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.ORG2 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