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A pneumonia outbreak associated with a new coronavirus of probable bat origin

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Since the SARS outbreak 18 years ago, a large number of severe acute respiratory syndrome-related coronaviruses (SARSr-CoV) have been discovered in their natural reservoir host, bats1–4. Previous studies indicated that some of those bat SARSr-CoVs have the potential to infect humans5–7. Here we report the identification and characterization of a novel coronavirus (2019-nCoV) which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started from 12 December 2019, has caused 2,050 laboratory-confirmed infections with 56 fatal cases by 26 January 2020. Full-length genome sequences were obtained from five patients at the early stage of the outbreak. They are almost identical to each other and share 79.5% sequence identify to SARS-CoV. Furthermore, it was found that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. The pairwise protein sequence analysis of seven conserved non-structural proteins show that this virus belongs to the species of SARSr-CoV. The 2019-nCoV virus was then isolated from the bronchoalveolar lavage fluid of a critically ill patient, which can be neutralized by sera from several patients. Importantly, we have confirmed that this novel CoV uses the same cell entry receptor, ACE2, as SARS-CoV.
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270 | Nature | Vol 579 | 12 March 2020
Article
A pneumonia outbreak associated with a
new coronavirus of probable bat origin
Peng Zhou1,5, Xing-Lou Yang1,5, Xian-Guang Wang2,5, Ben Hu1, Lei Zhang1, Wei Zhang1,
Hao-Rui Si1,3, Yan Zhu1, Bei Li1, Chao-Lin Huang2, Hui-Dong Chen2, Jing Chen1,3, Yun Luo1,3,
Hua Guo1,3, Ren-Di Jiang1,3, Mei-Qin Liu1,3, Ying Chen1,3, Xu-Rui Shen1,3, Xi Wang1,3,
Xiao-Shuang Zheng1,3, Kai Zhao1,3, Quan-Jiao Chen1, Fei Deng1, Lin-Lin Liu4, Bing Yan1,
Fa-Xian Zhan4, Yan-Yi Wang1, Geng-Fu Xiao1 & Zheng-Li Shi1 ✉
Since the outbreak of severe acute respiratory syndrome (SARS) 18years ago, a large
number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their
natural reservoir host, bats1–4. Previous studies have shown that some bat SARSr-CoVs
have the potential to infect humans5–7. Here we report the identication and
characterization of a new coronavirus (2019-nCoV), which caused an epidemic of
acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started
on 12 December 2019, had caused 2,794 laboratory-conrmed infections including 80
deaths by 26 January 2020. Full-length genome sequences were obtained from ve
patients at an early stage of the outbreak. The sequences are almost identical and
share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is
96% identical at the whole-genome level to a bat coronavirus. Pairwise protein
sequence analysis of seven conserved non-structural proteins domains show that this
virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from
the bronchoalveolar lavage uid of a critically ill patient could be neutralized by sera
from several patients. Notably, we conrmed that 2019-nCoV uses the same cell entry
receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV.
Coronaviruses have caused two large-scale pandemics in the past
twodecades, SARS and Middle East respiratory syndrome (MERS)
8,9
.
It has generally been thought that SARSr-CoV—which is mainly found
in bats—could cause a future disease outbreak
10,11
. Here we report
on a series of cases caused by an unidentified pneumonia disease
outbreak in Wuhan, Hubei province, central China. This disease out-
break—which started from a local seafood market—has grown sub-
stantially to infect 2,761people in China, is associated with 80deaths
and has led to the infection of 33people in 10additional countries
as of 26 January 2020
12
. Typical clinical symptoms of these patients
are fever, dry cough, breathing difficulties (dyspnoea), headache
and pneumonia. Disease onset may result in progressive respiratory
failure owing to alveolar damage (as observed by transverse chest
computerized-tomography images) and even death. The disease was
determined to be caused by virus-induced pneumonia by clinicians
according to clinical symptoms and other criteria, including a rise in
body temperature, decreases in the number of lymphocytes and white
blood cells (although levels of the latter were sometimes normal), new
pulmonary infiltrates on chest radiography and no obvious improve-
ment after treatment with antibiotics for three days. It appears that
most of the early cases had contact history with the original seafood
market; however, the disease has now progressed to be transmitted
by human-to-human contact.
Samples from seven patients with severe pneumonia (six of whom are
sellers or deliverymen from the seafood market), who were admitted to
the intensive care unit of Wuhan Jin Yin-Tan Hospital at the beginning
of the outbreak, were sent to the laboratory at the Wuhan Institute of
Virology (WIV) for the diagnosis of the causative pathogen (Extended
Data Table1). As a laboratory investigating CoV, we first used pan-CoV
PCR primers to test these samples
13
, given that the outbreak occurred in
winter and in a market—the same environment as SARS infections. We
found five samples to be PCR-positive for CoVs. One sample (WIV04),
collected from the bronchoalveolar lavage fluid (BALF), was analysed by
metagenomics analysis using next-generation sequencing to identify
potential aetiological agents. Of the 10,038,758total reads—of which
1,582total reads were retained after filtering of reads from the human
genome—1,378 (87.1%) sequences matched the sequence of SARSr-
CoV (Fig.1a). By denovo assembly and targeted PCR, we obtained a
29,891-base-pair CoV genome that shared 79.6% sequence identity
to SARS-CoV BJ01 (GenBank accession number AY278488.2). High
genome coverage was obtained by remapping the total reads to this
genome (Extended Data Fig.1). This sequence has been submitted to
GISAID (https://www.gisaid.org/) (accession number EPI_ISL_402124).
Following the name given by the World Health Organization (WHO),
we tentatively call it novel coronavirus 2019 (2019-nCoV). Four more
full-length genome sequences of 2019-nCoV (WIV02, WIV05, WIV06 and
https://doi.org/10.1038/s41586-020-2012-7
Received: 20 January 2020
Accepted: 29 January 2020
Published online: 3 February 2020
Open access
Check for updates
1CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China. 2Wuhan Jin Yin-Tan Hospital, Wuhan,
China. 3University of Chinese Academy of Sciences, Beijing, China. 4Hubei Provincial Center for Disease Control and Prevention, Wuhan, China. 5These authors contributed equally: Peng Zhou,
Xing-Lou Yang, Xian-Guang Wang. e-mail: zlshi@wh.iov.cn
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Nature | Vol 579 | 12 March 2020 | 271
WIV07) (GISAID accession numbers EPI_ISL_402127–402130) that were
more than 99.9% identical to each other were subsequently obtained
from four additional patients using next-generation sequencing and
PCR (Extended Data Table2).
The virus genome consists of six major open-reading frames (ORFs)
that are common to coronaviruses and a number of other accessory
genes (Fig.1b). Further analysis indicates that some of the 2019-nCoV
genes shared less than 80% nucleotide sequence identity to SARS-CoV.
However, the amino acid sequences of the seven conserved replicase
domains in ORF1ab that were used for CoV species classification were
94.4% identical between 2019-nCoV and SARS-CoV, suggesting that
the two viruses belong to the same species, SARSr-CoV.
We then found that a short region of RNA-dependent RNA polymerase
(RdRp) from a bat coronavirus (BatCoV RaTG13)—which was previously
detected in Rhinolophus affinis from Yunnan province—showed high
sequence identity to 2019-nCoV. We carried out full-length sequencing
on this RNA sample (GISAID accession number EPI_ISL_402131). Simplot
analysis showed that 2019-nCoV was highly similar throughout the
genome to RaTG13 (Fig.1c), with an overall genome sequence identity
of 96.2%. Using the aligned genome sequences of 2019-nCoV, RaTG13,
SARS-CoV and previously reported bat SARSr-CoVs, no evidence for
recombination events was detected in the genome of 2019-nCoV. Phy-
logenetic analysis of the full-length genome and the gene sequences of
RdRp and spike (S) showed that—for all sequences—RaTG13 is the clos-
est relative of 2019-nCoV and they form a distinct lineage from other
SARSr-CoVs (Fig.1d and Extended Data Fig.2). The receptor-binding
spike protein encoded by the S gene was highly divergent from other
CoVs (Extended Data Fig.2), with less than 75% nucleotide sequence
identity to all previously described SARSr-CoVs, except for a 93.1%
nucleotide identity to RaTG13 (Extended Data Table3). The S genes of
2019-nCoV and RaTG13 are longer than other SARSr-CoVs. The major
differences in the sequence of the S gene of 2019-nCoV are the three
short insertions in the N-terminal domain as well as changes in four out
of five of the key residues in the receptor-binding motif compared with
the sequence of SARS-CoV (Extended Data Fig.3). Whether the inser-
tions in the N-terminal domain of the S protein of 2019-nCoV confer
sialic-acid-binding activity as it does in MERS-CoV needs to be further
studied. The close phylogenetic relationship to RaTG13 provides evi-
dence that 2019-nCoV may have originated in bats.
We rapidly developed a qPCR-based detection method on the basis
of the sequence of the receptor-binding domain of the S gene, which
was the most variable region of the genome (Fig.1c). Our data show
that the primers could differentiate 2019-nCoV from all other human
coronaviruses including bat SARSr-CoV WIV1, which shares 95% identity
with SARS-CoV (Extended Data Fig.4a, b). Of the samples obtained from
the seven patients, we found that six BALF and five oral swab samples
were positive for 2019-nCoV during the first sampling, as assessed
by qPCR and conventional PCR. However, we could no longer detect
virus-positive samples in oral swabs, anal swabs and blood samples
taken from these patients during the second sampling (Fig.2a). How-
ever, we recommend that other qPCR targets, including the RdRp or
envelope (E) genes are used for the routine detection of 2019-nCoV.
On the basis of these findings, we propose that the disease could be
transmitted by airborne transmission, although we cannot rule out
other possible routes of transmission, as further investigation, includ-
ing more patients, is required.
5,000 10,000 15,000
Genome nucleotide position
20,000 25,000 30,000
ORF1a ORF1b S3a
E
M7b
7a
68
N
SARS-CoV BJ01
Bat CoV RaTG13
Bat CoV ZC45
Bat SARSr-CoV WIV1
Bat SARSr-CoV HKU3-1
0 5,000 10,000 15,000 20,000 25,000 30,000
Genome nucleotide position
40
50
60
70
80
90
100
Nucleotide identity (%)
a
b
d
c
0.4
MERS-CoV
Human CoV 229E
Bat SARSr-CoV ZXC21
TGEV
Bat SARSr-CoV Rf1
Mink CoV
Bat SARSr-CoV ZC45
Bat Hp BetaCoV Zhejiang2013
PEDV
Bat SARSr-CoV SC2018
Rousettus bat CoV HKU9
Bat SARSr-CoV Rs672
MHV
Miniopterus bat CoV HKU8
2019-nCoV BetaCoV/Wuhan/WIV05
Bat CoV GCCDC1
Human CoV OC43
SARS-CoV SZ3
Bat SARSr-CoV BM48-31
Bat SARSr-CoV HKU3-1
2019-nCoV BetaCoV/Wuhan/WIV04
Scotophilus bat CoV 512
Bat SARSr-CoV YNLF31C
Bat SARSr-CoV WIV1
Bat SARSr-CoV LYRa11
Bat SARSr-CoV GX2013
SARS-CoV BJ01
Bat SARSr-CoV Longquan-140
Bat SARSr-CoV SHC014
Bat SARSr-CoV SX2013
Bat CoV RaTG13
Human CoV NL63
2019-nCoV BetaCoV/Wuhan/WIV07
2019-nCoV BetaCoV/Wuhan/WIV02
Bat SARSr-CoV HuB2013
2019-nCoV BetaCoV/Wuhan/WIV06
Human CoV HKU1
Miniopterus bat CoV 1
Bat SARSr-CoV Rp3
Tylonycteris bat CoV HKU4
Pipistrellus bat CoV HKU5
Rhinolophus bat CoV HKU2
100
99
100
85
86
100
100
100
100
76
100
100
100
100
63
100
99
92
100
100
86
96
100
100
100
100
100
96
100
93
100
100
99
100
89
100
BetaCoVAlphaCoV
Bat SARSr-CoV Rs4231
Bat SARSr-CoV WIV16
SARSr-CoV (1,378)
Hyposoter fugitivus ichnovirus
segment B5, complete sequence (24)
Proteus phage VB_PmiS-Isfahan,
complete genome (28)
Dulcamara mottle virus,
complete genome (28)
Glypta fumiferanae ichnovirus
segment C10, complete sequence (36)
Glypta fumiferanae ichnovirus
segment C9, complete sequence (36)
Saccharomyces cerevisiae
killer virus M1, complete genome (52)
Fig. 1 | Geno me charact erization o f 2019-nCoV. a, Metagenomic s analysis of
next-generatio n sequencing o f BALF from patie nt ICU06. b, Geno mic
organiza tion of 2019-nCoV WIV0 4. M, membrane. c, S imilarity plo t based on
the full-leng th genome se quence of 2019-nCoV WI V04. Full-length gen ome
sequenc es of SARS- CoV BJ01, bat SARSr-CoV WI V1, bat coronav irus RaTG13 and
ZC45 were used as r eference seque nces. d, Phyloge netic tree bas ed on
nucleotid e sequence s of complete genom es of coronavir uses. MHV, murine
hepatiti s virus; PEDV, porcine epide mic diarrhoea v irus; TGEV, porcine
transmis sible gastroe nteritis vi rus.The scale bar s represent 0.1 su bstitution s
per nucleo tide position . Descript ions of the set tings and sof tware that was
used are inc luded in theMethod s.
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272 | Nature | Vol 579 | 12 March 2020
Article
For serological detection of 2019-nCoV, we used a previously devel-
oped nucleocapsid (N) protein from bat SARSr-CoV Rp3 as antigen
for IgG and IgM enzyme-linked immunosorbent assays (ELISAs), as
this protein shared 92% amino acid identity to N protein of 2019-nCoV
(Extended Data Fig.5) and showed no cross-reactivity against other
human coronaviruses except SARSr-CoV
7
. We were only able to obtain
five serum samples from the seven patients with viral infections. We
monitored viral antibody levels in one patient (ICU-06) 7, 8, 9 and
18 days after the onset of disease (Extended Data Table2). A clear trend
was observed in the IgG and IgM titres, which increased over time,
except that the IgM titre was decreased in the last sample (Fig.2b).
As a second analysis, we tested samples from 5 of the 7 virus-positive
patients around 20 days after disease onset for the presence of viral
antibodies (Extended Data Tables1, 2). All patient samples—but not
samples from healthy individuals—were strongly positive for viral IgG
(Fig.2b). There were also three IgM-positive samples, indicating an
acute infection.
We next successfully isolated the virus (called 2019-nCoV BetaCoV/
Wuhan/WIV04/2019) from both Vero E6 and Huh7 cells using the BALF
sample of patient ICU-06. Clear cytopathogenic effects were observed
in cells after incubation for three days (Extended Data Fig.6a, b). The
identity of the strain WIV04 was verified in Vero E6 cells by immuno-
fluorescence microscopy using the cross-reactive viral N antibody
(Extended Data Fig.6c, d) and by metagenomics sequencing, most of
the reads of which mapped to 2019-nCoV, and qPCR analysis showed
that the viral load increased from day1 to day3 (Extended Data Fig.6e, f).
Viral particles in ultrathin sections of infected cells displayed a typi-
cal coronavirus morphology, as visualized by electron microscopy
(Extended Data Fig.6g). To further confirm the neutralization activity
of the viral IgG-positive samples, we conducted serum-neutralization
assays in Vero E6 cells using the five patient sera that were IgG-positive.
We demonstrate that all samples were able to neutralize 100 TCID
50
(50% tissue-culture-infective dose) of 2019-nCoV at a dilution of
1:40–1:80. We also show that this virus could be cross-neutralized by
horse anti-SARS-CoV serum (gift from L.-F. Wang) at dilutions of 1:40;
however, the potential for cross-reactivity with SARS-CoV antibod-
ies needs to be confirmed with anti-SARS-CoV serum from humans
(Extended Data Table4).
ACE2 is known to be a cell receptor for SARS-CoV14. To determine
whether 2019-nCoV also uses ACE2 as a cellular entry receptor, we
conducted virus infectivity studies using HeLa cells that expressed or
did not express ACE2 proteins from humans, Chinese horseshoe bats,
civets, pigs and mice. We show that 2019-nCoV is able to use all ACE2
proteins, except for mouse ACE2, as an entry receptor to enter ACE2-
expressing cells, but not cells that did not express ACE2, indicating that
ACE2 is probably the cell receptor through which 2019-nCoV enters cells
(Fig.3). We also show that 2019-nCoV does not use other coronavirus
receptors, such as aminopeptidase N (APN) and dipeptidyl peptidase
4 (DPP4) (Extended Data Fig.7).
The study provides a detailed report on 2019-nCoV, the likely aetio-
logical agent responsible for the ongoing epidemic of acute respiratory
syndrome in China and other countries. Virus-specific nucleotide-
positive and viral-protein seroconversion was observed in all patients
tested and provides evidence of an association between the disease and
the presence of this virus. However, there are still many urgent ques-
tions that remain to be answered. The association between 2019-nCoV
and the disease has not been verified by animal experiments to fulfil
the Koch’s postulates to establish a causative relationship between a
microorganism and a disease. We do not yet know the transmission
routine of this virus among hosts. It appears that the virus is becom-
ing more transmissible between humans. We should closely monitor
whether the virus continues to evolve to become more virulent. Owing
to a shortage of specific treatments and considering the relatedness of
2019-nCoV to SARS-CoV, some drugs and pre-clinical vaccines against
a
b
c
BALF OS OS AS Blood
0
2
4
6
8
log(copy perml)
WIV01
WIV02
WIV03
WIV04
WIV05
WIV06
WIV07
First sampling
30-12-2019
Second sampling
10-01-2020
30-12-2019
31-12-2019
01
-01-2020
10
-01-2020
0
0.1
0.2
0.3
0.4
0
0.5
1.0
1.5
2.0
ELISAODratio (IgM)
ELISAODratio (IgG)
IgG
IgM
0
0.05
0.10
0.10
0.30
0.50
0
0.05
0.10
1.00
2.00
3.00
ELISAODratio
Ig
MI
gG
PatientHealthy PatientHealthy
Fig. 2 | Mole cular and ser ological inve stigation o f patient sam ples.
a, Molecular d etection of 2 019-nCoV in seven patient s. Patient in formation can
be found in Ext ended Data Tables1, 2 . Detectio n methods are de scribed in
theMethods . AS, anal swab; OS , oral swab. b, Dynami cs of 2019-nCoV antibo dy
levels in one pat ient who showed si gns of diseas e on 23 Decem ber 2019 (ICU-
06). OD ratio, optic al density at 450 –630nm. The ri ght and left y axe s indicate
ELISA OD rat ios for IgM and IgG , respecti vely. c, Serological t est of 2019-nCoV
antibodi es in five pati ents (Exten ded Data Table2). The asteri sk indicates d ata
collecte d from patient I CU-06 on 10 Janua ry 2020. b, c, The cut-off wa s to 0.2
for the IgM anal ysis and to 0.3 for the I gG analysis, ac cording to the levels of
healthy controls.
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Nature | Vol 579 | 12 March 2020 | 273
SARS-CoV could probably be used to treat this virus. Finally, consider-
ing the wide spread of SARSr-CoV in their natural reservoirs, future
research should be focused on active surveillance of these viruses
for broader geographical regions. In the long term, broad-spectrum
antiviral drugs and vaccines should be prepared for emerging infec-
tious diseases that are caused by this cluster of viruses in the future.
Most importantly, strict regulations against the domestication and
consumption of wildlife should be implemented.
Note added in proof: Since this paper was accepted, the ICTV has desig-
nated the virus as SARS-CoV-215; in addition, the WHO has released the
official name of the disease caused by this virus, which is COVID-19 16.
Online content
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maries, source data, extended data, supplementary information,
acknowledgements, peer review information; details of author con-
tributions and competing interests; and statements of data and code
availability are available at https://doi.org/10.1038/s41586-020-2012-7.
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© The Author(s) 2020
DAPI ACE2–FITC N–Cy3 Merge
hACE2bACE2sACE2cACE2mACE2Untransfected
Fig. 3 | Anal ysis of the rece ptor use of 2019-n CoV. Determination o f virus
infectiv ity in HeLa c ells that express ed or did not expre ss (untransfecte d)
ACE2. Th e expression of ACE 2 plasmid with S t ag was detec ted using mous e
anti-S tag monoclonal antibody. hACE2, human ACE2; bACE2, ACE2 of
Rhinolophus sinicus (bat); cACE 2, civet ACE2; sACE2 , swine ACE2 (pi g); mACE2,
mouse ACE2 . Green, ACE2; re d, viral protein (N); blu e, DAPI (nuclei). Scale bar s,
10μm.
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Article
Methods
Data reporting
No statistical methods were used to predetermine sample size. The
experiments were not randomized and the investigators were not
blinded to allocation during experiments and outcome assessment.
Sample collection
Human samples, including oral swabs, anal swabs, blood and BALF
samples were collected by Jinyintan hospital (Wuhan, China) with
the consent of all patients and approved by the ethics committee of
the designated hospital for emerging infectious diseases. Patients
were sampled without gender or age preference unless indicated.
For swabs, 1.5ml DMEM containing 2% FBS was added to each tube.
The supernatant was collected after centrifugation at 2,500rpm,
vortexing for 60s and a standing period of 15–30min. The superna-
tant from swabs or BALF (no pre-treatment) was added to either lysis
buffer for RNA extraction or to viral transport medium for isolation
of the virus. The viral transport medium was composed of Hank’s
balanced salt solution (pH7.4) containing BSA (1%), amphotericin
(15μgml
−1
), penicillin G (100unitsml
−1
) and streptomycin (50μgml
−1
).
Serum was separated by centrifugation at 3,000g for 1 5min within
24h of collection, followed by inactivation at 56 °C for 1h, and was
then stored at 4 °C until use.
Virus isolation, cell infection, electron microscopy and
neutralization assay
The following cell lines were used for virus isolation in this study: Vero
E6 and Huh7 cells, which were cultured in DMEM containing 10% FBS. All
cell lines were tested and free of mycoplasma contamination, submitted
for species identification and authenticated by morphological evalua-
tion by microscopy. None of the cell lines was on the list of commonly
misidentified cell lines (by ICLAC).
Cultured cell monolayers were maintained in their respective medium.
The PCR-positive BALF sample from ICU-06 patient was spun at 8,000g
for 15min, filtered and diluted 1:2 with DMEM supplemented with
16μgml−1 trypsin before it was added to the cells. After incubation at
37 °C for 1h, the inoculum was removed and replaced with fresh culture
medium containing antibiotics (see below) and 16μgml
−1
trypsin. The
cells were incubated at 37 °C and observed daily for cytopathogenic
effects. The culture supernatant was examined for the presence of virus
by qRT–PCR methods developed in this study, and cells were examined
by immunofluorescence microscopy using the anti-SARSr-CoV Rp3 N
antibody that was generated in-house (1:1,000). Penicillin (100unitsml
−1
)
and streptomycin (15μgml−1) were included in all tissue culture media.
Vero E6 cells were infected with the new virus at a multiplicity of
infection (MOI) of 0.5 and collected 48h after infection. Cells were fixed
with 2.5% (w/v) glutaraldehyde and 1% osmium tetroxide, dehydrated
through a graded series of ethanol concentrations (from 30 to 100%)
and embedded with epoxy resin. Ultrathin sections (80nm) of embed-
ded cells were prepared, deposited onto Formvar-coated copper grids
(200mesh), stained with uranyl acetate and lead citrate, and analysed
using a 200-kV Tecnai G2 electron microscope.
The virus neutralization test was carried out in a 96-well plate.
The patient serum samples were heat-inactivated by incubation at
56 °C for 1h before use. The serum samples were diluted to 1:10, 1:20,
1:40 or 1:80, and then an equal volume of virus stock was added and
incubated at 37°C for 60min in a 5% CO
2
incubator. Diluted horse
anti-SARS-CoV serum or serum samples from healthy individuals
were used as control. After incubation, 100μl mixtures were inocu-
lated onto a monolayer of Vero E6 cells in a 96-well plate for 1h. Each
serum was assessed in triplicate. After removing the supernatant, the
plate was washed twice with DMEM medium. Cells were incubated
with DMEM supplemented with 2% FBS for 3days. Subsequently,
the cells were checked for cytopathogenic effec ts.
RNA extraction and PCR
Whenever commercial kits were used, the manufacturer’s instructions
were followed without modification. RNA was extracted from 200μl
of samples with the High Pure Viral RNA kit (Roche). RNA was eluted in
50μl of elution buffer and used as the template for RT–PCR.
For qPCR analysis, primers based on the S gene of 2019-nCoV were
designed: RBD-qF1, 5′-CAATGGTTTAACAGGCACAGG-3′; RBD-qR1,
5′-CTCAAGTGTCTGTGGATCACG-3′. RNA extracted as described
above was used for qPCR using the HiScript II One Step qRT–PCR
SYBR Green Kit (Vazyme Biotech). Conventional PCRs were also
performed using the following primer pairs: ND-CoVs-951F, 5′-TGT-
KAGRTTYCCTAAYATTAC-3′; ND-CoVs-1805R, 5′-ACATCYTGATAN-
ARAACAGC-3′. The 20-μl qPCR reaction mix contained 10μl 2× One
Step SYBR Green mix, 1μl One Step SYBR Green Enzyme mix, 0.4μl
50× ROX Reference Dye 1, 0.4μl of each primer (10μM) and 2μl
template RNA. Amplification was performed as follows: 50 °C for
3min, 95 °C for 30s followed by 40cycles consisting of 95 °C for 10s
and 60 °C for 30s, and a default melting curve step in an ABI 7500
Real-time PCR machine.
Serological test
In-house anti-SARSr-CoV IgG and IgM ELISA kits were developed using
SARSr-CoV Rp3 N protein as antigen, which shared more than 90%
amino acid identity to all SARSr-CoVs
2
. For IgG analyses, MaxiSorp
Nunc-immuno 96-well ELISA plates were coated (100ng per well) over-
night with recombinant N protein. Human sera were used at a dilution
of 1:20 for 1h at 37 °C. An anti-human IgG HRP-conjugated monoclonal
antibody (Kyab Biotech) was used at a dilution of 1:40,000. The OD
value (450–630nm) was calculated. For IgM analyses, MaxiSorp Nunc-
immuno 96-well ELISA plates were coated (500ng per well) overnight
with anti-human IgM (μ chain). Human sera were used at a 1:100 dilu-
tion for 40min at 37 °C, followed by incubation with an anti-Rp3 N
HRP-conjugated antibody (Kyab Biotech) at a dilution of 1:4,000. The
OD value (450–630nm) was calculated.
Examination of ACE2 receptor for 2019-nCoV infection
HeLa cells transiently expressing ACE2 were prepared using Lipofectamine
3000 (Thermo Fisher Scientific) in a 96-well plate; mock-transfec ted cells
were used as controls. 2019-nCoV grown in Vero E6 cells was used for
infection at a MOI of 0.5. APN and DPP4 were analysed in the same way.
The inoculum was removed after absorption for 1h and washed twice
with PBS and supplemented with medium. At 24h after infection, cells
were washed with PBS and fixed with 4% formaldehyde in PBS (pH7.4)
for 20min at room temperature. ACE2 expression was detected using
a mouse anti-S tag monoclonal antibody and a FITC-labelled goat anti-
mouse IgG H&L (Abcam, ab96879). Viral replication was detected using
a rabbit antibody against the Rp3 N protein (generated in-house, 1:1,000)
and a Cy3-conjugated goat anti-rabbit IgG (1:200, Abcam, ab6939). Nuclei
were stained with DAPI (Beyotime). Staining patterns were
examined using confocal microscopy on a FV1200 microscope (Olympus).
High-throughput sequencing, pathogen screening and genome
assembly
Samples from patient BALF or from the supernatant of virus cul-
tures were used for RNA extraction and next-generation sequencing
(NGS) using BGI MGISEQ2000 and Illumina MiSeq 3000 sequencers.
Metagenomic analysis was carried out mainly based on the bio-
informatics platform MGmapper (PE_2.24 and SE_2.24). The raw
NGS reads were first processed by Cutadapt (v.1.18) with minimum
read length of 30base pairs. BWA (v.0.7.12-r1039) was used to align
reads to a local database with a filter hits parameter of 0.8FMM
((match+mismatch)/read length≥fraction] value and minimum
alignment score of 30. Parameters for post-processing of assigned
reads were set to a minimum size normalized abundance of 0.01,
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minimum read count of 20 and were otherwise set to default param-
eters. A local nucleic acid database for human and mammals was
used to filter reads of host genomes before mapping reads to the
virus database. The results of the metagenomic analysis were dis-
played as pie charts using Microsoft Office 2010. NGS reads were
assembled into genomes using Geneious (v.11.0.3) and MEGAHIT
(v.1.2.9). PCR and Sanger sequencing was performed to fill gaps in
the genome. 5′-rapid amplification of cDNA ends (RACE) was per-
formed to determine the 5′-end of the genomes using a SMARTer
RACE 5′/3′ kit (Takara). Genomes were annotated using the Clone
Manager Professional Suite 8 (Sci-Ed Software).
Phylogenetic analysis
Routine sequence management and analysis was carried out using
DNAStar. The sequence alignment of complete genome sequences
was performed using MAFFT (v.7.307) with default parameters. The
codon alignments of full-length S and RdRp gene sequences were con-
verted from the corresponding protein alignments by PAL2NAL (v.14);
the protein alignments were created by Clustal Omega (v.1.2.4) using
default parameters. Maximum likelihood phylogenetic trees were
generated using RAxML (v.0.9.0) with GTR+G substitution model and
1,000 bootstrap replicates.
Reporting summary
Further information on research design is available in theNature
Research Reporting Summary linked to this paper.
Data availability
Sequence data that support the findings of this study have been depos-
ited in GISAID (https://www.gisaid.org/) with accession numbers
EPI_ISL_402124, EPI_ISL_402127–EPI_ISL_402130 and EPI_ISL_402131;
GenBank with accession numbers MN996527MN996532; National
Genomics Data Center, Beijing Institute of Genomics, Chinese Academy
of Sciences (https://bigd.big.ac.cn/databases?lang=en) with accession
numbers SAMC133236–SAMC133240 and SAMC133252.
Acknowledgements We thank P. Zhang and A. Du from the WIV core facility centre for their
help with producing transmission electron microscopy micrographs; H.-Z. Liu and P. Yu from
WIV for bioinformatics analysis. This work was jointly supported by the Strategic Priority
Research Program of the Chinese Academy of Sciences (CAS) (XDB29010101 to Z.-L.S. and
XDB29010104 to P.Z.), China Natural Science Foundation for excellent scholars (81822028 to
P.Z., 31770175 to Z.-L.S. and 31800142 to B.H.), Mega-Project for Infectious Disease from
Minister of Science and Technology of the People’s Republic of China (2018ZX10305409-004-
001 to P.Z.), Youth innovation promotion association of CAS (2019328 to X.-L.Y.).
Author contributions Z.-L.S., P.Z., Y.-Y.W. and G.-F.X. conceived the study. X.-G.W., C.-L.H., H.-D.C.,
F.D., Q.-J.C., F.-X.Z. and L.-L.L. collected patient samples. X.-L .Y., B.Y., W.Z., B.L., J.C., X.-S.Z., Y.L.,
H.G., R.-D.J., M.-Q.L., Y.C., X.W., X.-R.S. and K.Z. performed qPCR, serology and virus culturing
experiments. L.Z., Y.Z., H.-R.S. and B.H. performed genome sequencing and annotations.
Competing interests The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020-
2012-7.
Correspondence and requests for materials should be addressed to Z.-L.S.
Peer review information Nature thanks the anonymous reviewers for their contribution to the
peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Article
Extende d Data Fig. 1 | NG S raw reads of sam ple WIV04 map ping to the 2019-n CoV sequence. T he x axis indic ates the genom e nucleotide po sition and the y ax is
represen ts the read dept h of the mapping.
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Extende d Data Fig. 2 | Phylo genetic tre es based o n the complete S a nd
RdRp gene s equence s of coronaviru ses. a, b, Phylogeneti c trees on the ba sis
of the gene se quences of S (a) and RdRp (b) are show n. 2019-nCoV and bat CoV
RaTG13 are shown in bol d and in red. The tre es were constr ucted using t he
maximum likel ihood metho d using the GTR + G sub stitution mo del with
bootst rap values deter mined by 1,000 r eplicates. B ootstrap s values of more
than 50% are sho wn.
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Article
Extende d Data Fig. 3 | Am ino acid seq uence align ment of the S1 pro tein of
the 2019-nCoV to SAR S-CoV and se lected bat SA RSr-CoVs. The receptor-
binding mot if of SARS-C oV and the homologous re gion of other co ronaviruses
are indicate d by the red box. Th e key amino acid residu es involved in the
interac tion with human ACE 2 are numbered a t the top of the align ed
sequenc es. The shor t insertio ns in the N-termi nal domain of the 2019-n CoV are
indicate d by the blue boxes. Ba t CoV RaTG13 was obtained fro m R. affi nis,
found in Yunnan provi nce. Bat CoV ZC45 was obt ained from R. sinicu s, found in
Zhejiang province.
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Extende d Data Fig. 4 | Mol ecular dete ction met hod used to de tect
2019- nCoV. a, Standard cu rve for qPCR prime rs. The PCR pro duct of the S gene
that was ser ial diluted in the r ange of 108 to 101 (lines f rom left to rig ht) was used
as a template . Primer seq uences and exp erimental c onditions are de scribed in
theMethods . b, Specificity o f the qPCR prime rs. Nucleoti de samples from t he
indicate d pathogens were u sed.
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Article
Extende d Data Fig. 5 | Am ino acid seq uence align ment of the nuc leocapsi d protein of 2019- nCoV to bat SARSr- CoV Rp3 and SARS -CoV BJ01. Bat SA RSr-CoV
Rp3 was obt ained from R. sinicu s, which is found in Guan gxi province .
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Extende d Data Fig. 6 | Is olation and a ntigenic ch aracteriz ation of
2019- nCoV. a, b, Vero E6 cells are shown at 24h af ter infectio n with mock viru s
(a) or 2019-nCoV (b). c, d, Mock-virus-infecte d (c) or 2019-nCoV-infected (d)
samples we re stained wit h rabbit serum r aised agains t recombinant SA RSr-CoV
Rp3 N protein (red) a nd DAPI (blue). The expe riment was con ducted tw ice
indepen dently with sim ilar results. e, T he ratio of the num ber of reads rela ted
to 2019-nCoV among th e total number o f virus-related re ads in metage nomics
analysis of supernatants from Vero E6 cell cultures. f, Virus growth in Vero E6
cells. g, Viral particl es in the ultrath in sections we re imaged using el ectron
microscopy a t 200kV. The sample was fro m virus-infecte d Vero E6 cells. The
inset shows t he viral part icles in an intra- cytosolic va cuole.
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Article
Extende d Data Fig. 7 | An alysis of 2019-nC oV receptor usa ge. Determination
of virus infe ctivity in H eLa cells wi th or without the ex pression of hum an APN
and DPP4. The expre ssion of ACE2, A PN and DPP4 plasmid s with S tag were
detect ed using mouse a nti-S tag monoc lonal antibod y. ACE2, APN and DPP4
proteins ( green), viral protein (red ) and nuclei (blue) are sh own. Scale bar s,
10μm.
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Extended Data Table 1 | Patient information and diagnosis history
Note, some records are missing. All patients are sellers or deliverymen at the seafood market except ICU-01, whose contact history is unclear. All patients were admitted to intensive care unit
(ICU) during the irst investigation and were now in stable condition. Blood IgM tests have been performed for the following respiratory pathogens for all patients: Legionella pneumophilia,
Mycoplasma pneumoniae, Chlamydia pneumoniae, respiratory syncytial virus, adenovirus, Rickettsia, inluenza A virus, inluenza B virus and parainluenza virus.
*This patient reported fever on 12December 2019 and then recovered without medical treatment. He came back to the hospital on 27December 2019 with a fever. His wife was also ill and
admitted to the hospital. Both individuals recovered.
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Article
Extended Data Table 2 | Laboratory results
Samples from two patients (ICU-01 and ICU-08) were not available during the second investigation. They had been discharged from the hospital. We did a serial test for patient ICU-06 on the
following dates: 30December 2019, 31December 2019, 1January 2020 and 10January 2020, corresponding to 7, 8, 9 and 18days after disease onset (23December 2019). Molecular and
serological (IgM and IgG) virus-detection results for 2019-nCoV are shown. NA, not available.
*Virus isolated.
#A full-length genome was obtained.
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Extended Data Table 3 | Genomic comparison of 2019-nCoV WIV04 with SARS-CoVs and bat SARSr-CoVs
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Article
Extended Data Table 4 | Virus neutralization test of serum samples
Each serum sample was tested in triplicate. Serum samples from two healthy individuals from Wuhan and ive patients as well as a horse anti-SARS-CoV anti-serum were used.
We used 120 TCID50 viruses per well. Serum samples were used at dilutions of 1:10, 1:20, 1:40 and 1:80. neg, negative; VNT, virus neutralization test.
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1
nature research | reporting summary October 2018
Corresponding author(s): Zheng-Li Shi
Reporting Summary
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Data collection No software was used.
Data analysis BWA (v0.712-r1039), Cutadapt (v1.18), Geneious (v11.0.3), MEGAHIT (v1.2.9), Clone Manager Professional Suite 8, MAFFT (v7.307),
MGmapper (PE2.24 and SE2.24), PAL2NAL (version 14), Clustal Omega (version 1.2.4), RAxML (version 0.9.0)
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
Last updated by author(s): Feb 21, 2020
Sequence data that support the findings of this study have been deposited in GISAID with the accession no. EPI_ISL_402124 and EPI_ISL_402127-402131.
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size Samples of seven pneumonia patients are available from the clinical hospital to be sent to Wuhan Institute of Virology for pathogen
identification. The coronavirus genome sequences were obtained from 5 different patients and shared >99.9% identity, suggesting they were
infected by the same virus. Therefore, the sample size is sufficient for conducting the following study which aims to identify and characterize
the causative agent of this pneumonia outbreak.
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Antibodies
Antibodies used
Validation The house-made SARSr-CoV Rp3 NP antibodies and anti-S tag monoclonal antibody were validated in a WB. The cy3-conjugated
anti-rabbit IgGs were validated in IFA. The FITC-labelled goat anti-mouse IgG H&L was validated in IHC.
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ATCC.
1. African green monkey origin, Vero E6 cell; 2. Human lung cell Huh7 ; 3. Human HeLa cells. All cell lines werefrom
(Abcam,ab6939, dilution: 1:200); 6. mouse anti-S tag monoclonal antibody made in house
4 .FITC-labelled goat anti-mouse IgG H&L (Abcam, ab96879, dlilution 1:100); 5. cyanin 3-conjugated goat anti-rabbit IgG
Ltd,Wuhan, China, dilution: 1:40000); 3. Anti-Rp3 NP-HRP conjugated (Kyab Biotech Co.,Ltd, Wuhan, China, dilution: 1:4000);
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... Since December 2019, the world pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to human health and world economy [1,2]. As of August 2022, SARS-CoV-2 has accounted for more than 590 million infections and more than six million deaths worldwide according to data released by the WHO (https://www.who.int/emergencies/diseases/novel-coronavirus-2019 ...
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All coronaviruses are characterized by spike glycoproteins whose S1 subunit contains the receptor binding domain anchoring the virus to the host cellular membrane and regulating virus transmissibility and infectious process. Although the protein/receptor interaction depends on the spike secondary-conformation, in particular to its S1 unit, few is known about the secondary-structure of different coronaviruses. In this paper the S1 conformation is investigated for MERS-CoV, SARS-CoV and SARS-CoV-2 in serological condition, by measuring their Amide I infrared vibrational absorption bands. The SARS-CoV-2 secondary structure reveals a strong difference in comparison to MERS-CoV and SARS-CoV ones, with a higher amount of intermolecular β-sheet content. Moreover, the conformation of SARS-CoV-2 S1 shows a significant change by moving from serological pH and mild acidic to alkaline pH conditions close to the bat ecological niche. Both results suggest a huge capability of SARS-CoV-2 S1 glycoprotein to adapt its secondary structure to different environments.
... 56 Random-amplification deep sequencing methods played a major role in the initial identification of SARS-CoV-2. [57][58][59][60][61] Deep sequencing molecular methods such as next generation sequencing and metagenomic next generation sequencing will continue to be needed to determine future mutations of SARS CoV-2 but are currently impractical for diagnosing COVID-19 infections. ...
... The high sequence identity of the two coronavirus genes (96.2% for Rhinolophus affinis -RaTG13 and 93.3% for Rhinolophus malayanus -RmYN02) of bats suggests that bats may be the natural reservoir of SARS-CoV-2 (Chan et al., 2013;Nguyen et al., 2022;Ren et al., 2020;Zhou et al., 2020aZhou et al., , 2020b. Although direct infection of humans with bat viruses has not yet been documented, it could, however, occur in mixed unsanitary environments such as bazaars, where people mix with domestic or wild animals that carry viruses and exchange them with each other, making them more contagious, and easier to be transmitted to humans (Menachery et al., 2015). ...
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Non-technical summary Humans have the tendency to damage the natural environment in many ways. Deforestation and conversion of forests for residential, industrial development, and expansion of agricultural crops, as well as the burning of fossil fuels, are some activities that disrupt natural ecosystems and wildlife and contribute to climate change. As a result, the life cycles of pathogens and intermediate hosts (insects, rodents, mammals) as well as biodiversity are affected. Through these activities, humans meet wild animals that transmit pathogens, resulting in their infection by zoonoses and causing epidemics–pandemics, the effects of which have as their final recipient himself and his activities. Technical summary This article aims to highlight the two-way relationship between those human activities and the occurrence of epidemics–pandemics. We will try to elaborate this two-way relationship, through the overview of the current pandemic (origin of SARS-CoV-2, modes of transmission, clinical picture of the disease of COVID-19, influence of weather and air pollution on prevalence and mortality, pandemic effects, and treatments). They are used as primary sources, scientific articles, literature, websites, and databases (Supplementary appendix) to analyze factors involved in the occurrence and transmission of zoonotic diseases in humans (Ebola, influenza, Lyme disease, dengue fever, cholera, AIDS/HIV, SARS-CoV, MERS-CoV). The present paper concluded that humanity today faces two major challenges: controlling the COVID-19 pandemic and minimizing the risk of a new global health crisis occurring in the future. The first can be achieved through equitable access to vaccines and treatments for all people. The second needs the global community to make a great change and start protecting the natural environment and its ecosystems through the adoption of prevention policies. Summary of social media Two-way relationship between human activities and epidemics highlighted, through review of the COVID-19 pandemic.
... Coronavirus disease 2019 (COVID-19) is a newly emerging infectious disease caused by a positive single-stranded RNA virus -severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1][2][3][4]. Variants of SARS-CoV-2 escape from the immune system of human hosts, causing cracks in vaccination efforts to reach herd immunity [5][6][7][8][9]. However, the majority of variants that develop within hosts are eventually eliminated or not transmitted, and their significance is thus far, has been largely overlooked. ...
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Objectives Variants of SARS-CoV-2 frequently arise within infected individuals. Here, we explored the level and pattern of intra-host viral diversity in association with disease severity. Then, we analyzed information underlying these nucleotide changes to infer the impetus including mutational signatures and immune selection from neutralizing antibody or T cell recognition. Methods From 23rd January to 31st March 2020, a set of cross-sectional samples were collected from individuals with homogenous founder virus regardless of disease severity. Intra-host single-nucleotide variants (iSNVs) were enumerated using deep sequencing. HLA alleles were genotyped by sanger sequencing. Medical records were collected and reviewed by attending physicians. Results A total of 836 iSNVs (3-106 per sample) were identified and distributed in a highly individualized pattern. The number of iSNVs paced with infection duration peaked within days and declined thereafter. These iSNVs did not stochastically arise due to a strong bias towards C>U/G>A and U>C/A>G substitutions in reciprocal proportion with escalating disease severity. Eight nonsynonymous iSNVs in the receptor-binding domain could escape from neutralization and 18 iSNVs were significantly associated with specific HLA alleles. Conclusion The level and pattern of iSNVs reflect in vivo viral-host interaction and the disease pathogenesis.
... The understanding of immune responses against SARS-CoV-1 provided information for the prophylactic and therapeutic treatments against SARS-CoV-2 infection. For the above reasons, anti-SARS-CoV-2 antibody responses were explored soon after the outbreak of COVID-19 and indeed detected in the sera of COVID-19 patients [3,4]. Thus, the anti-SARS-CoV-2 antibody levels have been used as indicators of infection or efficacy of vaccination afterwards. ...
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As an important part of adaptive immunity, T cells are indispensable in the defense against pathogens including viruses. SARS-CoV-2 is a new human coronavirus that occurred at the end of 2019 and has caused the COVID-19 pandemic. Nevertheless, most of the infected patients recovered without any antiviral therapies, suggesting an effective immunity developed in the bodies. T cell immunity responds upon SARS-CoV-2 infection or vaccination and plays crucial roles in eliminating the viruses and generating T cell memory. Specifically, a subpopulation of CD4 ⁺ T cells could support the production of anti-SARS-CoV-2 antibodies, and cytotoxic CD8 ⁺ T cells are also protective against the infection. SARS-CoV-2–recognizing T cells could be detected in SARS-CoV-2–unexposed donors, but the role of these cross-reactive T cells is still in debate. T cell responses could be diverse across individuals, mainly due to the polymorphism of HLAs. Thus, compared to antibodies, T cell responses are generally less affected by the mutations of SARS-CoV-2 variants. Up to now, a huge number of studies on SARS-CoV-2–responsive T cells have been published. In this review, we introduced some major findings addressing the questions in the main aspects about T cell responses elicited by SARS-CoV-2, to summarize the current understanding of COVID-19.
Objectives We aimed to determine the incidences of neuroimaging findings (NIF) and investigate the relationship between the course of pneumonia severity and neuroimaging findings. Materials and Methods Our study was a retrospective analysis of 272 (>18 years) COVID-19 patients who were admitted between “March 11, 2021, and September 26, 2022". All patients underwent both chest CT and neuroimaging. The patient's chest CTs were evaluated for pneumonia severity using a severity score system (CT-SS). The incidence of NIF was calculated. NIF were categorized into two groups; neuroimaging positive (NIP) and neuroimaging negative (NIN). Consecutive CT-SS changes in positive and negative NIF patients were analyzed. Results The median age of total patients was 71; IQR, 57-80. Of all patients, 56/272 (20.6%) were NIP. There was no significant relationship between NIP and mortality (p= 0.815) and ICU admission (p= 0.187). The incidences of NIF in our patients were as follows: Acute-subacute ischemic stroke: 47/272 (17.3%); Acute spontaneous intracranial hemorrhage: 13/272 (4.8%); Cerebral microhemorrhages: 10/272 (3.7%) and Cerebral venous sinus thrombosis: 3/25 (10.7%). Temporal change of CT-SSs, there was a statistically significant increase in the second and third CT-SSs compared to the first CT-SS in both patients with NIP and NIN. Conclusion Our results showed that since neurological damage can be seen in the late period and neurological damage may develop regardless of pneumonia severity.
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The COVID-19 pandemic has resulted in great morbidity and mortality worldwide and human genetic factors have been implicated in the susceptibility and severity of COVID-19. However, few replicate researches have been performed, and studies on associated genes mainly focused on genic regions while regulatory regions were a lack of in-depth dissection. Here, based on previously reported associated variants and genes, we designed a capture panel covering 1,238 candidate variants and 25 regulatory regions of 19 candidate genes and targeted-sequenced 96 mild and 145 severe COVID-19 patients. Genetic association analysis was conducted between mild and severe COVID-19 patients, between all COVID-19 patients and general population, or between severe COVID-19 patients and general population. A total of 49 variants were confirmed to be associated with susceptibility or severity of COVID-19 (p < 0.05), corresponding to 18 independent loci. Specifically, rs1799964 in the promoter of inflammation-related gene TNF, rs9975538 in the intron of interferon receptor gene IFNAR2, rs429358 in the exon of APOE, rs1886814 in the intron of FOXP4-AS1 and a list of variants in the widely reported 3p21.31 and ABO gene were confirmed. It is worth noting that, for the confirmed variants, the phenotypes of the cases and controls were highly consistent between our study and previous reports, and the confirmed variants identified between mild and severe patients were quite different from those identified between patients and general population, suggesting the genetic basis of susceptibility and severity of SARS-CoV-2 infection might be quite different. Moreover, we newly identified 67 significant associated variants in the 12 regulatory regions of 11 candidate genes (p < 0.05). Further annotation by RegulomeDB database and GTEx eQTL data filtered out two variants (rs11246060 and rs28655829) in the enhancer of broad-spectrum antiviral gene IFITM3 that might affect disease severity by regulating the gene expression. Collectively, we confirmed a list of previously reported variants and identified novel regulatory variants associated with susceptibility and severity of COVID-19, which might provide biological and clinical insights into COVID-19 pathogenesis and treatment.
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The coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread over the world, resulting in a global severe pneumonia pandemic. Both the cell receptor angiotensin-converting enzyme 2 (ACE2) and the breakdown of S protein by transmembrane serine protease 2 (TMPRSS2) are required by SARS-CoV-2 to enter the host cells. Similarly, the expression level of viral receptor genes in various organs determines the likelihood of viral infection. Several animal species have been found to be infected by the SARS-CoV-2, such as minks, posing an enormous threat to humans. Because the mice and rats were closely related to human and the fact that rats and mice have a risk of infection by SARS-CoV-2 with specific variants, we investigated the expression patterns of 79 receptor genes from 107 viruses ,including SARS-CoV-2, in 14 organs of the rat and mouse, and 5 organs of the muskrat, to find the most likely host organs to become infected with certain viruses. The findings of this study are anticipated to aid in prevention of zoonotic infections spread by rats, mice, muskrats, and other rodents.
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The present outbreak of lower respiratory tract infections, including respiratory distress syndrome, is the third spillover, in only two decades, of an animal coronavirus to humans resulting in a major epidemic. Here, the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the official classification of viruses and taxa naming (taxonomy) of the Coronaviridae family, assessed the novelty of the human pathogen tentatively named 2019-nCoV. Based on phylogeny, taxonomy and established practice, the CSG formally recognizes this virus as a sister to severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus and designates it as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To facilitate communication, the CSG further proposes to use the following naming convention for individual isolates: SARS-CoV-2/Isolate/Host/Date/Location. The spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined. The independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying the entire (virus) species to complement research focused on individual pathogenic viruses of immediate significance. This research will improve our understanding of virus-host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.
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During the past two decades, three zoonotic coronaviruses have been identified as the cause of large-scale disease outbreaks–Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and Swine Acute Diarrhea Syndrome (SADS). SARS and MERS emerged in 2003 and 2012, respectively, and caused a worldwide pandemic that claimed thousands of human lives, while SADS struck the swine industry in 2017. They have common characteristics, such as they are all highly pathogenic to humans or livestock, their agents originated from bats, and two of them originated in China. Thus, it is highly likely that future SARS- or MERS-like coronavirus outbreaks will originate from bats, and there is an increased probability that this will occur in China. Therefore, the investigation of bat coronaviruses becomes an urgent issue for the detection of early warning signs, which in turn minimizes the impact of such future outbreaks in China. The purpose of the review is to summarize the current knowledge on viral diversity, reservoir hosts, and the geographical distributions of bat coronaviruses in China, and eventually we aim to predict virus hotspots and their cross-species transmission potential.
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Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are two highly transmissible and pathogenic viruses that emerged in humans at the beginning of the 21st century. Both viruses likely originated in bats, and genetically diverse coronaviruses that are related to SARS-CoV and MERS-CoV were discovered in bats worldwide. In this Review, we summarize the current knowledge on the origin and evolution of these two pathogenic coronaviruses and discuss their receptor usage; we also highlight the diversity and potential of spillover of bat-borne coronaviruses, as evidenced by the recent spillover of swine acute diarrhoea syndrome coronavirus (SADS-CoV) to pigs.
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A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.
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Significance The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV highlights the continued risk of cross-species transmission leading to epidemic disease. This manuscript describes efforts to extend surveillance beyond sequence analysis, constructing chimeric and full-length zoonotic coronaviruses to evaluate emergence potential. Focusing on SARS-like virus sequences isolated from Chinese horseshoe bats, the results indicate a significant threat posed by WIV1-CoV. Both full-length and chimeric WIV1-CoV readily replicated efficiently in human airway cultures and in vivo, suggesting capability of direct transmission to humans. In addition, while monoclonal antibody treatments prove effective, the SARS-based vaccine approach failed to confer protection. Together, the study indicates an ongoing threat posed by WIV1-related viruses and the need for continued study and surveillance.
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The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.
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The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.
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