A pneumonia outbreak associated with a new coronavirus of probable bat origin — TEI-tagged view

Peng ZhoupersName, Xing‐Lou YangpersName, Xian-Guang WangpersName, Ben HupersName, Lei ZhangpersName, Wei ZhangpersName, Hao-Rui SipersName, Yan ZhupersName, Bei LipersName, Chao-Lin HuangpersName, Huidong ChenpersName, Jing ChenpersName, Yun LuopersName, Hua GuopersName, Ren-Di JiangpersName, Mei-Qin LiupersName, Ying ChenpersName, Xu-Rui ShenpersName, Xi WangpersName, Xiao-Shuang ZhengpersName, Kai ZhaopersName, Quan-Jiao ChenpersName, Fēi DèngpersName, Linlin LiupersName, Bing YanpersName, Fa-Xian ZhanpersName, Yanyi WangpersName, Gengfu XiaopersName, Zheng‐Li ShipersName (2020date). “A pneumonia outbreak associated with a new coronavirus of probable bat origin.” Naturetitle·venue.

Object of study — a virus with no name yet - identified, sequenced, classified and provisionally named in one paper, and RENAMED BY A COMMITTEE BEFORE THE PAPER WAS PRINTED

Research question — what the Wuhan pneumonia outbreak was caused by, where it sits taxonomically, and whether it uses the SARS-CoV receptor

Contribution — five full-length genomes from early patients, the 96.2% match to a batterm coronavirus, the species assignment, and the confirmation that the virus enters cells through ACE2term

Full text — tagged

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Abstract. Abstract Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs)term have been discovered in their natural reservoir hostterm, batsterm 1 – 4 . Previous studies have shown that some batterm SARSr-CoVsterm have the potential to infect humansterm 5 – 7 . Here we report the identification and characterization of a new coronavirus (2019-nCoVterm), which caused an epidemic of acute respiratory syndrometerm in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infectionsterm including 80 deaths by 26 January 2020. Full-length genome sequencesterm were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identityterm to SARS-CoV. Furthermore, we show that 2019-nCoVterm is 96% identical at the whole-genome levelterm to a batterm coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoVterm. In addition, 2019-nCoVterm virus isolated from the bronchoalveolar lavage fluidterm of a critically ill patient could be neutralized by seraterm from several patients. Notably, we confirmed that 2019-nCoVterm uses the same cell entry receptorterm—angiotensin converting enzyme II (ACE2term)—as SARS-CoV. Subject terms: Pathogens, Virology, SARS-CoV-2term Characterization of full-length genome sequencesterm from patients infected with a new coronavirus (2019-nCoVterm) shows that the sequences are nearly identical and indicates that the virus is related to a batterm coronavirus. Main Coronaviruses have caused two large-scale pandemics in the past two decades, SARS and Middle East respiratory syndrome (MERS) 8 , 9 . It has generally been thought that SARSr-CoVterm—which is mainly found in batsterm—could cause a future disease outbreak 10 , 11 . Here we report on a series of cases caused by an unidentified pneumonia disease outbreakterm in Wuhan, Hubei province, central China. This disease outbreak—which started from a local seafood marketterm—has grown substantially to infect 2,761 people in China, is associated with 80 deaths and has led to the infection of 33 people in 10 additional countries as of 26 January 2020 12 . Typical clinical symptoms of these patients are feverterm, dry coughterm, breathing difficulties (dyspnoea)term, headache and pneumonia. Disease onset may result in progressive respiratory failureterm owing to alveolar damageterm (as observed by transverse chest computerized-tomography images) and even death. The disease was determined to be caused by virus-induced pneumoniaterm by clinicians according to clinical symptoms and other criteria, including a rise in body temperature, decreases in the number of lymphocytes and white bloodterm cells (although levels of the latter were sometimes normal), new pulmonary infiltratesterm on chest radiography and no obvious improvement after treatment with antibiotics for three daysterm. It appears that most of the early cases had contact history with the original seafood market; however, the disease has now progressed to be transmitted by human-to-human contactterm. Samples from seven patients with severe pneumonia (six of whom are sellers or deliverymen from the seafood market), who were admitted to the intensive care unit of Wuhan Jin Yin-Tan Hospital at the beginning of the outbreak, were sent to the laboratory at the Wuhan Institute of Virology (WIV) for the diagnosis of the causative pathogenterm (Extended Data Table 1 ). As a laboratory investigating CoV, we first used pan-CoV PCR primersterm to test these samplesterm 13 , given that the outbreak occurred in winter and in a market—the same environment as SARS infections. We found five samples to be PCR-positive for CoVsterm. One sample (WIV04), collected from the bronchoalveolar lavage fluidterm (BALF), was analysed by metagenomics analysisterm using next-generation sequencingterm to identify potential aetiological agentsterm. Of the 10,038,758 total reads—of which 1,582 total reads were retained after filtering of reads from the human genometerm—1,378 (87.1%) sequences matched the sequence of SARSr-CoVterm (Fig. 1a ). By de novo assemblyterm and targeted PCR, we obtained a 29,891-base-pair CoV genometerm that shared 79.6% sequence identityterm to SARS-CoV BJ01 (GenBank accession number AY278488.2 ). High genome coverage was obtained by remapping the total reads to this genome (Extended Data Fig. 1 ). This sequence has been submitted to GISAIDterm ( https://www.gisaidterm.org/ ) (accession number EPI_ISL_402124). Following the name given by the World Health Organization (WHO), we tentatively call it novel coronavirus 2019term (2019-nCoVterm). Four more full-length genome sequences of 2019-nCoVterm (WIV02, WIV05, WIV06 and WIV07) (GISAIDterm accession numbers EPI_ISL_402127–402130) that were more than 99.9% identical to each otherterm were subsequently obtained from four additional patients using next-generation sequencingterm and PCR (Extended Data Table 2 ). Extended Data Table 1 Patient information and diagnosis history Note, some records are missing. All patients are sellers or deliverymen at the seafood market except ICU-01, whose contact history is unclear. All patients were admitted to intensive care unit (ICU) during the first investigation and were now in stable condition. Bloodterm IgM tests have been performed for the following respiratory pathogens for all patients: Legionella pneumophilia , Mycoplasma pneumoniae , Chlamydia pneumoniae , respiratory syncytial virus, adenovirus, Rickettsia , influenza A virus, influenza B virus and parainfluenza virus. *This patient reported feverterm on 12 December 2019 and then recovered without medical treatment. He came back to the hospital on 27 December 2019 with a feverterm. His wife was also ill and admitted to the hospital. Both individuals recovered. Fig. 1 Genome characterization of 2019-nCoVterm. a , Metagenomics analysisterm of next-generation sequencingterm of BALF from patient ICU06. b , Genomic organization of 2019-nCoVterm WIV04. M, membrane. c , Similarity plot based on the full-length genome sequenceterm of 2019-nCoVterm WIV04. Full-length genome sequencesterm of SARS-CoV BJ01, batterm SARSr-CoVterm WIV1, batterm coronavirus RaTG13term 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 . Extended Data Fig. 1 NGS raw reads of sample WIV04 mapping to the 2019-nCoVterm sequence. The x axis indicates the genome nucleotide position and the y axis represents the read depth of the mapping. Extended Data Table 2 Laboratory results Samples from two patients (ICU-01 and ICU-08) were not available during the second investigation. They had been discharged from the hospital. We did a serial test for patient ICU-06 on the following dates: 30 December 2019, 31 December 2019, 1 January 2020 and 10 January 2020, corresponding to 7, 8, 9 and 18 days after disease onset (23 December 2019). Molecular and serological (IgM and IgG) virus-detection results for 2019-nCoVterm are shown. NA, not available. *Virus isolated. # A full-length genome was obtained. The virus genometerm consists of six major open-reading frames (ORFs)term that are common to coronaviruses and a number of other accessory genesterm (Fig. 1b ). Further analysis indicates that some of the 2019-nCoVterm genes shared less than 80% nucleotide sequence identity to SARS-CoV. However, the amino acid sequences of the seven conserved replicase domainsterm in ORF1ab that were used for CoV species classificationterm were 94.4% identicalterm between 2019-nCoVterm and SARS-CoV, suggesting that the two viruses belong to the same species, SARSr-CoVterm. We then found that a short region of RNA-dependent RNA polymerase (RdRp)term from a batterm coronavirus (BatCoV RaTG13term)—which was previously detected in Rhinolophus affinisterm from Yunnan province—showed high sequence identity to 2019-nCoVterm. We carried out full-length sequencingterm on this RNA sample (GISAIDterm accession number EPI_ISL_402131). Simplot analysisterm showed that 2019-nCoVterm was highly similar throughout the genome to RaTG13term (Fig. 1c ), with an overall genome sequence identity of 96.2%term. Using the aligned genome sequences of 2019-nCoVterm, RaTG13term, SARS-CoV and previously reported batterm SARSr-CoVsterm, no evidence for recombination eventsterm was detected in the genome of 2019-nCoVterm. Phylogenetic analysisterm of the full-length genome and the gene sequences of RdRp and spike ( S ) showed that—for all sequences—RaTG13term is the closest relative of 2019-nCoVterm and they form a distinct lineageterm from other SARSr-CoVsterm (Fig. 1d and Extended Data Fig. 2 ). The receptor-binding spike proteinterm encoded by the S gene was highly divergent from other CoVs (Extended Data Fig. 2 ), with less than 75% nucleotide sequence identity to all previously described SARSr-CoVsterm, except for a 93.1% nucleotide identity to RaTG13term (Extended Data Table 3 ). The S genes of 2019-nCoVterm and RaTG13term are longer than other SARSr-CoVsterm. The major differences in the sequence of the S gene of 2019-nCoVterm are the three short insertionsterm in the N-terminal domainterm as well as changes in four out of five of the key residues in the receptor-binding motifterm compared with the sequence of SARS-CoV (Extended Data Fig. 3 ). Whether the insertions in the N-terminal domainterm of the S protein of 2019-nCoVterm confer sialic-acid-binding activityterm as it does in MERS-CoV needs to be further studied. The close phylogenetic relationship to RaTG13term provides evidence that 2019-nCoVterm may have originated in batsterm. 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-nCoVterm and batterm CoV RaTG13term 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 Table 3 Genomic comparison of 2019-nCoVterm WIV04 with SARS-CoVs and batterm SARSr-CoVsterm Extended Data Fig. 3 Amino acid sequence alignment of the S1 protein of the 2019-nCoVterm to SARS-CoV and selected batterm SARSr-CoVsterm. The receptor-binding motifterm 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 ACE2term are numbered at the top of the aligned sequences. The short insertions in the N-terminal domainterm of the 2019-nCoVterm are indicated by the blue boxes. Batterm CoV RaTG13term was obtained from R. affinis , found in Yunnan province. Batterm CoV ZC45 was obtained from R. sinicus , found in Zhejiang province. We rapidly developed a qPCR-based detection methodterm on the basis of the sequence of the receptor-binding domain of the S geneterm, which was the most variable region of the genome (Fig. 1c ). Our data show that the primers could differentiate 2019-nCoVterm from all other human coronavirusesterm including batterm SARSr-CoVterm WIV1, which shares 95% identity with SARS-CoV (Extended Data Fig. 4a, b ). Of the samples obtained from the seven patients, we found that six BALF and five oral swab samplesterm were positive for 2019-nCoVterm during the first sampling, as assessed by qPCR and conventional PCR. However, we could no longer detect virus-positive samples in oral swabsterm, anal swabsterm and bloodterm samples taken from these patients during the second sampling (Fig. 2a ). However, we recommend that other qPCR targets, including the RdRp or envelope ( E ) genes are used for the routine detectionterm of 2019-nCoVterm. On the basis of these findings, we propose that the disease could be transmitted by airborne transmissionterm, although we cannot rule out other possible routes of transmissionterm, as further investigation, including more patients, is required. Extended Data Fig. 4 Molecular detection method used to detect 2019-nCoVterm. a , Standard curve for qPCR primers. The PCR product of the S gene that was serial diluted in the range of 10 8 to 10 1 (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. Fig. 2 Molecular and serological investigation of patient samples. a , Molecular detection of 2019-nCoVterm in seven patients. Patient information can be found in Extended Data Tables 1 , 2 . Detection methods are described in the Methods . AS, anal swabterm; OS, oral swabterm. b , Dynamics of 2019-nCoVterm 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-nCoVterm 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. For serological detection of 2019-nCoVterm, we used a previously developed nucleocapsid (N) proteinterm from batterm SARSr-CoVterm Rp3 as antigen for IgG and IgM enzyme-linked immunosorbent assays (ELISAs)term, as this protein shared 92% amino acid identity to N protein of 2019-nCoVterm (Extended Data Fig. 5 ) and showed no cross-reactivityterm against other human coronaviruses except SARSr-CoVterm 7 . We were only able to obtain five serum samples from the seven patients with viral infections. We monitored viral antibody levelsterm in one patient (ICU-06) 7, 8, 9 and 18 days after the onset of disease (Extended Data Table 2 ). A clear trend was observed in the IgG and IgM titresterm, which increased over time, except that the IgM titre was decreased in the last sample (Fig. 2b ). As a second analysis, we tested samples from 5 of the 7 virus-positive patients around 20 days after disease onset for the presence of viral antibodies (Extended Data Tables 1 , 2 ). All patient samples—but not samples from healthy individualsterm—were strongly positive for viral IgG (Fig. 2b ). There were also three IgM-positive samples, indicating an acute infectionterm. Extended Data Fig. 6 Isolation and antigenic characterization of 2019-nCoVterm. a , b , Vero E6term cells are shown at 24 h after infection with mock virus ( a ) or 2019-nCoVterm ( b ). c , d , Mock-virus-infected ( c ) or 2019-nCoV-infected ( d ) samples were stained with rabbit serum raised against recombinant SARSr-CoVterm 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-nCoVterm among the total number of virus-related reads in metagenomics analysisterm of supernatants from Vero E6term cell cultures. f , Virus growth in Vero E6term cells. g , Viral particles in the ultrathin sectionsterm were imaged using electron microscopyterm at 200 kV. The sample was from virus-infected Vero E6term cells. The inset shows the viral particles in an intra-cytosolic vacuole. We next successfully isolated the virus (called 2019-nCoVterm BetaCoV/Wuhan/WIV04/2019) from both Vero E6term and Huh7 cellsterm using the BALF sample of patient ICU-06. Clear cytopathogenic effectsterm were observed in cells after incubation for three days (Extended Data Fig. 6a, b ). The identity of the strain WIV04 was verified in Vero E6term cells by immunofluorescence microscopyterm using the cross-reactive viral N antibody (Extended Data Fig. 6c, d ) and by metagenomics sequencing, most of the reads of which mapped to 2019-nCoVterm, and qPCR analysis showed that the viral loadterm increased from day 1 to day 3 (Extended Data Fig. 6e, f ). Viral particles in ultrathin sectionsterm of infected cells displayed a typical coronavirus morphologyterm, as visualized by electron microscopyterm (Extended Data Fig. 6g ). To further confirm the neutralization activity of the viral IgG-positive samplesterm, we conducted serum-neutralization assaysterm in Vero E6term cells using the five patient seraterm that were IgG-positive. We demonstrate that all samples were able to neutralize 100 TCID 50 (50% tissue-culture-infective doseterm) of 2019-nCoVterm at a dilution of 1:40–1:80. We also show that this virus could be cross-neutralized by horse anti-SARS-CoV serumterm (gift from L.-F. WangpersName) at dilutions of 1:40; however, the potential for cross-reactivityterm with SARS-CoV antibodies needs to be confirmed with anti-SARS-CoV serum from humans (Extended Data Table 4 ). Extended Data Table 4 Virus neutralization testterm of serum samples Each serum sample was tested in triplicateterm. Serum samples from two healthy individualsterm from Wuhan and five patients as well as a horse anti-SARS-CoV anti-serum were used. We used 120 TCID 50 viruses per well. Serum samples were used at dilutions of 1:10, 1:20, 1:40 and 1:80. neg, negative; VNT, virus neutralization testterm. ACE2term is known to be a cell receptor for SARS-CoVterm 14 . To determine whether 2019-nCoVterm also uses ACE2term as a cellular entry receptorterm, we conducted virus infectivity studiesterm using HeLa cellsterm that expressed or did not express ACE2 proteinsterm from humans, Chinese horseshoe batsterm, civets, pigs and mice. We show that 2019-nCoVterm is able to use all ACE2 proteinsterm, except for mouse ACE2term, as an entry receptorterm to enter ACE2-expressing cells, but not cells that did not express ACE2term, indicating that ACE2term is probably the cell receptor through which 2019-nCoVterm enters cells (Fig. 3 ). We also show that 2019-nCoVterm does not use other coronavirus receptorsterm, such as aminopeptidase N (APN)term and dipeptidyl peptidase 4 (DPP4)term (Extended Data Fig. 7 ). Fig. 3 Analysis of the receptor use of 2019-nCoVterm. Determination of virus infectivity in HeLa cellsterm that expressed or did not express (untransfected) ACE2term. The expression of ACE2term plasmid with S tag was detected using mouse anti-S tag monoclonal antibody. hACE2, human ACE2term; bACE2, ACE2term of Rhinolophus sinicus (batterm); cACE2, civet ACE2term; sACE2, swine ACE2term (pig); mACE2, mouse ACE2term. Green, ACE2term; red, viral protein (N); blue, DAPI (nuclei). Scale bars, 10 μm. Extended Data Fig. 7 Analysis of 2019-nCoVterm receptor usage. Determination of virus infectivity in HeLa cellsterm with or without the expression of human APN and DPP4. The expression of ACE2term, APN and DPP4 plasmids with S tag were detected using mouse anti-S tag monoclonal antibody. ACE2term, APN and DPP4 proteins (green), viral protein (red) and nuclei (blue) are shown. Scale bars, 10 μm. The study provides a detailed report on 2019-nCoVterm, the likely aetiological agentterm responsible for the ongoing epidemic of acute respiratory syndrometerm in China and other countries. Virus-specific nucleotide-positive and viral-protein seroconversionterm was observed in all patients tested and provides evidence of an association between the disease and the presence of this virusterm. However, there are still many urgent questions that remain to be answered. The association between 2019-nCoV and the diseaseterm has not been verified by animal experimentsterm to fulfil the Koch’s postulatesterm to establish a causative relationshipterm between a microorganism and a disease. We do not yet know the transmission routineterm of this virus among hosts. It appears that the virus is becoming more transmissible between humansterm. We should closely monitor whether the virus continues to evolve to become more virulent. Owing to a shortage of specific treatmentsterm and considering the relatedness of 2019-nCoVterm to SARS-CoV, some drugs and pre-clinical vaccines against SARS-CoV could probably be used to treat this virus. Finally, considering the wide spread of SARSr-CoVterm in their natural reservoirs, future researchterm should be focused on active surveillanceterm of these viruses for broader geographical regions. In the long term, broad-spectrum antiviral drugs and vaccinesterm should be prepared for emerging infectious diseases that are caused by this cluster of viruses in the future. Most importantly, strict regulationsterm against the domestication and consumption of wildlife should be implemented. Note added in proof: Since this paper was accepted, the ICTV has designated the virus as SARS-CoV-2term 15 ; in addition, the WHO has released the official name of the disease caused by this virus, which is COVID-19term 16 . Methods Data reporting No statistical methods were used to predetermine sample sizeterm. The experiments were not randomizedterm and the investigators were not blinded to allocationterm during experiments and outcome assessment. Sample collection Human samples, including oral swabsterm, anal swabsterm, bloodterm and BALF samples were collected by Jinyintan hospital (Wuhan, China) with the consent of all patients and approved by the ethics committee of the designated hospital for emerging infectious diseases. Patients were sampled without gender or age preference unless indicated. For swabs, 1.5 ml DMEM containing 2% FBS was added to each tube. The supernatant was collected after centrifugation at 2,500 rpm, vortexing for 60 s and a standing period of 15–30 min. The supernatant from swabs or BALF (no pre-treatment) was added to either lysis buffer for RNA extraction or to viral transport mediumterm for isolation of the virus. The viral transport mediumterm was composed of Hank’s balanced salt solution (pH 7.4) containing BSA (1%), amphotericin (15 μg ml −1 ), penicillin G (100 units ml −1 ) and streptomycin (50 μg ml −1 ). Serum was separated by centrifugation at 3,000 g for 15 min within 24 h of collection, followed by inactivation at 56 °C for 1 h, and was then stored at 4 °C until use. Virus isolation, cell infection, electron microscopyterm and neutralization assay The following cell lines were used for virus isolation in this study: Vero E6term and Huh7 cellsterm, which were cultured in DMEM containing 10% FBS. All cell lines were tested and free of mycoplasma contamination, submitted for species identification and authenticated by morphological evaluation by microscopy. None of the cell lines was on the list of commonly misidentified cell lines (by ICLAC). Cultured cell monolayers were maintained in their respective medium. The PCR-positive BALF sample from ICU-06 patient was spun at 8,000 g for 15 min, filtered and diluted 1:2 with DMEM supplemented with 16 μg ml −1 trypsin before it was added to the cells. After incubation at 37 °C for 1 h, the inoculum was removed and replaced with fresh culture medium containing antibiotics (see below) and 16 μg ml −1 trypsin. The cells were incubated at 37 °C and observed daily for cytopathogenic effectsterm. The culture supernatant was examined for the presence of virus by qRT–PCR methods developed in this study, and cells were examined by immunofluorescence microscopyterm using the anti-SARSr-CoV Rp3 N antibody that was generated in-house (1:1,000). Penicillin (100 units ml −1 ) and streptomycin (15 μg ml −1 ) were included in all tissue culture media. Vero E6term cells were infected with the new virus at a multiplicity of infection (MOI) of 0.5term and collected 48 h after infection. Cells were fixed with 2.5% (w/v) glutaraldehyde and 1% osmium tetroxide, dehydrated through a graded series of ethanol concentrations (from 30 to 100%) and embedded with epoxy resin. Ultrathin sectionsterm (80 nm) of embedded cells were prepared, deposited onto Formvar-coated copper grids (200 mesh), stained with uranyl acetate and lead citrate, and analysed using a 200-kV Tecnai G2 electron microscope. The virus neutralization testterm was carried out in a 96-well plate. The patient serum samples were heat-inactivated by incubation at 56 °C for 1 h before use. The serum samples were diluted to 1:10, 1:20, 1:40 or 1:80, and then an equal volume of virus stock was added and incubated at 37 °C for 60 min in a 5% CO 2 incubator. Diluted horse anti-SARS-CoV serumterm or serum samples from healthy individualsterm were used as control. After incubation, 100 μl mixtures were inoculated onto a monolayer of Vero E6term cells in a 96-well plate for 1 h. Each serum was assessed in triplicateterm. After removing the supernatant, the plate was washed twice with DMEM medium. Cells were incubated with DMEM supplemented with 2% FBS for 3 days. Subsequently, the cells were checked for cytopathogenic effectsterm. RNA extraction and PCR Whenever commercial kits were used, the manufacturer’s instructions were followed without modification. RNA was extracted from 200 μl of samples with the High Pure Viral RNA kit (Roche). RNA was eluted in 50 μl of elution buffer and used as the template for RT–PCR. For qPCR analysis, primers based on the S gene of 2019-nCoVterm were designed: RBD-qF1, 5′-CAATGGTTTAACAGGCACAGG-3′; RBD-qR1, 5′-CTCAAGTGTCTGTGGATCACG-3′. RNA extracted as described above was used for qPCR using the HiScript II One Step qRT–PCR SYBR Green Kit (Vazyme Biotech). Conventional PCRs were also performed using the following primer pairs: ND-CoVs-951F, 5′-TGTKAGRTTYCCTAAYATTAC-3′; ND-CoVs-1805R, 5′-ACATCYTGATANARAACAGC-3′. The 20-μl qPCR reaction mix contained 10 μl 2× One Step SYBR Green mix, 1 μl One Step SYBR Green Enzyme mix, 0.4 μl 50× ROX Reference Dye 1, 0.4 μl of each primer (10 μM) and 2 μl template RNA. Amplification was performed as follows: 50 °C for 3 min, 95 °C for 30 s followed by 40 cycles consisting of 95 °C for 10 s and 60 °C for 30 s, and a default melting curve step in an ABI 7500 Real-time PCR machine. Serological test In-house anti-SARSr-CoV IgG and IgM ELISA kits were developed using SARSr-CoVterm Rp3 N protein as antigen, which shared more than 90% amino acid identity to all SARSr-CoVsterm 2 . For IgG analyses, MaxiSorp Nunc-immuno 96-well ELISA plates were coated (100 ng per well) overnight with recombinant N protein. Human sera were used at a dilution of 1:20 for 1 h at 37 °C. An anti-human IgG HRP-conjugated monoclonal antibody (Kyab Biotech) was used at a dilution of 1:40,000. The OD value (450–630 nm) was calculated. For IgM analyses, MaxiSorp Nunc-immuno 96-well ELISA plates were coated (500 ng per well) overnight with anti-human IgM (μ chain). Human sera were used at a 1:100 dilution for 40 min at 37 °C, followed by incubation with an anti-Rp3 N HRP-conjugated antibody (Kyab Biotech) at a dilution of 1:4,000. The OD value (450–630 nm) was calculated. Examination of ACE2term receptor for 2019-nCoVterm infection HeLa cellsterm transiently expressing ACE2term were prepared using Lipofectamine 3000 (Thermo Fisher Scientific) in a 96-well plate; mock-transfected cells were used as controls. 2019-nCoVterm grown in Vero E6term cells was used for infection at a MOI of 0.5. APN and DPP4 were analysed in the same way. The inoculum was removed after absorption for 1 h and washed twice with PBS and supplemented with medium. At 24 h after infection, cells were washed with PBS and fixed with 4% formaldehyde in PBS (pH 7.4) for 20 min at room temperature. ACE2term expression was detected using a mouse anti-S tag monoclonal antibody and a FITC-labelled goat anti-mouse IgG H&L (Abcam, ab96879). Viral replication was detected using a rabbit antibody against the Rp3 N protein (generated in-house, 1:1,000) and a Cy3-conjugated goat anti-rabbit IgG (1:200, Abcam, ab6939). Nuclei were stained with DAPI (Beyotime). Staining patterns were examined using confocal microscopy on a FV1200 microscope (Olympus). High-throughput sequencingterm, pathogen screening and genome assembly Samples from patient BALF or from the supernatant of virus cultures were used for RNA extraction and next-generation sequencingterm (NGS) using BGI MGISEQ2000 and Illumina MiSeq 3000 sequencers. Metagenomic analysis was carried out mainly based on the bioinformatics platform MGmapper (PE_2.24 and SE_2.24). The raw NGS reads were first processed by Cutadaptterm (v.1.18) with minimum read length of 30 base pairs. BWA (v.0.7.12-r1039) was used to align reads to a local database with a filter hits parameter of 0.8 FMM ((match + mismatch)/read length ≥ fraction] value and minimum alignment score of 30. Parameters for post-processing of assigned reads were set to a minimum size normalized abundance of 0.01, minimum read count of 20 and were otherwise set to default parameters. A local nucleic acid database for human and mammals was used to filter reads of host genomesterm before mapping reads to the virus database. The results of the metagenomic analysis were displayed as pie charts using Microsoft Office 2010. NGS reads were assembled into genomes using Geneious (v.11.0.3) and MEGAHITterm (v.1.2.9). PCR and Sanger sequencingterm was performed to fill gaps in the genome. 5′-rapid amplification of cDNA ends (RACE) was performed to determine the 5′-end of the genomes using a SMARTer RACE 5′/3′ kit (Takara). Genomes were annotated using the Clone Manager Professional Suite 8 (Sci-Ed Software). Phylogenetic analysisterm Routine sequence management and analysis was carried out using DNAStar. The sequence alignment of complete genome sequences was performed using MAFFTterm (v.7.307) with default parameters. The codon alignments of full-length S and RdRp gene sequences were converted from the corresponding protein alignments by PAL2NALterm (v.14); the protein alignments were created by Clustal Omega (v.1.2.4) using default parameters. Maximum likelihood phylogenetic treesterm were generated using RAxML (v.0.9.0) with GTR+G substitution modelterm and 1,000 bootstrap replicatesterm. 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/s41586-020-2012-7. Supplementary information Reporting Summary Acknowledgements We thank P. ZhangpersName and A. Du from the WIV core facility centre for their help with producing transmission electron microscopyterm micrographs; H.-Z. LiupersName and P. Yu from WIV for bioinformatics analysis. This work was jointly supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS) (XDB29010101 to Z.-L.S. and XDB29010104 to P.Z.), China Natural Science Foundation for excellent scholars (81822028 to P.Z., 31770175 to Z.-L.S. and 31800142 to B.H.), Mega-Project for Infectious Disease from Minister of Science and Technology of the People’s Republic of China (2018ZX10305409-004-001 to P.Z.), Youth innovation promotion association of CAS (2019328 to X.-L.Y.). Extended data figures and tables Extended Data Fig. 5 Amino acid sequence alignment of the nucleocapsid protein of 2019-nCoVterm to batterm SARSr-CoVterm Rp3 and SARS-CoV BJ01. Batterm SARSr-CoVterm Rp3 was obtained from R. sinicus , which is found in Guangxi province. Author contributions Z.-L.S., P.Z., Y.-Y.W. and G.-F.X. conceived the study. X.-G.W., C.-L.H., H.-D.C., F.D., Q.-J.C., F.-X.Z. and L.-L.L. collected patient samples. X.-L.Y., B.Y., W.Z., B.L., J.C., X.-S.Z., Y.L., H.G., R.-D.J., M.-Q.L., Y.C., X.W., X.-R.S. and K.Z. performed qPCR, serology and virus culturing experiments. L.Z., Y.Z., H.-R.S. and B.H. performed genome sequencing and annotations. Data availability Sequence data that support the findings of this study have been deposited in GISAIDterm ( https://www.gisaidterm.org/ ) with accession numbers EPI_ISL_402124, EPI_ISL_402127–EPI_ISL_402130 and EPI_ISL_402131; GenBank with accession numbers MN996527 – MN996532 ; National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences ( https://bigd.big.ac.cn/databases?lang=en ) with accession numbers SAMC133236–SAMC133240 and SAMC133252. Competing interests The authors declare no competing interests. Footnotes Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Peng ZhoupersName, Xing-Lou YangpersName, Xian-Guang WangpersName Change history 9/28/2020 This article was amended to correct the Peer review information. Extended data is available for this paper at 10.1038/s41586-020-2012-7. Supplementary information is available for this paper at 10.1038/s41586-020-2012-7.