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Abstract

The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures. Article Summary Scientists have isolated virus from the first US COVID-19 patient. The isolation and reagents described here will serve as the US reference strain used in research, drug discovery and vaccine testing.
Isolation and characterization of SARS-CoV-2 from the first US COVID-19 patient
Jennifer Harcourt Ph.D1*, Azaibi Tamin Ph.D1*, Xiaoyan Lu1, Shifaq Kamili2, Senthil Kumar.
Sakthivel3, Lijuan Wang2, Janna Murray2, Krista Queen Ph.D.1, Brian Lynch3, Brett Whitaker1,
Ying Tao Ph.D.1, Clinton R. Paden Ph.D.1, Jing Zhang3, Yan Li1, Anna Uehara Ph.D.5, Haibin
Wang3, Cynthia Goldsmith Ph.D.1, Hannah A. Bullock Ph.D2, Rashi Gautam Ph.D1, Craig
Schindewolf 6, Kumari G. Lokugamage Ph.D6, Dionna Scharton7, Jessica A. Plante Ph.D7, Divya
Mirchandani7, Steven G. Widen Ph.D.8, Krishna Narayanan Ph.D.6, Shinji Makino Ph.D.6,
Thomas G. Ksiazek DVM, Ph.D7,9, Kenneth S. Plante Ph.D.7, Scott C. Weaver Ph.D.6,7,9,
Stephen Lindstrom Ph.D1, Suxiang Tong Ph.D1,Vineet D. Menachery Ph.D7,9+, Natalie J.
Thornburg1+
1 Centers for Disease Control and Prevention, Atlanta, GA, USA
2 Synergy America Inc., Atlanta GA, USA
3Eagle Contracting, Atlanta GA, USA
4 IHRC, Atlanta GA, USA
5 ORISE, Oak Ridge TN
6 Department of Microbiology and Immunology, 7World Reference Center for Emerging Viruses
and Arboviruses, 8Department of Biochemistry & Molecular Biology, 9Department of Pathology,
Institute for Human Infection and Immunity, University of Texas Medical Branch, Galveston
TX, USA
*Authors contributed equally
+These senior authors contributed equally
Corresponding author
Natalie Thornburg
1600 Clifton Rd. NE MS G-18
Atlanta GA 30329
(404)639-3797
Nax3@cdc.gov
Article Summary: Scientists have isolated replication competent virus from the first US
COVID-19 patient. The isolated virus described here will serve as the US reference strain to be
used in research, drug discovery and vaccine testing.
Running title: Isolation of SARS-CoV-2 USA-WA1/2020
Keywords: Coronavirus, SARS-CoV-2, COVID-19, virus isolation
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ABSTRACT
The etiologic agent of the outbreak of pneumonia in Wuhan China in January-2020, was
identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) . The
first US patient was diagnosed by the State of Washington and the US Centers for Disease
Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and
oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell
culture tropism. We found that the virus replicated to high titers in Vero-CCL81 cells and Vero
E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making
it broadly available to the public health and research communities. We hope that open access to
this important reagent will expedite development of medical countermeasures.
BACKGROUND
A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),
has been identified as the source of a pneumonia outbreak in Wuhan China in late 2019 (1, 2).
The virus was found to be a member of the beta coronavirus family, in the same species as
SARS-CoV and SARS-related bat CoVs (3, 4). Patterns of spread indicate that SARS-CoV-2 can
be transmitted person-to-person, and may be more transmissible than SARS-CoV (5-7). The
spike protein, one of the structural outer proteins of coronaviruses, mediates virus binding and
cell entry. Initial characterization of SARS-CoV-2 spike protein indicate that it binds the same
receptor (ACE2) as SARS-CoV, which is expressed in both the upper and lower human
respiratory tracts (8). The unprecedented rapidity of spread of this outbreak presents a critical
need for reference reagents for further research on SARS-CoV-2. The public health community
requires viral lysates to serve as diagnostic references, and the research community needs virus
isolates to test anti-viral compounds, develop new vaccines, and perform basic research. In this
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manuscript, we describe isolation of virus from the first US COVID-19 patient and described its
genomic sequence and replication characteristics. We have made the virus isolate available to the
public health community by depositing into two virus reagent repositories, BEI resources and
The World Reference Center for Emergine Viruses and Arboviruses.
RESULTS and DISCUSSION
A patient with confirmed COVID-19 was identified in Washington State on January 22,
2020 with cycle threshold (Cts) of 18-20 (nasopharyngeal (NP)) and 21-22 (oropharyngeal (OP))
(1). The positive clinical specimens were aliquoted and refrozen, then inoculated into cell culture
on January 22, 2020. We first observed cytopathic effect (CPE) 2 days post inoculation and
harvested viral lysate on day 3 post inoculation (Figure 1B and 1C). Fifty µl of first passage (P1)
viral lysates were used for nucleic acid extraction to confirm the presence of SARS-CoV-2 using
the CDC molecular diagnostic assay, which tests three regions of the nucleocapsid (N) gene by
reverse transcription real time PCR (rRT-PCR) (1). The Cts of three different rRT-PCRs ranged
from 16.0-17.1 for N1, 15.9-17.1 for N2 and 16.2-17.3 for N3, confirming isolation of SARS-
CoV-2. A Ct of less than 40 is considered positive (10). The extracts were also tested for the
presence of 33 additional different viral and bacterial respiratory pathogens with the Fast Track
33 assay (FTD diagnostics, Silema, Malta). No other pathogens were detected. Identification was
additionally supported by thin section electron microscopy (Figure 1D). We observed the
morphology and morphogenesis characteristic of coronaviruses.
Isolates from the first passage of an OP and an NP specimen were used for whole genome
sequencing. The genomes from the NP specimen (Genbank accession MT020880) and OP
specimen (Genbank accession MT020881) matched each other 100%. The isolates also matched
the corresponding OP clinical specimen 100% (Genbank accession MN985325).
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After the second passage, OP and NP specimens were not cultured separately, and the
isolate was named SARS-CoV-2 USA-WA1/2020. Virus isolate was passaged two more times in
Vero CCL-81 cells, and titrated by TCID50. The titers of the third and fourth passages were 8.65
x 106 and 7.65 x 106 TCID50 per mL, respectively.
Of note, we passaged this virus in the absence of trypsin. The spike protein coding
sequence of SARS-CoV-2 has an RRAR insertion at the S1-S2 interface that may be cleaved by
furin (11). Highly pathogenic avian influenza viruses have highly basic furin cleavage sites at the
hemagglutinin protein HA1-HA2 interface that permit intracellular maturation of virions and
more efficient viral (12). The RRAR insertion in SARS-CoV-2 may serve a similar function.
We subsequently generated a fourth passage stock of SARS-CoV-2 on VeroE6 cells,
another fetal rhesus monkey kidney cell line. Viral RNA from SARS-CoV-2 passage four stock
was sequenced and confirmed to have no nucleotide mutations compared with the original
reference sequence (Genbank accession MN985325). Both SARS-CoV and MERS-CoV had
been found to grow well on VeroE6 and Vero CCL81 respectively (13, 14). To establish a plaque
assay and determine the preferred Vero cell type for quantification, we titered our passage four
stock on VeroE6 and VeroCCL81. Following infection with a dilution series, we found that
SARS-CoV-2 replicated in both Vero cell types; however, the viral titers were slightly higher in
VeroE6 cells than Vero CCL81 (Figure 2A). In addition, plaques were more distinct and visible
on Vero E6 (Figure 2B). As early as 2 days post inoculations, VeroE6 cells produced distinct
plaques visible with neutral red staining. In contrast, Vero CCL81 produced less clear plaques
and was most easily quantitated with neutral red 3 days post inoculation. On the individual
plaque monolayers, SARS-CoV-2 infection of Vero E6 cells produced cytopathic effect with
areas of cell clearance (Figure 2C). In contrast, Vero CCL81 had areas of dead cells that had
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fused to form plaques, but the cells did not clear. Together, the results suggest that VeroE6 may
be the best choice for amplification and quantification, but both Vero cell types support
amplification and replication of SARS-CoV-2.
As research is initiated to study and respond to SARS-CoV-2, information about cell line
tropism is needed. Therefore, we examined the capacity of SARS-CoV-2 to infect and replicate
in several common primate and human cell lines, including human adenocarcinoma cells (A549),
human liver cells (HUH7.0), and human embryonic kidney cells (HEK-293T), in addition to
Vero E6 and Vero CCL81. We also examined an available big brown bat kidney cell line
(EFK3B) for SARS-CoV-2 replication capacity. Each cell line was inoculated with at high MOI
and examined 24 hours post infection (Figure 3A). No cytopathic effect was observed in any of
the cell lines except in Vero cells which grew to >107 PFU at 24 hours post infection. In contrast,
both HUH7.0 and 293T cells showed only modest viral replication and A549 cells were
incompatible with SARS-CoV-2 infection. These results are consistent with previous
susceptibility findings for SARS-CoV(15). In addition, SARS-CoV-2 failed to replicate in the
bat EFK3B cells which are susceptible to MERS-CoV. Together, the results indicate that SARS-
CoV-2 maintain a similar profile to SARS-CoV in terms of susceptible cell lines.
Having established robust infection with SARS-CoV-2 in several cell types, we next
evaluated the cross reactivity of SARS-CoV antibodies against the SARS-CoV-2. Cell lysates
from infected cell lines were probed for protein analysis; we found that antibodies against the
SARS-CoV spike and nucleocapsid proteins recognize SARS-CoV-2 (Figure 3B & C). The N
protein, highly conserved across the group 2B family, retains >90% amino acid identity between
SARS-CoV and SARS-CoV-2. Consistent with the replication results (Figure 3A), SARS-CoV-
2 showed robust N protein in both Vero cell types, less protein in HUH7.0 and 293T, and
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minimal signal in A549 and EFK3B cells (Figure 3B). Similarly, the SARS-CoV spike antibody
also recognized SARS-CoV-2 spike protein, indicating cross reactivity (Figure 3C). Consistent
with SARS CoV, several cleaved and uncleaved forms of the SARS-CoV-2 spike protein.
Notably, the cleavage pattern to the the SARS spike positive control from Calu3 cells, a
respiratory cell line, varies slightly and could signal differences between proteolytic cleavage of
the spike proteins between the two viruses. However, differences in cell type and conditions
complicate this interpretation and indicate the need to further study in equivalent systems.
Overall, the protein expression data from SARS-CoV N and S antibodies recapitulate replication
findings and indicate that SARS-CoV reagents can be utilized to characterize SARS-CoV-2
infection.
The SARS-CoV-2 USA-WA1/2020 viral strain described above has been deposited into
BEI reagent resources (ATCC) and the World Reference Center for Emerging Viruses and
Arboviruses (WRCEVA, UTMB) to serve as the SARS-CoV-2 reference strain for the United
States. The SARS-CoV-2 fourth passage virus has been sequenced and the nucleotide sequence
is identical to that of the original US clinical strain. This deposit makes it available to the
domestic and international public health, academic, and pharmaceutical sectors for basic
research, diagnostic development, antiviral testing, and vaccine development. We hope broad
access will expedite countermeasure development and testing, in addition to facilitating a better
understanding of the transmissibility and pathogenesis of this novel emerging virus.
METHODS
Specimen collection
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Virus isolation from patient samples was deemed to be non-human subjects research by CDC
National Center for Immunizations and Respiratory Diseases (research determination
0900f3eb81ab4b6e). Clinical specimens from the first identified US case of COVID-19 acquired
during travel to China, were collected as described (1). Nasopharyngeal (NP) and oropharyngeal
(OP) swabs in 2 to 3 mL viral transport media were collected on day 3 post-symptom onset for
molecular diagnosis (https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-
specimens.html) , shipped on cold packs, extracted, used for molecular diagnostics, and frozen.
Confirmed rRT-PCR- positive specimens were aliquoted and refrozen until virus isolation was
initiated.
Cell culture, limiting dilution, and isolation
Vero CCL-81 cells were used for isolation and initial passage. Vero E6, Vero CCL-81, HUH 7.0,
293T, A549, and EFKB3 cells were cultured in Dulbecco’s minimal essential medium (DMEM)
supplemented with heat inactivated fetal bovine serum (5 or 10%) and anti/anti antibiotic
(GIBCO). Both NP an OP swabs were used for virus isolation. For the isolation, limiting
dilution, and passage 1 of the virus, 50 µl serum free DMEM was pipetted into columns 2-12 of
a 96-well tissue culture plate. One-hundred µl clinical specimens were pipetted into column 1,
and then serially diluted 2-fold across the plate. Vero cells were trypsinized and resuspended in
DMEM + 10% FBS + 2X Penicillin-Streptomycin + 2X antibiotic – antimycotic + 2 X
amphotericin B at 2.5 x 105 cells / ml. One hundred µl of cell suspension were added directly to
the clinical specimen dilutions and mixed gently by pipetting. The inoculated cultures were
grown in a humidified 37°C incubator with 5% CO2 and observed for cytopathic effect (CPE)
daily. Standard plaque assays were used for SARS-CoV-2 based on both SARS-CoV and
MERS-CoV protocols (16, 17).
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When CPE were observed, the cell monolayers were scraped with the back of a pipette tip. Fifty
µl of the viral lysate were used for total nucleic acid extraction for confirmatory testing and
sequencing. Fifty µl of virus lysate was used to inoculate a well of a 90% confluent 24-well
plate.
Inclusivity / Exclusivity testing
From the wells in which CPE were observed, confirmatory testing was performed using CDC’s
SARS-CoV-2 molecular diagnostic assay (1) The CDC molecular diagnostic assay targets three
portions of the N gene, and all three must be positive to be considered as positive result
(https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-detection-instructions.html) and
(https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html). To confirm
that no other respiratory viruses were present, Fast Track respiratory pathogen 33 testing was
performed (FTD diagnostics, Silema, Malta).
Whole genome sequencing. Thirty-seven pairs of nested PCR assays spanning the genome were
designed based on the reference sequence, Genbank Accession No. NC045512. Nucleic acid was
extracted from isolates and amplified by the 37 individual nested PCR assays. Positive PCR
amplicons were used individually for subsequent Sanger sequencing and also pooled for library
preparation using a ligation sequencing kit (Oxford Nanopore Technologies, Oxford, UK),
subsequently for Oxford Nanopore MinION sequencing. Consensus Nanopore sequences were
generated using minimap 2.17 and samtools 1.9 (18, 19). Consensus sequences by Sanger
sequences were generated from both directions using Sequencher 5.4.6 (Gene Codes
Corporation, Ann Arbor, MI), and were further confirmed by consensus sequences generated
from nanopore sequencing.
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To sequence passage four stock, libraries for sequencing were prepared with the NEB Next Ultra
II RNA Prep Kit (New England BioLabs, Inc., Ipswich, MA) following the manufacturer’s
protocol. Briefly, ~70-100 ng of RNA was fragmented for 15 minutes, followed by cDNA
synthesis, end repair and adapter ligation. After 6 rounds of PCR the libraries were analyzed on
an Agilent Bioanalyzer and quantified by qPCR. Samples were pooled and sequenced with a
paired-end 75 base protocol on an Illumina (Illumina, Inc, San Diego, CA) MiniSeq instrument
using the High-Output kit. Reads were processed with Trimmomatic v0.36 (20) to remove low
quality base calls and any adapter sequences. The de novo assembly program ABySS (21) was
used to assemble the reads into contigs, using several different sets of reads, and kmer values
from 20 to 40. Contigs greater than 400 bases long were compared against the NCBI nucleotide
collection using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A nearly full length viral
contig was obtained in each sample with 100% identity to the 2019-nCoV/USA-WA1/2020
strain (MN985325.1). All the remaining contigs mapped to either host cell ribosomal RNA or
mitochondria. The trimmed reads were mapped to the MN985325.1 reference sequence with
BWA v0.7.17 (19) and visualized with the Integrated Genomics Viewer (22) to confirm the
identity to the USA-WA1/2020 strain.
Electron microscopy
Infected Vero cells were scraped from the flask, pelleted by low speed centrifugation, rinsed with
0.1M phosphate buffer, pelleted again and fixed for 2 hours in 2.5% buffered glutaraldehyde.
Specimens were post fixed with 1% osmium tetroxide, en bloc staining with 4% uranyl acetate,
dehydrated and embedded in epoxy resin. Ultrathin sections were cut, stained with 4% uranyl
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acetate and lead citrate, and examined with a Thermo Fisher/FEI Tecnai Spirit electron
microscope.
Protein Analysis and Western Blot. Cell lysates were harvested with Laemmli SDS-PAGE
sample buffer (Bio RAD, Hercules CA) containing a final concentration of 2% SDS and 5% -
mercaptoethanol. Cell lysates were the boiled and removed from the BSL3. The lysates were
then loaded onto a poly-acrylamide gel, and SDS-PAGE followed by transfer to polyvinylidene
difluoride PVDF membrane. The membrane was then blocked in 5% nonfat dry milk dissolved
in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 hour, followed by a short TBS-T
wash. Overnight incubation with primary antibody, either SARS-CoV spike antibody (Sino
Biological #40150-T52), -Actin antibody (Cell Signaling Technology #4970), or a custom
SARS-CoV nucleocapsid antibody, was then performed. After primary antibody incubation, the
membrane was washed 3x with TBS-T, and then horseradish peroxidase-conjugated secondary
antibody was applied for 1 hour. Subsequently, the membrane was washed 3x with TBS-T, and
incubated with Clarity Western ECL Substrate (Bio-Rad #1705060S), and imaged with a multi-
purpose imaging system.
Generation of SARS Nucleocapsid antibodies. The plasmid, pBM302 (23), was used to
express SARS-CoV N protein, with a C-terminal His6 tag, to high levels within the inclusion
bodies of E.coli and the recombinant protein was purified from the inclusion bodies by nickel-
affinity column chromatography under denaturing conditions. The recombinant SARS-CoV N
protein was refolded by stepwise dialysis against Tris/phosphate buffer with decreasing
concentrations of urea to renature the protein. The renatured, full-length, SARS-CoV N protein
was used to immunize rabbits to generate an affinity-purified rabbit anti-SARS-CoV N
polyclonal antibody.
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REFERENCES
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author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under
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ACKNOWLEDGEMENTS
The reagent described is available through BEI Resources, NIAID, NIH: SARS-related
coronavirus 2, Isolate USA-WA1/2020, NR-52281. We thank Dr. Mavanur R. Suresh for
providing the plasmid, pBM302, expressing the SARS-CoV N protein. Research was supported
by grants from NIA and NIAID of the NIH (U19AI100625 and R00AG049092 to VDM;
R24AI120942 to SCW; AI99107 and AI114657 to SM). Research was also supported by STARs
Award provided by the University of Texas System to VDM, funds from the Institute for Human
Infections and Immunity at UTMB to SM, and trainee funding provided by the McLaughlin
Fellowship Fund at UTMB
DISCLAIMERS
The findings and conclusions in this report are those of the author(s) and do not necessarily
represent the official position of the Centers for Disease Control and Prevention. Names of
specific vendors, manufacturers, or products are included for public health and informational
purposes; inclusion does not imply endorsement of the vendors, manufacturers, or products by
the Centers for Disease Control and Prevention or the US Department of Health and Human
Services.
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author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under
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author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under
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... Viral infection was carried out according to the method described earlier. The infected cells were regularly monitored for cytopathic effects (CPE) (Harcourt et al., 2020b). Then, 72 h post-infection (hpi), the culture supernatants were collected, and the clarified supernatants (at 3,000 rpm for 5 min) were used as inoculum for the subsequent (second) passage of virus in naïve Vero E6 cells. ...
... Hence, attempts were made to isolate the SARS-CoV-2 virus from the OP samples of COVID-19 patients in the current study. Based on previous reports, Vero E6 cells were used for virus propagation (Harcourt et al., 2020b). The clarified supernatant collected after the 10th passage was used as viral stock for all the experiments in the current study. ...
... Similar to previous studies, different cell lines were used to check the susceptibility of SARS-CoV-2 isolates in this study (Caccuri et al., 2020;Harcourt et al., 2020b;Wurtz et al., 2021). Various cell lines were infected with the respective isolates, and the culture supernatants were collected at 24 hpi. ...
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... While SARS-CoV-2 is thought to exit the cell through continuous budding rather than being lytic (31), it is possible that by 72 hpi the virus has exhausted the ability of the cells to continuously manufacture new viruses at the same rate. Others groups have suggested time from absorption to release of new viral particles is between 8 (32) and 36 (33) hpi, which would explain the differences observed at 24 hpi. We propose that exploring time points after 24 hpi is not useful for detecting differences in growth. ...
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The recent emergence of Wuhan coronavirus (2019-nCoV) puts the world on alert. 2019-nCoV is reminiscent of the SARS-CoV outbreak in 2002 to 2003. Our decade-long structural studies on the receptor recognition by SARS-CoV have identified key interactions between SARS-CoV spike protein and its host receptor angiotensin-converting enzyme 2 (ACE2), which regulate both the cross-species and human-to-human transmissions of SARS-CoV. One of the goals of SARS-CoV research was to build an atomic-level iterative framework of virus-receptor interactions to facilitate epidemic surveillance, predict species-specific receptor usage, and identify potential animal hosts and animal models of viruses. Based on the sequence of 2019-nCoV spike protein, we apply this predictive framework to provide novel insights into the receptor usage and likely host range of 2019-nCoV. This study provides a robust test of this reiterative framework, providing the basic, translational, and public health research communities with predictive insights that may help study and battle this novel 2019-nCoV.
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Background: In late December, 2019, patients presenting with viral pneumonia due to an unidentified microbial agent were reported in Wuhan, China. A novel coronavirus was subsequently identified as the causative pathogen, provisionally named 2019 novel coronavirus (2019-nCoV). As of Jan 26, 2020, more than 2000 cases of 2019-nCoV infection have been confirmed, most of which involved people living in or visiting Wuhan, and human-to-human transmission has been confirmed. Methods: We did next-generation sequencing of samples from bronchoalveolar lavage fluid and cultured isolates from nine inpatients, eight of whom had visited the Huanan seafood market in Wuhan. Complete and partial 2019-nCoV genome sequences were obtained from these individuals. Viral contigs were connected using Sanger sequencing to obtain the full-length genomes, with the terminal regions determined by rapid amplification of cDNA ends. Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses was used to determine the evolutionary history of the virus and help infer its likely origin. Homology modelling was done to explore the likely receptor-binding properties of the virus. Findings: The ten genome sequences of 2019-nCoV obtained from the nine patients were extremely similar, exhibiting more than 99·98% sequence identity. Notably, 2019-nCoV was closely related (with 88% identity) to two bat-derived severe acute respiratory syndrome (SARS)-like coronaviruses, bat-SL-CoVZC45 and bat-SL-CoVZXC21, collected in 2018 in Zhoushan, eastern China, but were more distant from SARS-CoV (about 79%) and MERS-CoV (about 50%). Phylogenetic analysis revealed that 2019-nCoV fell within the subgenus Sarbecovirus of the genus Betacoronavirus, with a relatively long branch length to its closest relatives bat-SL-CoVZC45 and bat-SL-CoVZXC21, and was genetically distinct from SARS-CoV. Notably, homology modelling revealed that 2019-nCoV had a similar receptor-binding domain structure to that of SARS-CoV, despite amino acid variation at some key residues. Interpretation: 2019-nCoV is sufficiently divergent from SARS-CoV to be considered a new human-infecting betacoronavirus. Although our phylogenetic analysis suggests that bats might be the original host of this virus, an animal sold at the seafood market in Wuhan might represent an intermediate host facilitating the emergence of the virus in humans. Importantly, structural analysis suggests that 2019-nCoV might be able to bind to the angiotensin-converting enzyme 2 receptor in humans. The future evolution, adaptation, and spread of this virus warrant urgent investigation. Funding: National Key Research and Development Program of China, National Major Project for Control and Prevention of Infectious Disease in China, Chinese Academy of Sciences, Shandong First Medical University.
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Background: In December, 2019, a pneumonia associated with the 2019 novel coronavirus (2019-nCoV) emerged in Wuhan, China. We aimed to further clarify the epidemiological and clinical characteristics of 2019-nCoV pneumonia. Methods: In this retrospective, single-centre study, we included all confirmed cases of 2019-nCoV in Wuhan Jinyintan Hospital from Jan 1 to Jan 20, 2020. Cases were confirmed by real-time RT-PCR and were analysed for epidemiological, demographic, clinical, and radiological features and laboratory data. Outcomes were followed up until Jan 25, 2020. Findings: Of the 99 patients with 2019-nCoV pneumonia, 49 (49%) had a history of exposure to the Huanan seafood market. The average age of the patients was 55·5 years (SD 13·1), including 67 men and 32 women. 2019-nCoV was detected in all patients by real-time RT-PCR. 50 (51%) patients had chronic diseases. Patients had clinical manifestations of fever (82 [83%] patients), cough (81 [82%] patients), shortness of breath (31 [31%] patients), muscle ache (11 [11%] patients), confusion (nine [9%] patients), headache (eight [8%] patients), sore throat (five [5%] patients), rhinorrhoea (four [4%] patients), chest pain (two [2%] patients), diarrhoea (two [2%] patients), and nausea and vomiting (one [1%] patient). According to imaging examination, 74 (75%) patients showed bilateral pneumonia, 14 (14%) patients showed multiple mottling and ground-glass opacity, and one (1%) patient had pneumothorax. 17 (17%) patients developed acute respiratory distress syndrome and, among them, 11 (11%) patients worsened in a short period of time and died of multiple organ failure. Interpretation: The 2019-nCoV infection was of clustering onset, is more likely to affect older males with comorbidities, and can result in severe and even fatal respiratory diseases such as acute respiratory distress syndrome. In general, characteristics of patients who died were in line with the MuLBSTA score, an early warning model for predicting mortality in viral pneumonia. Further investigation is needed to explore the applicability of the MuLBSTA score in predicting the risk of mortality in 2019-nCoV infection. Funding: National Key R&D Program of China.