《内科学》课程教学资源（新冠文献）A Novel Coronavirus from Patients with Pneumonia in China, 2019.pdf_大学文库

The new england journal o f medicine n engl j med nejm.org 1 Brief Report Summary In December 2019, a cluster of patients with pneumonia of unknown cause was linked to a seafood wholesale market in Wuhan, China. A previously unknown betacoronavirus was discovered through the use of unbiased sequencing in samples from patients with pneumonia. Human airway epithelial cells were used to isolate a novel coronavirus, named 2019-nCoV, which formed a clade within the subgenus sarbecovirus, Orthocoronavirinae subfamily. Different from both MERS-CoV and SARS-CoV, 2019-nCoV is the seventh member of the family of coronaviruses that infect humans. Enhanced surveillance and further investigation are ongoing. (Funded by the National Key Research and Development Program of China and the National Major Project for Control and Prevention of Infectious Disease in China.) Emerging and reemerging pathogens are global challenges for public health.1 Coronaviruses are enveloped RNA viruses that are distributed broadly among humans, other mammals, and birds and that cause respiratory, enteric, hepatic, and neurologic diseases.2,3 Six coronavirus species are known to cause human disease.4 Four viruses — 229E, OC43, NL63, and HKU1 — are prevalent and typically cause common cold symptoms in immunocompetent individuals.4 The two other strains — severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) — are zoonotic in origin and have been linked to sometimes fatal illness.5 SARS-CoV was the causal agent of the severe acute respiratory syndrome outbreaks in 2002 and 2003 in Guangdong Province, China.6-8 MERS-CoV was the pathogen responsible for severe respiratory disease outbreaks in 2012 in the Middle East.9 Given the high prevalence and wide distribution of coronaviruses, the large genetic diversity and frequent recombination of their genomes, and increasing human–animal interface activities, novel coronaviruses are likely to emerge periodically in humans owing to frequent cross-species infections and occasional spillover events.5,10 In late December 2019, several local health facilities reported clusters of patients with pneumonia of unknown cause that were epidemiologically linked to a seafood and wet animal wholesale market in Wuhan, Hubei Province, China.11 On December 31, 2019, the Chinese Center for Disease Control and Prevention (China CDC) dispatched a rapid response team to accompany Hubei provincial and Wuhan city health authorities and to conduct an epidemiologic and etiologic investigation. We report the results of this investigation, identifying the source of the pneumonia From the NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (N.Z., W.W., J.S., X.Z., B.H., R.L., P.N., X.M., D.W., W.X., G.W., G.F.G., W.T.), and the Department of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University (X.L.) — both in Beijing; Wuhan Jinyintan Hospital (D.Z.), the Division for Viral Disease Detection, Hubei Provincial Center for Disease Control and Prevention (B.Y., F.Z.), and the Center for Biosafety Mega-Science, Chinese Academy of Sciences (W.T.) — all in Wuhan; and the Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China (W.S.). Address reprint requests to Dr. Tan at the NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, China CDC, 155 Changbai Road, Changping District, Beijing 102206, China; or at tanwj@ivdc.chinacdc.cn, Dr. Gao at the National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China, or at gaof@ im.ac.cn, or Dr. Wu at the NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 102206, China, or at wugz@ivdc.chinacdc.cn. Drs. Zhu, Zhang, W. Wang, Li, and Yang contributed equally to this article. This article was published on January 24, 2020, and updated on January 29, 2020, at NEJM.org. DOI: 10.1056/NEJMoa2001017 Copyright © 2020 Massachusetts Medical Society. A Novel Coronavirus from Patients with Pneumonia in China, 2019 Na Zhu, Ph.D., Dingyu Zhang, M.D., Wenling Wang, Ph.D., Xingwang Li, M.D., Bo Yang, M.S., Jingdong Song, Ph.D., Xiang Zhao, Ph.D., Baoying Huang, Ph.D., Weifeng Shi, Ph.D., Roujian Lu, M.D., Peihua Niu, Ph.D., Faxian Zhan, Ph.D., Xuejun Ma, Ph.D., Dayan Wang, Ph.D., Wenbo Xu, M.D., Guizhen Wu, M.D., George F. Gao, D.Phil., and Wenjie Tan, M.D., Ph.D., for the China Novel Coronavirus Investigating and Research Team

Thr NEW ENGLAND JOURNAL Of MEDICINE on plastic substrate cell mens were tested by the China CDC at an early 2.5x10 cells per well on ne stage of the outbreak.We also describe clinical COL (12-mm diameter)supports.Human airwa features of the pneumonia in two of these pa- epithelial cell cultures were generated in an tients. air-liquid interface for 4 to 6 weeks to form cultures resen METHODS mucociliary epi VIRAL DIAGNOSTIC METHODS Prior to infection.apical surfaces of the hu Four lower respiratory tract samples including man aira epithelial cells were washed three bronchoalyeolar-lavage fluid.were collected from times with phosphate-buffered saline:150 ul of patients with pneumonia of unknown cause who supernatant from bronchoalveolar-lavage fluid were identifie han on December 21,201 samples inoculated onto the apica r pres the Huana ter a 2 o the me yw. vere collected from nts in Beiiine utes.human airu pithelial cell mair hospitals with pneumonia of known cause to tained in an air-liquid interface incubated at serve as control samples.Extraction of nucleic 37C with 5%carbon dioxide.Every 48 hours acids from clinical samples gincluding uninfect 150 ul of phosphate-buffered saline was applied ed cultures th at serve d as negative to the apic cal surfaces of the human H Is,an arter 10 minutes or ested for ote mair riruses and hacteria by po chain re tained in this en nt tion (PCR),using the RespiFinderSmart22kit passaged in a 1:3 diluted vial stock to new cells (PathoFinder BV)and the LightCycler 480 real- The cells were monitored daily with light micros time PCR system,in accordance with manufac copy,for with RI-PCR.fo ample were an the presfte n the ruse r three high-thnga Ψ tron microscopy eprepared fo previous was used to discover microbial se quences not identifiable by the means describec TRANSMISSION ELECTRON MICROSCOPY above.A real-time reverse transcription PCR(RT Supernatant from human airway epithelial cell PCR)assay was used to detect viral RNA by tar cultures that showe cytopathic effects was e for at least natant was negatively stained on film-coated SOLATION OF VIRUS grids for examination human airway epithelial Bronchoalveolar-lavage fluid samples were col- lected in sterile transport and fixe 2%paraformaldehyde 2.59 we e then centr glutaraldehyde with 19 d to an ro. which had hee block and resected from patients undergoing surgery for uranvl acetate and lead citrate, rately The lung cancer and were confirmed to be special-negative stained grids and ultrathin sections pathogen-free by NGS. were observed under transmission electron mi Human airway epithelial cells were expanded croscopy. N ENGLJ MED NEJM.ORG

2 n engl j med nejm.org The new england journal o f medicine clusters, and describe a novel coronavirus detected in patients with pneumonia whose specimens were tested by the China CDC at an early stage of the outbreak. We also describe clinical features of the pneumonia in two of these patients. Methods Viral Diagnostic Methods Four lower respiratory tract samples, including bronchoalveolar-lavage fluid, were collected from patients with pneumonia of unknown cause who were identified in Wuhan on December 21, 2019, or later and who had been present at the Huanan Seafood Market close to the time of their clinical presentation. Seven bronchoalveolar-lavage fluid specimens were collected from patients in Beijing hospitals with pneumonia of known cause to serve as control samples. Extraction of nucleic acids from clinical samples (including uninfected cultures that served as negative controls) was performed with a High Pure Viral Nucleic Acid Kit, as described by the manufacturer (Roche). Extracted nucleic acid samples were tested for viruses and bacteria by polymerase chain reaction (PCR), using the RespiFinderSmart22kit (PathoFinder BV) and the LightCycler 480 realtime PCR system, in accordance with manufacturer instructions.12 Samples were analyzed for 22 pathogens (18 viruses and 4 bacteria) as detailed in the Supplementary Appendix. In addition, unbiased, high-throughput sequencing, described previously,13 was used to discover microbial sequences not identifiable by the means described above. A real-time reverse transcription PCR (RTPCR) assay was used to detect viral RNA by targeting a consensus RdRp region of pan β-CoV, as described in the Supplementary Appendix. Isolation of Virus Bronchoalveolar-lavage fluid samples were collected in sterile cups to which virus transport medium was added. Samples were then centrifuged to remove cellular debris. The supernatant was inoculated on human airway epithelial cells,13 which had been obtained from airway specimens resected from patients undergoing surgery for lung cancer and were confirmed to be specialpathogen-free by NGS.14 Human airway epithelial cells were expanded on plastic substrate to generate passage-1 cells and were subsequently plated at a density of 2.5×105 cells per well on permeable TranswellCOL (12-mm diameter) supports. Human airway epithelial cell cultures were generated in an air–liquid interface for 4 to 6 weeks to form well-differentiated, polarized cultures resembling in vivo pseudostratified mucociliary epithelium.13 Prior to infection, apical surfaces of the human airway epithelial cells were washed three times with phosphate-buffered saline; 150 μl of supernatant from bronchoalveolar-lavage fluid samples was inoculated onto the apical surface of the cell cultures. After a 2-hour incubation at 37°C, unbound virus was removed by washing with 500 μl of phosphate-buffered saline for 10 minutes; human airway epithelial cells were maintained in an air–liquid interface incubated at 37°C with 5% carbon dioxide. Every 48 hours, 150 μl of phosphate-buffered saline was applied to the apical surfaces of the human airway epithelial cells, and after 10 minutes of incubation at 37°C the samples were harvested. Pseudostratified mucociliary epithelium cells were maintained in this environment; apical samples were passaged in a 1:3 diluted vial stock to new cells. The cells were monitored daily with light microscopy, for cytopathic effects, and with RT-PCR, for the presence of viral nucleic acid in the supernatant. After three passages, apical samples and human airway epithelial cells were prepared for transmission electron microscopy. Transmission Electron Microscopy Supernatant from human airway epithelial cell cultures that showed cytopathic effects was collected, inactivated with 2% paraformaldehyde for at least 2 hours, and ultracentrifuged to sediment virus particles. The enriched supernatant was negatively stained on film-coated grids for examination. Human airway epithelial cells showing cytopathic effects were collected and fixed with 2% paraformaldehyde–2.5% glutaraldehyde and were then fixed with 1% osmium tetroxide dehydrated with grade ethanol embedded with PON812 resin. Sections (80 nm) were cut from resin block and stained with uranyl acetate and lead citrate, separately. The negative stained grids and ultrathin sections were observed under transmission electron microscopy

n engl j med nejm.org 3 Brief Report Viral Genome Sequencing RNA extracted from bronchoalveolar-lavage fluid and culture supernatants was used as a template to clone and sequence the genome. We used a combination of Illumina sequencing and nanopore sequencing to characterize the virus genome. Sequence reads were assembled into contig maps (a set of overlapping DNA segments) with the use of CLC Genomics software, version 4.6.1 (CLC Bio). Specific primers were subsequently designed for PCR, and 5′- or 3′-RACE (rapid amplification of cDNA ends) was used to fill genome gaps from conventional Sanger sequencing. These PCR products were purified from gels and sequenced with a BigDye Terminator v3.1 Cycle Sequencing Kit and a 3130XL Genetic Analyzer, in accordance with the manufacturers’ instructions. Multiple-sequence alignment of the 2019- nCoV and reference sequences was performed with the use of Muscle. Phylogenetic analysis of the complete genomes was performed with RAxML (13) with 1000 bootstrap replicates and a general time-reversible model used as the nucleotide substitution model. Results Patients Three adult patients presented with severe pneumonia and were admitted to a hospital in Wuhan on December 27, 2019. Patient 1 was a 49-year-old woman, Patient 2 was a 61-year-old man, and Patient 3 was a 32-year-old man. Clinical profiles were available for Patients 1 and 2. Patient 1 reported having no underlying chronic medical conditions but reported fever (temperature, 37°C to 38°C) and cough with chest discomfort on December 23, 2019. Four days after the onset of illness, her cough and chest discomfort worsened, but the fever was reduced; a diagnosis of pneumonia was based on computed tomographic (CT) scan. Her occupation was retailer in the seafood wholesale market. Patient 2 initially reported fever and cough on December 20, 2019; respiratory distress developed 7 days after the onset of illness and worsened over the next 2 days (see chest radiographs, Fig. 1), at which time mechanical ventilation was started. He had been a frequent visitor to the seafood wholesale market. Patients 1 and 3 recovered and were discharged from the hospital on January 16, 2020. Patient 2 died on January 9, 2020. No biopsy specimens were obtained. Figure 1. Chest Radiographs. Shown are chest radiographs from Patient 2 on days 8 and 11 after the onset of illness. The trachea was intubated and mechanical ventilation instituted in the period between the acquisition of the two images. Bilateral fluffy opacities are present in both images but are increased in density, profusion, and confluence in the second image; these changes are most marked in the lower lung fields. Changes consistent with the accumulation of pleural liquid are also visible in the second image. A B

Thr NEW ENGLAND JOURNAL Of MEDICINE A Mock 100m 2.0 athic Efferts in H ocuation with 19-nCoV DETECTION AND ISOLATION OF A NOVEL man airway epithelial cells:a lack of ciliun CORONAVIRUS beating was seen with light microcopy in the Three bronchoalveolar-lavage samples were col- center of the focus (Fig.2).No specific cyto- lected from Wuhan Jinyintan Hospital on De pathic effects were observed in the Vero E and cember 30,2019. Huh-7 cell lines until 6 days after inoculation 43,and 2019c negative-s 。 patients by the Resp with some phism 22kit RNA extracted from bronchoalyeolar-la ied from about 60 to 140 nm.Virus par ticles ha rage fluid from the patients was used as a tem quite distinctive spikes,about 9 to 12 nm,and plate to clone and sequence a genome using a gave virions the appearance of a solar corona combination of lllumina Extracellular free virus particles and inclusio d sicles in particles in me ed to the e found nhology is tent more than 85%identity with a bat SARS-like Coronaviridae family CoV (bat-SL-CoVZC45,MG772933.1)genome To further characterize the virus,de novo se- published previously.Positive results ere alsc quences of 2019-nCov genome from clinical spec imens (bronchoalv age fluid)and humar RN e tes we of pan p isolation from the clinical specimens was per Tvo nearly full-length coronavirus sequence formed with human airwa epithelial cells and were obtained from hronchoalyeolar-lavage fluid Vero E6 and Huh-7 cell lines.The isolated virus (BetaCoV/Wuhan/IVDC-HB-04/2020, BetaCoV was named 2019-n Wuhan/IVDC-HB-05/2020EPI_ISL_402 21),and insparticdeseouldbs one ful engt sequenc obtained rom mock-infa IVDC-HB-0120 the th examined with light microscopy daily and with were submitted to GISAID (BetaCov/Wuhan transmission electron microscopy 6days after IVDC-HB-01/2019,accessionID:EPI_ISL_402119 noculation.Cytopathic effects were observec BetaCoV/Wuhan/IVDC-HB-04/2020,accession ID nours after inoculation on surface layers of hu EPI_ISL_402120;BetaCoV/Wuhan/IVDC-HB-05/2019 N ENGLJ MED NEJM.ORG

4 n engl j med nejm.org The new england journal o f medicine Detection and Isolation of a Novel Coronavirus Three bronchoalveolar-lavage samples were collected from Wuhan Jinyintan Hospital on December 30, 2019. No specific pathogens (including HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1) were detected in clinical specimens from these patients by the RespiFinderSmart- 22kit. RNA extracted from bronchoalveolar-lavage fluid from the patients was used as a template to clone and sequence a genome using a combination of Illumina sequencing and nanopore sequencing. More than 20,000 viral reads from individual specimens were obtained, and most contigs matched to the genome from lineage B of the genus betacoronavirus — showing more than 85% identity with a bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1) genome published previously. Positive results were also obtained with use of a real-time RT-PCR assay for RNA targeting to a consensus RdRp region of pan β-CoV (although the cycle threshold value was higher than 34 for detected samples). Virus isolation from the clinical specimens was performed with human airway epithelial cells and Vero E6 and Huh-7 cell lines. The isolated virus was named 2019-nCoV. To determine whether virus particles could be visualized in 2019-nCoV–infected human airway epithelial cells, mock-infected and 2019-nCoV– infected human airway epithelial cultures were examined with light microscopy daily and with transmission electron microscopy 6 days after inoculation. Cytopathic effects were observed 96 hours after inoculation on surface layers of human airway epithelial cells; a lack of cilium beating was seen with light microcopy in the center of the focus (Fig. 2). No specific cytopathic effects were observed in the Vero E6 and Huh-7 cell lines until 6 days after inoculation. Electron micrographs of negative-stained 2019-nCoV particles were generally spherical with some pleomorphism (Fig. 3). Diameter varied from about 60 to 140 nm. Virus particles had quite distinctive spikes, about 9 to 12 nm, and gave virions the appearance of a solar corona. Extracellular free virus particles and inclusion bodies filled with virus particles in membranebound vesicles in cytoplasm were found in the human airway epithelial ultrathin sections. This observed morphology is consistent with the Coronaviridae family. To further characterize the virus, de novo sequences of 2019-nCoV genome from clinical specimens (bronchoalveolar-lavage fluid) and human airway epithelial cell virus isolates were obtained by Illumina and nanopore sequencing. The novel coronavirus was identified from all three patients. Two nearly full-length coronavirus sequences were obtained from bronchoalveolar-lavage fluid (BetaCoV/Wuhan/IVDC-HB-04/2020, BetaCoV/ Wuhan/IVDC-HB-05/2020|EPI_ISL_402121), and one full-length sequence was obtained from a virus isolated from a patient (BetaCoV/Wuhan/ IVDC-HB-01/2020|EPI_ISL_402119). Complete genome sequences of the three novel coronaviruses were submitted to GISAID (BetaCoV/Wuhan/ IVDC-HB-01/2019, accession ID: EPI_ISL_402119; BetaCoV/Wuhan/IVDC-HB-04/2020, accession ID: EPI_ISL_402120; BetaCoV/Wuhan/IVDC-HB-05/2019, Figure 2. Cytopathic Effects in Human Airway Epithelial Cell Cultures after Inoculation with 2019-nCoV. A Mock B HAE-CPE 100 µm

n engl j med nejm.org 5 Brief Report accession ID: EPI_ISL_402121) and have a 86.9% nucleotide sequence identity to a previously published bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1) genome. The three 2019-nCoV genomes clustered together within the sarbecovirus subgenus, which shows the typical betacoronavirus organization: a 5′ untranslated region (UTR), replicase complex (orf1ab), S gene, E gene, M gene, N gene, 3′ UTR, and several unidentified nonstructural open reading frames. Although 2019-nCoV is similar to some betacoronaviruses detected in bats (Fig. 4), it is distinct from SARS-CoV and MERS-CoV. The three 2019-nCoV coronaviruses from Wuhan, together with two bat-derived SARS-like strains, ZC45 and ZXC21, form a distinct clade. SARS-CoV strains from humans and genetically similar SARS-like coronaviruses from bats collected from southwestern China formed another clade within the subgenus sarbecovirus. Since the sequence identity in conserved replicase domains (ORF 1ab) is less than 90% between 2019-nCoV and other members of betacoronavirus, the 2019-nCoV — the likely causative agent of the viral pneumonia in Wuhan — is a novel betacoronavirus belonging to the sarbecovirus subgenus of Coronaviridae family. Discussion We report a novel CoV (2019-nCoV) that was identified in hospitalized patients in Wuhan, China, in December 2019 and January 2020. Evidence for the presence of this virus includes identification in bronchoalveolar-lavage fluid in three patients by whole-genome sequencing, direct PCR, and culture. The illness likely to have been caused by this CoV was named “novel coronavirus-infected pneumonia” (NCIP). Complete genomes were submitted to GISAID. Phylogenetic analysis revealed that 2019-nCoV falls into the genus betacoronavirus, which includes coronaviruses (SARS-CoV, bat SARS-like CoV, and others) discovered in humans, bats, and other wild animals.15 We report isolation of the virus and the initial description of its specific cytopathic effects and morphology. Molecular techniques have been used successfully to identify infectious agents for many years. Unbiased, high-throughput sequencing is Figure 3. Visualization of 2019-nCoV with Transmission Electron Microscopy. Negative-stained 2019-nCoV particles are shown in Panel A, and 2019-nCoV particles in the human airway epithelial cell ultrathin sections are shown in Panel B. Arrowheads indicate extracellular virus particles, arrows indicate inclusion bodies formed by virus components, and triangles indicate cilia. A 100 nm 1 µm B

6 n engl j med nejm.org The new england journal o f medicine Figure 4. Schematic of 2019-nCoV and Phylogenetic Analysis of 2019-nCoV and Other Betacoronavirus Genomes. Shown are a schematic of 2019-nCoV (Panel A) and full-length phylogenetic analysis of 2019-nCoV and other betacoronavirus genomes in the Orthocoronavirinae subfamily (Panel B). A B AY508724|SARS_coronavirus_NS-1 AY485277|SARS_coronavirus_Sino1-11 AY390556|SARS_coronavirus_GZ02 AY278489|SARS_coronavirus_GD01 KT444582|SARS-like_coronavirus_WIV16 KY417146|Bat_SARS-like_coronavirus_Rs4231 KY417151|Bat_SARS-like_coronavirus_Rs7327 KY417152|Bat_SARS-like_coronavirus_Rs9401 MK211376|Coronavirus_BtRs-BetaCoV/YN2018B MK211377|Coronavirus_BtRs-BetaCoV/YN2018C MK211374|Coronavirus_BtRl-BetaCoV/SC2018 BetaCoV-Wuhan-IVDC-HB-04-2020 BetaCoV/Wuhan/IVDC-HB-01/2019|EPI_ISL_402119 BetaCoV-WuhanI-VDC-HB-05-2019EPI_ISL_402121 KY770859|Bat_coronavirus|Anlong-112 KJ473816|BtRs_BetaCoV/YN2013 KJ473815|BtRs_BetaCoV/GX2013|BtRs-GX2013 JX993988|Bat_coronavirus_Cp/Yunnan2011 KY417145|Bat_SARS-like_coronavirus_Rf4092 KY417142|Bat_SARS-like_coronavirus|As6526 KY417148|Bat_SARS-like_coronavirus|Rs4247 MG772934|Bat_SARS-like_coronavirus|bat-SL-CoVZXC21 KF636752|Bat_Hp-betacoronavirus/Zhejiang2013 EF065513|Bat_coronavirus_HKU9-1|BF_005I EF065505|Bat_coronavirus_HKU4-1|B04f KU762338|Rousettus_bat_coronavirus|GCCDC1_356 EF065509|Bat_coronavirus_HKU5-1|LMH03f JX869059|Human_betacoronavirus_2c_EMC/2012|HCoV-EMC KC545386|Betacoronavirus_Erinaceus/VMC/DEU/2012| ErinaceusCoV/2012-216/GER/2012 KM349744|Betacoronavirus_HKU24|HKU24-R05010l AY391777|Human_coronavirus_OC43|ATCC_VR-759 MK167038|Human_coronavirus_HKU1|SC2521 FJ647223|Murine_coronavirus_MHV-1|MHV-1 MG772933|Bat_SARS-like_coronavirus|bat-SL-CoVZC45 SARS-CoV MERS-CoV Sarbecovirus Hibecovirus Nobecovirus Merbecovirus Embevovirus 0.2 100 100 100 100 83 66 84 60 90 94 100 100 100 100 100 100 100 100 100 96 99 100 Nsp1 Nsp2 Nsp3 Nsp4 Nsp7 Nsp9 Nsp12 Nsp8 Nsp6 Nsp13 Nsp14 Nsp15 Nsp16 Nsp10 Nsp5 >BetaCoV/Wuhan/IVDC-HB-01/2019|EPI_ISL_402119 (29892 bp) UTR 5´ polyA 3´ Spike Receptor binding Enzyme 3 E M 78 1314 8 10b 7 M E 3 10b 13 14 N ORF-1ab

n engl j med nejm.org 7 Brief Report a powerful tool for the discovery of pathogens.14,16 Next-generation sequencing and bioinformatics are changing the way we can respond to infectious disease outbreaks, improving our understanding of disease occurrence and transmission, accelerating the identification of pathogens, and promoting data sharing. We describe in this report the use of molecular techniques and unbiased DNA sequencing to discover a novel betacoronavirus that is likely to have been the cause of severe pneumonia in three patients in Wuhan, China. Although establishing human airway epithelial cell cultures is labor intensive, they appear to be a valuable research tool for analysis of human respiratory pathogens.13 Our study showed that initial propagation of human respiratory secretions onto human airway epithelial cell cultures, followed by transmission electron microscopy and whole genome sequencing of culture supernatant, was successfully used for visualization and detection of new human coronavirus that can possibly elude identification by traditional approaches. Further development of accurate and rapid methods to identify unknown respiratory pathogens is still needed. On the basis of analysis of three complete genomes obtained in this study, we designed several specific and sensitive assays targeting ORF1ab, N, and E regions of the 2019- nCoV genome to detect viral RNA in clinical specimens. The primer sets and standard operating procedures have been shared with the World Health Organization and are intended for surveillance and detection of 2019-nCoV infection globally and in China. More recent data show 2019-nCoV detection in 830 persons in China.17 Although our study does not fulfill Koch’s postulates, our analyses provide evidence implicating 2019-nCoV in the Wuhan outbreak. Additional evidence to confirm the etiologic significance of 2019-nCoV in the Wuhan outbreak include identification of a 2019-nCoV antigen in the lung tissue of patients by immunohistochemical analysis, detection of IgM and IgG antiviral antibodies in the serum samples from a patient at two time points to demonstrate seroconversion, and animal (monkey) experiments to provide evidence of pathogenicity. Of critical importance are epidemiologic investigations to characterize transmission modes, reproduction interval, and clinical spectrum resulting from infection to inform and refine strategies that can prevent, control, and stop the spread of 2019-nCoV. This work was supported by grants from the National Key Research and Development Program of China (2016YFD0500301) and the National Major Project for Control and Prevention of Infectious Disease in China (2018ZX10101002). Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. We thank Dr. Zhongjie Li, Dr. Guangxue He, Dr. Lance Rodewald, Yu Li, Fei Ye, Li Zhao, Weimin Zhou, Jun Liu, Yao Meng, Huijuan Wang, and many staff members at the China CDC for their contributions and assistance in this preparation and submission of an earlier version of the manuscript. References 1. Gao GF. From “A”IV to “Z”IKV: attacks from emerging and re-emerging pathogens. Cell 2018;172:1157-9. 2. Weiss SR, Leibowitz JL. Coronavirus pathogenesis. Adv Virus Res 2011;81:85- 164. 3. Masters PS, Perlman S. Coronaviridae. In: Knipe DM, Howley PM, eds. Fields virology. 6th ed. Lippincott Williams & Wilkins, 2013:825-58. 4. Su S, Wong G, Shi W, et al. Epidemiology, genetic recombination, and pathogenesis of coronaviruses. Trends Microbiol 2016;24:490-502. 5. Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 2019;17:181-92. 6. Zhong NS, Zheng BJ, Li YM, et al. Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People’s Republic of China, in February, 2003. Lancet 2003;362:1353-8. 7. Ksiazek TG, Erdman D, Goldsmith CS, et al. A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 2003;348:1953-66. 8. Drosten C, Günther S, Preiser W, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003;348:1967- 76. 9. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 2012;367:1814-20. 10. Wong G, Liu W, Liu Y, Zhou B, Bi Y, Gao GF. MERS, SARS, and Ebola: the role of super-spreaders in infectious disease. Cell Host Microbe 2015;18:398-401. 11. Report of clustering pneumonia of unknown etiology in Wuhan City. Wuhan Municipal Health Commission, 2019. (http://wjw.wuhan.gov.cn/front/web/ showDetail/2019123108989). 12. Liu GS, Li H, Zhao SC, Lu RJ, Niu PH, Tan WJ. Viral and bacterial etiology of acute febrile respiratory syndrome among patients in Qinghai, China. Biomed Environ Sci 2019;32:438-45. 13. Jonsdottir HR, Dijkman R. Coronaviruses and the human airway: a universal system for virus-host interaction studies. Virol J 2016;13:24. 14. Palacios G, Druce J, Du L, et al. A new arenavirus in a cluster of fatal transplantassociated diseases. N Engl J Med 2008; 358:991-8. 15. Tan WJ, Zhao X, Ma XJ, et al. A novel coronavirus genome identified in a cluster of pneumonia cases — Wuhan, China 2019−2020. China CDC Weekly 2020;2: 61-2. 16. Armstrong GL, MacCannell DR, Taylor J, et al. Pathogen genomics in public health. N Engl J Med 2019;381:2569-80. 17. Report of novel coronavirus-infected pneumonia in Wuhan City. Wuhan Municipal Health Commission, 2020 (http:// wjw.wuhan.gov.cn/front/web/showDetail/ 2020012009077). Copyright © 2020 Massachusetts Medical Society