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

The NEW ENGLAND JOURNAL Of MEDICINE BRIEF REPORT A Novel Coronavirus from Patients with Pneumonia in China,2019 NaZh,Ph.D..M.D.Wenling Wang.Ph.D.Li,M.D PhD Weifeng Shi,Ph.D..D Peihua Niu.Ph.zh 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 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 etacoronavirus was discovered t through the use of sequencing in sample from patients with pe d subfamily.Different from both MERS-CoV and SARS-CoV.2019-nCov is the seve nth 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.) public health s are en broadly among humans,other mammals,and birds and that cause respira- tory,enteric,hepatic,and neurologic diseases. Six coronavirus species are known to cause human disease.Four viruses- 229E,OC43,NL63,and HKU1 prevalent nd typically cause ommon o (SARS-C severe ac tory syn otic in igin and have h or at gaot the causal agent of the severe acute respiratory syndrome outhreaks in 2002 and for severe respirat ing 10226.China. prevalence and wide distr tion of coronaviruses,the large genetic diversity and frequen adon of t omes,and increasin Yang hmans owing 02 of pa tients with pneumonia of unknown cause that were epidemiologically linked to seafood and wet animal wholesale market in Wuhan,Hubei Province,China."On December 31,2019,the Chinese Center for Disease Control and Prevention (C hina CDC dispatched a rapid sponse team to accompany Hub or report the source ofthe pneumon N ENGLJ MED NEJM.ORC

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 respira￾tory, 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 indi￾viduals.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 pa￾tients 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 Biosafe￾ty, 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; Wu￾han Jinyintan Hospital (D.Z.), the Divi￾sion for Viral Disease Detection, Hubei Provincial Center for Disease Control and Prevention (B.Y., F.Z.), and the Center for Biosafety Mega-Science, Chinese Acade￾my of Sciences (W.T.) — all in Wuhan; and the Shandong First Medical Univer￾sity and Shandong Academy of Medical Sciences, Jinan, China (W.S.). Address reprint requests to Dr. Tan at the NHC Key Laboratory of Biosafety, National In￾stitute for Viral Disease Control and Pre￾vention, China CDC, 155 Changbai Road, Changping District, Beijing 102206, Chi￾na; 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 Labo￾ratory 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 de￾tected in patients with pneumonia whose speci￾mens 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 pa￾tients. 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 uninfect￾ed 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 reac￾tion (PCR), using the RespiFinderSmart22kit (PathoFinder BV) and the LightCycler 480 real￾time PCR system, in accordance with manufac￾turer instructions.12 Samples were analyzed for 22 pathogens (18 viruses and 4 bacteria) as de￾tailed in the Supplementary Appendix. In addition, unbiased, high-throughput sequencing, described previously,13 was used to discover microbial se￾quences not identifiable by the means described above. A real-time reverse transcription PCR (RT￾PCR) assay was used to detect viral RNA by tar￾geting a consensus RdRp region of pan β-CoV, as described in the Supplementary Appendix. Isolation of Virus Bronchoalveolar-lavage fluid samples were col￾lected in sterile cups to which virus transport medium was added. Samples were then centri￾fuged 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 special￾pathogen-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 Transwell￾COL (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 resem￾bling in vivo pseudostratified mucociliary epi￾thelium.13 Prior to infection, apical surfaces of the hu￾man 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 min￾utes; human airway epithelial cells were main￾tained 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 epi￾thelial cells, and after 10 minutes of incubation at 37°C the samples were harvested. Pseudostrat￾ified mucociliary epithelium cells were main￾tained in this environment; apical samples were passaged in a 1:3 diluted vial stock to new cells. The cells were monitored daily with light micros￾copy, for cytopathic effects, and with RT-PCR, for the presence of viral nucleic acid in the superna￾tant. 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% paraformalde￾hyde for at least 2 hours, and ultracentrifuged to sediment virus particles. The enriched super￾natant 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 mi￾croscopy

BRIEF REPORT VIRAL GENOME SEQUENCING hospital on lanuary 16.2020.Patient 2 died or RNA extracted from bronchoalveolar-lavage flu-January 9,2020.No biopsy specimens were ob- id and culture supernatants was used as a tem-tained. plate to clone and sequence the genome. used a combination of Illumina sequencing and nanopore sequencing t ing DNA segm with the use of CLC Genomics software.version 4.6.1 (CLC Bio).Specific primers were subse quently designed for PCR, and 5-or 3'-RAC PCR s and sear nator v3.1 Cycle Sequencing Kit and a 3130XI Genetic Analyzer,in accordance with the manu facturers'instructions 2019 eswas performe RAxML (13)wi1OO0bo a general time-reversible model used as the nu cleotide substitution model. RESULTS Three adult ted with s monia and were admitted ro a hospital in wu han on December 27,2019.Patient 1 was a 49-year-old woman,Patient 2 was a 61-year-old 1 an y 38C) and cough chest discomfort on December 23.2019.Fou days after the onset of illness,her cough and chest discomfort worsened,but the fever was reduced;a diagnosis of pneumonia was based Figure 1.Chest Radiographs. ( .Pa d and r r the onse l ventila on i dis tress develoned 7 days after the onset of illnes nt in both es but aren and worsened over the next 2 days (see ches cond i radiographs,Fig.1),at which time mechanica lung was tarte had been a trequen nd w mage. N ENGLJ MED NEJM.ORC

n engl j med nejm.org 3 Brief Report Viral Genome Sequencing RNA extracted from bronchoalveolar-lavage flu￾id and culture supernatants was used as a tem￾plate 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 subse￾quently designed for PCR, and 5′- or 3′-RACE (rapid amplification of cDNA ends) was used to fill genome gaps from conventional Sanger se￾quencing. These PCR products were purified from gels and sequenced with a BigDye Termi￾nator v3.1 Cycle Sequencing Kit and a 3130XL Genetic Analyzer, in accordance with the manu￾facturers’ 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 nu￾cleotide substitution model. Results Patients Three adult patients presented with severe pneu￾monia and were admitted to a hospital in Wu￾han 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 occu￾pation was retailer in the seafood wholesale market. Patient 2 initially reported fever and cough on December 20, 2019; respiratory dis￾tress 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 ob￾tained. Figure 1. Chest Radiographs. Shown are chest radiographs from Patient 2 on days 8 and 11 after the onset of illness. The trachea was intu￾bated and mechanical ventilation instituted in the peri￾od between the acquisition of the two images. Bilateral fluffy opacities are present in both images but are in￾creased in density, profusion, and confluence in the second image; these changes are most marked in the lower lung fields. Changes consistent with the accu￾mulation 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 col￾lected from Wuhan Jinyintan Hospital on De￾cember 30, 2019. No specific pathogens (includ￾ing 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-la￾vage fluid from the patients was used as a tem￾plate to clone and sequence a genome using a combination of Illumina sequencing and nano￾pore sequencing. More than 20,000 viral reads from individual specimens were obtained, and most contigs matched to the genome from lin￾eage 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 per￾formed 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 hu￾man airway epithelial cells; a lack of cilium beating was seen with light microcopy in the center of the focus (Fig. 2). No specific cyto￾pathic 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 var￾ied 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 membrane￾bound 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 se￾quences of 2019-nCoV genome from clinical spec￾imens (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 ge￾nome 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

BRIEF REPORT Figure 3.Visualization of 2019-nCoy with Transmission Electron Mic sion bodies formed by virus components,and triangles indicate cilia acc on ID:EPI_ISL_402121)and havea86.9% MG772933.1)genome.The three 2019-nCoV ge DISCUSSION nomes clustered together within the sarbecovirus subgenus,which shows the typical betacorona- We report a novel Cov (2019-nCov)that was rins.oeganizatioaSlntnshcdtegonTRl identified in hospitalized patients in Wuhan, gene, three whole- age fluid coronavi ses detected in bats (Fig.4),it is dis-rect PCR.and culture.The illness likely to have tinct from SARS-CoV and MERS-CoV.The three been caused by this Cov was named "novel coro- 2019-nCov coronaviruses from Wuhan,together navirus-infected pneumonia"(NCIP).Complete with two bat-derived SARS-like strains,ZC45 genomes were submitted to GISAlD.Phyloge and ZXC21 form a distinct cla SA netic analysis revealed that 20 mans and gene from southwestern China formed another clade others)discovered in humans,bats,and other within the subgenus sarhecovirus since the se wild animals '5 we report isolation of the virus quence identity in conserved replicase domains and the initial description of its specific cyto- (ORF 1ab)is les than 90%between 2019-nCov pathic effects and morphology. of betacoronavirus, cular tec niques have been used suc pneumonia el beta cessfully ed,high -th N ENGLJ MED NEJM.ORG

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 pub￾lished bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1) genome. The three 2019-nCoV ge￾nomes clustered together within the sarbecovirus subgenus, which shows the typical betacorona￾virus organization: a 5′ untranslated region (UTR), replicase complex (orf1ab), S gene, E gene, M gene, N gene, 3′ UTR, and several unidentified non￾structural open reading frames. Although 2019-nCoV is similar to some beta￾coronaviruses detected in bats (Fig. 4), it is dis￾tinct 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 se￾quence 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 beta￾coronavirus belonging to the sarbecovirus sub￾genus 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. Evi￾dence for the presence of this virus includes identification in bronchoalveolar-lavage fluid in three patients by whole-genome sequencing, di￾rect PCR, and culture. The illness likely to have been caused by this CoV was named “novel coro￾navirus-infected pneumonia” (NCIP). Complete genomes were submitted to GISAID. Phyloge￾netic analysis revealed that 2019-nCoV falls into the genus betacoronavirus, which includes coro￾naviruses (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 cyto￾pathic effects and morphology. Molecular techniques have been used suc￾cessfully 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 inclu￾sion bodies formed by virus components, and triangles indicate cilia. A 100 nm 1 µm B

Thr NEW ENGLAND JOURNAL Of MEDICINE BetaCoV/Wuhan/IVDC-HB-01/2019EPI_ISL_402119(29892bp) ORF-lab 10h Nsp5 Nsp7 Nsp9 EM78 h31 TR Nsp2 Nsp3 Nsp13 Nspl4 Nspl5 Nsp16 Spike Nsp4 Receptor binding 3 E■+ M■ 7■+ 8 10b m 13■+ 14■ S-CoV 2018 113 G7729348*t SARS-lik 21 _HKU5 corona 9744 05010 AY391777 99 02 -月647n20 Figure4.Schematic of 2019-nCoV and Phylogenetic Analysis of 2019-nCoV and Other Betacoronavirus Genomes. N ENGLJ MED NEJM.ORC

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

BRIEF REPORT a powerful tool for the discovery of patho- Next-generatic sequencing and bioin can our one ance an nd e China.M nt dat understanding of disease occurrence and trans. show 2019-nCov detection in 830 persons in mission,accelerating the identification of patho-China. Although our study does not fulfill Koch's postul 01 nce im novel beracoronirus that is likely to have been ditional evidence to confirm the etiologic sig the cause of severe pneumonia in three patients nificance of 2019-nCoV in the Wuhan outbreak clude identification of a 2019-nCov antigen in is g human airway epith ng tissue of patients man respiratory pathogens.Our study showed a patient at two time points to demonstrate se- that initial propagation of human respiratory se roconversion,and animal (monkey)experiments ce to provide evidence of pat ogenicity.Of critica of culture characterize tra ssion modes. tion in terval,and clinical spectrum resulting from infec tion and detection of new human coronavirus tion to inform and refine strategies that can pre that can possibly elude identification by tradi- vent,control,and stop the spread of 2019-nCov of accurate and rapid methods to identify unknown re l Maio n of gens is still needed.On the basis of analysis of three complete genomes obtained in this study. w th of this the as ue He,Dr.L ce Kod nd E 10 Yu Li nCo genome to detect viral RNA in cinical ne fo ration and s specimens.The primer sets and standard oper- o GE. .G 3.0 shost inter rome.N E Med20034-1967. uceJ.Du Let al.A ne Wilkins..2013.82 acnmrccaNEngljMcd20o8 ie and 15318398-40 g NS,Zhe guM,tal.pi Ith.N 91231 icipal Heat Co Viral and al e 77 N ENGLJ MED NM.ORC

n engl j med nejm.org 7 Brief Report a powerful tool for the discovery of patho￾gens.14,16 Next-generation sequencing and bioin￾formatics are changing the way we can respond to infectious disease outbreaks, improving our understanding of disease occurrence and trans￾mission, accelerating the identification of patho￾gens, 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 epithe￾lial cell cultures is labor intensive, they appear to be a valuable research tool for analysis of hu￾man respiratory pathogens.13 Our study showed that initial propagation of human respiratory se￾cretions onto human airway epithelial cell cul￾tures, followed by transmission electron micros￾copy and whole genome sequencing of culture supernatant, was successfully used for visualiza￾tion and detection of new human coronavirus that can possibly elude identification by tradi￾tional approaches. Further development of accurate and rapid methods to identify unknown respiratory patho￾gens 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 oper￾ating procedures have been shared with the World Health Organization and are intended for surveillance and detection of 2019-nCoV infec￾tion 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 impli￾cating 2019-nCoV in the Wuhan outbreak. Ad￾ditional evidence to confirm the etiologic sig￾nificance of 2019-nCoV in the Wuhan outbreak include identification of a 2019-nCoV antigen in the lung tissue of patients by immunohisto￾chemical analysis, detection of IgM and IgG antiviral antibodies in the serum samples from a patient at two time points to demonstrate se￾roconversion, and animal (monkey) experiments to provide evidence of pathogenicity. Of critical importance are epidemiologic investigations to characterize transmission modes, reproduction in￾terval, and clinical spectrum resulting from infec￾tion to inform and refine strategies that can pre￾vent, 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 Rode￾wald, 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 sub￾mission of an earlier version of the manuscript. References 1. Gao GF. From “A”IV to “Z”IKV: attacks from emerging and re-emerging patho￾gens. Cell 2018;172:1157-9. 2. 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Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isola￾tion 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 Envi￾ron Sci 2019;32:438-45. 13. Jonsdottir HR, Dijkman R. Coronavi￾ruses 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 transplant￾associated 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 clus￾ter of pneumonia cases — Wuhan, China 2019−2020. China CDC Weekly 2020;2: 61-2. 16. Armstrong GL, MacCannell DR, Tay￾lor 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 Mu￾nicipal Health Commission, 2020 (http:// wjw.wuhan.gov.cn/front/web/showDetail/ 2020012009077). Copyright © 2020 Massachusetts Medical Society