison of the N-terminal domain of the spike protein.

Amino acid sequence comparison of the N-terminal domain of the spike protein of WHCV, bovine coronavirus (BCoV), mouse hepatitis virus (MHV) and human coronaviruses (HCoV OC43 and HKU1) that can bind to sialic acid and the SARS-CoVs that cannot (SZ3, WH20, BJ0 and Tor2). The key residues for sialic acid binding on BCoV, MHV, and HCoV OC43 and HKU1 are highlighted by orange squares.

Extended Data Fig. 9

Recombination events in WHCV. The sequence similarity plot of WHCV, SARS-like CoVs and bat SARS-like CoVs reveals putative recombination events.