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.ORG4 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