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LETTER doi:10.1038/nature12711
Isolation and characterization of a bat SARS-like
coronavirus that uses the ACE2 receptor
Xing-Yi Ge
1
*, Jia-Lu Li
1
*, Xing-Lou Yang
1
*, Aleksei A. Chmura
2
, Guangjian Zhu
2
, Jonathan H. Epstein
2
, Jonna K. Mazet
3
, Ben Hu
1
,
Wei Zhang
1
, Cheng Peng
1
, Yu-Ji Zhang
1
, Chu-Ming Luo
1
, Bing Tan
1
, Ning Wang
1
, Yan Zhu
1
, Gary Crameri
4
, Shu-Yi Zhang
5
,
Lin-Fa Wang
4,6
, Peter Daszak
2
& Zheng-Li Shi
1
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
1
. An ongoing outbreak of Middle East respira-
tory syndrome coronavirus
2
suggests that this group of viruses remains
a key threat and that their distribution is wider than previously recog-
nized. Although bats have been suggested to be the natural reservoirs
of both viruses
3–5
, attempts to isolate the progenitor virus of SARS-
CoV from bats have been unsuccessful. Diverse SARS-like corona-
viruses (SL-CoVs) have now been reported from bats in China,
Europe and Africa
5–8
, but none is considered a direct progenitor
of SARS-CoV because of theirphylogenetic disparity from this virus
and the inabilityof their spike proteins to use theSARS-CoV cellular
receptor molecule, the human angiotensin converting enzyme II
(ACE2)
9,10
. 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 coro-
naviruses, 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
necessaryfor direct human infectionby 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.
The 2002–3 pandemic of SARS
1
and the ongoing emergence of the
Middle East respiratory syndrome coronavirus (MERS-CoV)
2
demon-
strate that CoVs are a significant public health threat. SARS-CoV was
shown to use the human ACE2 molecule as its entry receptor, and this
is considered a hallmark of its cross-species transmissibility
11
. The receptor
binding domain (RBD) located in the amino-terminal region (amino
acids 318–510) of the SARS-CoV spike (S) protein is directly involved
in binding to ACE2 (ref. 12). However, despite phylogenetic evidence
that SARS-CoV evolved from bat SL-CoVs, all previously identified
SL-CoVs have major sequence differences from SARS-CoVin the RBD
of their S proteins, including one or two deletions
6,9
. Replacing the RBD
of one SL-CoV S protein with SARS-CoV S conferred the ability to use
human ACE2 and replicate efficiently in mice
9,13
. However, to date, no
SL-CoVs have been isolated from bats,and no wild-type SL-CoV of bat
origin has been shown to use ACE2.
We conducted a 12-month longitudinal survey (April 2011–September
2012) of SL-CoVs in a colony of Rhinolophus sinicus at a single location
in Kunming, Yunnan Province,China (Extended Data Table 1). A total
of 117 anal swabs or faecal samples were collected from individual bats
using a previously published method
5,14
. A one-step reverse transcrip-
tion (RT)-nested PCR was conducted to amplify the RNA-dependent
RNA polymerase (RdRP) motifs A and C, which are conserved among
alphacoronaviruses and betacoronaviruses
15
.
Twenty-seven of the 117 samples (23%) were classed as positive by
PCR and subsequently confirmed by sequencing. The species origin of
all positive samples was confirmed to be R. sinicus by cytochrome b
sequence analysis, as described previously
16
. A higher prevalence was
observed in samples collected in October (30% in 2011 and 48.7% in
2012) than those in April (7.1% in 2011) or May (7.4% in 2012) (Extended
Data Table 1). Analysis of the S protein RBD sequences indicated the
presence of seven different strains of SL-CoVs (Fig. 1a and Extended
Data Figs1 and 2). In additionto RBD sequences, which closely matched
previously described SL-CoVs (Rs672, Rf1 and HKU3)
5,8,17,18
, two novel
strains (designated SL-CoV RsSHC014 and Rs3367) were discovered.
Their full-length genome sequences were determined, and both were
found to be 29,787 base pairs in size (excluding the poly(A) tail). The
overall nucleotide sequence identity of these two genomes with human
SARS-CoV (Tor2 strain) is 95%, higher than that observed previously
for bat SL-CoVs in China (88–92%)
5,8,17,18
or Europe (76%)
6
(Extended
Data Table 2 and Extended Data Figs 3 and 4). Higher sequence iden-
tities were observed at the protein level between these new SL-CoVs
and SARS-CoVs (Extended Data Tables 3 and 4). To understand the
evolutionary origin of these two novel SL-CoV strains, we conducted
recombination analysis with the Recombination Detection Program
4.0 package
19
using available genome sequences of bat SL-CoV strains
(Rf1, Rp3, Rs672, Rm1, HKU3 and BM48-31) and human and civet
representative SARS-CoV strains (BJ01, SZ3, Tor2 and GZ02). Three
breakpoints were detected with strong Pvalues (,10
220
) and supported
by similarity plot and bootscan analysis (Extended Data Fig. 5a, b). Break-
points were located at nucleotides 20,827, 26,553 and 28,685 in the
Rs3367 (and RsSHC014) genome, and generated recombination frag-
ments covering nucleotides 20,827–26,533 (5,727 nucleotides) (inclu-
ding partial open reading frame (ORF) 1b, full-length S, ORF3, E and
partial M gene) and nucleotides 26,534–28,685 (2,133 nucleotides)
(including partial ORF M, full-length ORF6, ORF7, ORF8 and partial
N gene). Phylogenetic analysis using the major and minor parental regions
suggested that Rs3367, or RsSHC014, is the descendent of a recombination
of lineages that ultimately lead to SARS-CoV and SL-CoV Rs672 (Fig. 1b).
The most notable sequence differences between these two new SL-
CoVs and previously identified SL-CoVs is in the RBD regions of their
S proteins. First,they have higheramino acid sequence identity to SARS-
CoV (85% and 96% for RsSHC014 and Rs3367, respectively). Second,
there are no deletions and they have perfect sequence alignment with
the SARS-CoV RBD region (Extended Data Figs 1 and 2). Structural
*These authors contributed equally to this work.
1
Center for Emerging Infectious Diseases, State Key Laboratory of Virology, Wuhan Institute of Virology of the Chinese Academy of Sciences, Wuhan 430071, China.
2
EcoHealth Alliance, New York, New York
10001, USA.
3
One Health Institute, School of Veterinary Medicine, University of California, Davis, California 95616, USA.
4
CSIRO Australian Animal Health Laboratory, Geelong, Victoria 3220, Australia.
5
College of Life Sciences, East China Normal University, Shanghai 200062, China.
6
Emerging Infectious Diseases Program, Duke-NUS Graduate Medical School, Singapore 169857.
00 MONTH 2013 | VOL 000 | NATURE | 1
Macmillan Publishers Limited. All rights reserved
©2013
and mutagenesis studies have previously identified five key residues
(amino acids 442, 472, 479, 487 and 491) in the RBD ofthe SARS-CoV
S protein that have a pivotal role in receptor binding
20,21
. Although all
five residues in the RsSHC014 S protein were found to be different
from those of SARS-CoV, two of the five residues in the Rs3367 RBD
were conserved (Fig. 1 and Extended Data Fig. 1).
Despite the rapid accumulation of bat CoV sequences in the last
decade, there has been no report of successful virus isolation
6,22,23
.We
attempted isolation from SL-CoV PCR-positive samples. Using an
optimized protocol and Vero E6 cells, we obtained one isolate which
caused cytopathic effect during the second blind passage. Purified virions
displayed typical coronavirus morphology under electron microscopy
(Fig. 2). Sequence analysis using a sequence-independent amplifica-
tion method
14
to avoid PCR-introduced contamination indicated that
the isolate was almost identical to Rs3367, with 99.9% nucleotide genome
sequence identity and 100% amino acid sequence identity for the S1
region. The new isolate was named SL-CoV-WIV1.
To determine whether WIV1 can use ACE2 as a cellular entry receptor,
we conducted virus infectivity studies using HeLa cells expressing or
not expressing ACE2 from humans, civets or Chinese horseshoe bats.
We found that WIV1 is able to use ACE2 of differentorigins as an entry
receptor and replicated efficiently in the ACE2-expressing cells (Fig.3).
This is, to our knowledge, the first identification of a wild-type bat SL-
CoV capable of using ACE2 as an entry receptor.
To assessits cross-species transmissionpotential, we conducted infec-
tivity assays in cell lines from a range ofspecies. Our results (Fig. 4 and
Extended Data Table 5) indicate that bat SL-CoV-WIV1 can grow in
human alveolar basal epithelial (A549), pig kidney 15 (PK-15) and
Rhinolophus sinicus kidney (RSKT) cell lines, but not in human cervix
(HeLa), Syrian golden hamster kidney (BHK21), Myotis davidii kidney
(BK), Myotis chinensis kidney (MCKT), Rousettus leschenaulti kidney
(RLK) or Pteropus alecto kidney (PaKi) cell lines. Real-time RT–PCR
indicated that WIV1 replicated much less efficiently in A549, PK-15
and RSKT cells than in Vero E6 cells (Fig. 4).
0.02 0.01
Tor2
BJ01
SZ3
GZ02
Rf1
HKU3
Rp3
Rs627
Rm1
3367
SHC014
SARS CoV
SL-CoV
Rf1
Rp3
Rm1
HKU3
BJ01
GZ02
SZ3
Rs672
Tor2
SHC014
3367
442 472 479 487 491
YL NTY
YL NTY
YL NTY
YL KSY
SF NNY
WP RAH
S—SVY
S—SIY
S—SVY
RL ASF
0.2
Human SARS CoV Tor2
Human SARS CoV BJ01
Human SARS CoV GZ02
Civet SARS CoV SZ3
Bat SL-CoV Rs4087-1
Bat SL-CoV Rs4110
Bat SL-CoV Rs4090
Bat SL-CoV Rs4079
Bat SL-CoV Rs3367
Bat SL-CoV Rs4105
Bat SL-CoV RsSHC014
Bat SL-CoV Rs4084
Bat SL-CoV Rs3267-1
Bat SL-CoV Rs3369
Bat SL-CoV Rf1
Bat SL-CoV HKU3-1
Bat SL-CoV Rm1
Bat SL-CoV Rs672
Bat SL-CoV Rp3
Bat SL-CoV Rs3262-1
Bat SL-CoV Rs4092
Bat SL-CoV Rs4075
Bat SL-CoV Rs3262-2
Bat SL-CoV Rs4085
Bat SL-CoV Rs3267-2
Bat SL-CoV Rs4108
Bat SL-CoV Rs4081
Bat SL-CoV Rs4096
Bat SL-CoV Rs4087-2
Bat SL-CoV Rs4097
Bat SL-CoV Rs4080
Bat SARS-related CoV BM48-31
Bat CoV HKU9-1
a
b
94
85
80
92
64 99
99
98
90
100
100
52
100
100
100
100
100
86
100
100
100
100
71
86
95
51
97
68
98
Figure 1
|
Phylogenetic tree based on amino acid sequences of the S RBD
region and the two parental regions of bat SL-CoV Rs3367 or RsSHC014.
a, SARS-CoV S protein amino acid residues 310–520 were aligned with
homologous regions of bat SL-CoVs using theClustalWsoftware. A maximum-
likelihood phylogenetic tree was constructed using a Poisson model with
bootstrap valuesdetermined by 1,000 replicates in the MEGA5 software package.
The RBD sequences identified in this study are in bold and named by the sample
numbers. The key amino acid residues involved in interacting with the human
ACE2 molecule are indicated on theright of the tree. SARS-CoV GZ02,BJ01 and
Tor2 wereisolated from patients in the early, middle and late phase, respectively,
of the SARS outbreak in 2003. SARS-CoV SZ3 was identified from Paguma
larvata in 2003collected inGuangdong,China. SL-CoVRp3, Rs672and HKU3-1
were identified from R. sinicus collected in China (respectively: Guangxi, 2004;
Guizhou, 2006; Hong Kong, 2005). Rf1 and Rm1 were identified from
R. ferrumequinum and R. macrotis, respectively, collected in Hubei, China, in
2004. Bat SARS-related CoV BM48-31 was identified from R. blasii collected in
Bulgaria in 2008. Bat CoV HKU9-1 was identified from Rousettus leschenaultii
collected in Guangdong, China in 2005/2006 and used as an outgroup. All
sequences in bold and italics were identified in the current study. Filled triangles,
circles and diamonds indicate samples with co-infection by two different
SL-CoVs. ‘–’ indicates the amino acid deletion. b, Phylogenetic origins of the two
parental regions of Rs3367 or RsSHC014. Maximum likelihood phylogenetic
trees were constructed from alignments of two fragments covering nucleotides
20,827–26,533 (5,727 nucleotides) and 26,534 –28,685 (2,133 nucleotides) of the
Rs3367 genome, respectively. For display purposes, the trees were midpoint
rooted. The taxa were annotated according to strain names: SARS-CoV, SARS
coronavirus;SARS-likeCoV, bat SARS-like coronavirus. The twonovel SL-CoVs,
Rs3367 and RsSHC014, are in bold and italics.
RESEARCH LETTER
2 | NATURE | VOL 000 | 00 MONTH 2013
Macmillan Publishers Limited. All rights reserved
©2013
To assess the cross-neutralization activity of human SARS-CoV sera
against WIV1, we conducted serum-neutralization assays using nine
convalescent sera from SARS patients collected in 2003. The results
showed that seven of these were able to completely neutralize 100 tissue
culture infectious dose 50 (TCID
50
) WIV1 at dilutions of 1:10 to 1:40,
further confirming the close relationship between WIV1 and SARS-CoV.
Our findings have important implications for public health. First,
they providethe clearest evidenceyet that SARS-CoV originated in bats.
Our previous work provided phylogenetic evidence of this
5
,butthelack
of an isolate or evidence that bat SL-CoVs can naturally infect human
cells, until now, had cast doubt on this hypothesis. Second, the lack of
capacity of SL-CoVs to use of ACE2 receptors has previously been
considered as the key barrier for their direct spillover into humans, suppor-
ting the suggestion that civets were intermediate hosts for SARS-CoV
adaptation to human transmission during the SARS outbreak
24
. However,
the ability of SL-CoV-WIV1 to use human ACE2 argues against the
necessity of this step for SL-CoV-WIV1 and suggests that direct bat-
to-human infection is a plausible scenario for some bat SL-CoVs. This
has implicationsfor public health control measuresin the face of poten-
tial spilloverof a diverse and growing pool of recently discovered SARS-
like CoVs with a wide geographic distribution.
Our findings suggest that the diversity of bat CoVs is substantially
higher than that previously reported. In this study we were able to demon-
strate the circulation of at least seven different strains of SL-CoVs within a
single colony of R. sinicus during a 12-month period. The high genetic
diversity of SL-CoVs within this colony was mirrored by high pheno-
typic diversity in the differential use of ACE2 by different strains. It
would therefore not be surprising if further surveillance reveals a broad
diversity of bat SL-CoVs that are able to use ACE2, some of whichmay
have even closer homology to SARS-CoV than SL-CoV-WIV1. Our
results—in addition to the recent demonstration of MERS-CoV in a
Saudi Arabian bat
25
, and of bat CoVs closely related to MERS-CoV in
China, Africa, Europe and North America
3,26,27
—suggest that bat coro-
naviruses remain a substantial global threat to public health.
Finally, this study demonstrates the public health importance of path-
ogen discovery programs targeting wildlife that aim to identify the ‘known
unknowns’—previously unknown viral strains closely related to known
pathogens. These programs, focused on specific high-risk wildlife groups
and hotspots of disease emergence, may be a critical part of future global
strategies to predict, prepare for, and prevent pandemic emergence
28
.
HeLa-
hACE2
HeLa-
cACE2
HeLa-
bACE2
HeLa
DAPI FITC Cy3 Merged
0 12 24 48
0 12 24 48
1 × 10
3
1 × 10
4
0 12 24 48
0 12 24 48
TCID50 ml–1
1 × 10
3
1 × 10
4
1 × 10
5
1 × 10
6
TCID50 ml–1
1 × 10
3
1 × 10
4
1 × 10
5
1 × 10
6
TCID50 ml–1
1 × 10
3
1 × 10
4
1 × 10
5
1 × 10
6
1 × 10
7
TCID50 ml–1
Time after
infection (h)
128 µm
Figure 3
|
Analysis of receptor usage of SL-CoV-WIV1 determined by
immunofluorescence assay and real-time PCR. Determination of virus
infectivity in HeLa cells with and without the expression of ACE2. b, bat;
c, civet; h, human. ACE2 expression was detected with goat anti-humanACE2
antibody followed by fluorescein isothiocyanate (FITC)-conjugated donkey
anti-goat IgG. Virus replication was detected with rabbit antibody against the
SL-CoV Rp3 nucleocapsid protein followed by cyanine 3 (Cy3)-conjugated
mouse anti-rabbit IgG. Nuclei were stained with DAPI (49,6-diamidino-2-
phenylindole). The columns (from left to right) show staining of nuclei (blue),
ACE2 expression (green), virus replication (red), merged triple-stained
images and real-time PCR results, respectively. (n53); error bars represent
standard deviation.
200 nm
Figure 2
|
Electron micrograph of purified virions. Virions from a 10-ml
culture were collected, fixed and concentrated/purified by sucrose gradient
centrifugation. The pelleted viral particles were suspended in 100 mlPBS,
stained with 2% phosphotungstic acid (pH 7.0) and examined directly using a
Tecnai transmission electron microscope (FEI) at 200kV.
LETTER RESEARCH
00 MONTH 2013 | VOL 000 | NATURE | 3
Macmillan Publishers Limited. All rights reserved
©2013
METHODS SUMMARY
Throat and faecal swabs or fresh faecal samples were collected in viral transport
medium as described previously
14
. All PCR was conducted withthe One-Step RT–
PCR kit (Invitrogen). Primers targeting the highly conserved regions of the RdRP
gene were used for detection of all alphacoronaviruses and betacoronaviruses as
described previously
15
. Degenerate primers were designed on the basis of all avail-
able genomic sequences of SARS-CoVs and SL-CoVs and used for amplification of
the RBD sequences of S genes or full-length genomic sequences. Degenerate primers
were used for amplification of the bat ACE2 gene as described previously
29
. PCR
products were gel purified and cloned into pGEM-T Easy Vector (Promega). At
least fourindependent cloneswere sequenced to obtaina consensus sequence.PCR-
positivefaecal samples (in 200 ml buffer) were gradient centrifuged at 3,000–12,000g
and supernatant dilutedat 1:10 in DMEM before beingadded to Vero E6 cells. After
incubation at 37 uC for 1 h, inocula were removed and replaced with fresh DMEM
with 2% FCS. Cells were incubated at 37 uC and checkeddaily for cytopathic effect.
Cell lines from different origins were grown on coverslips in 24-well plates and
inoculated with the novel SL-CoV at a multiplicity of infection of 10. Virus repli-
cation was detected at 24h after infection using rabbit antibodies against the SL-
CoV Rp3 nucleocapsid protein followed by Cy3-conjugated goat anti-rabbit IgG.
Online Content Any additional Methods, ExtendedData display items and Source
Data are available in the online version of the paper; references unique to these
sections appear only in the online paper.
Received 16 May; accepted 18 September 2013.
Published online 30 October 2013.
1. Ksiazek, T. G. et al. A novel coronavirus associated with severe acute respiratory
syndrome. N. Engl. J. Med. 348, 1953–1966 (2003).
2. Zaki, A. M., van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D. & Fouchier, R. A.
Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.
N. Engl. J. Med. 367, 1814–1820 (2012).
3. Anthony, S. J. et al. Coronaviruses in bats from Mexico. J. Gen. Virol. 94, 1028–1038 (2013).
4. Raj, V. S. et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging
human coronavirus-EMC. Nature 495, 251–254 (2013).
5. Li, W. et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 310,
676–679 (2005).
6. Drexler, J. F. et al. Genomic characterization of severe acute respiratory
syndrome-related coronavirus in European bats and classification of
coronaviruses based on partial RNA-dependent RNA polymerase gene
sequences. J. Virol. 84, 11336–11349 (2010).
7. Tong, S. et al. Detection of novel SARS-like and other coronaviruses in bats from
Kenya. Emerg. Infect. Dis. 15, 482–485 (2009).
8. Lau, S. K. P. et al. Severe acute respiratory syndrome coronavirus-like virus in
Chinese horseshoe bats. Proc. Natl Acad. Sci. USA 102, 14040–14045 (2005).
9. Ren, W. et al. Difference in receptor usage between severe acute respiratory
syndrome (SARS) coronavirus andSARS-like coronavirus of batorigin. J. Virol. 82,
1899–1907 (2008).
10. Hon, C. C. et al. Evidence of the recombinant originof a bat severe acute respiratory
syndrome (SARS)-like coronavirus and its implications on the direct ancestor of
SARS coronavirus. J. Virol. 82, 1819–1826 (2008).
11. Li, W. et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS
coronavirus. Nature 426, 450–454 (2003).
12. Wong, S. K., Li, W., Moore,M. J., Choe, H. & Farzan, M.A 193-amino acid fragmentof
the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme
2. J. Biol. Chem. 279, 3197–3201 (2004).
13. Becker, M. M. et al. Synthetic recombinant bat SARS-like coronavirus is
infectious in cultured cells and in mice. Proc. Natl Acad. Sci. USA 105,
19944–19949 (2008).
14. Li, Y. et al. Host range, prevalence, and genetic diversity of adenoviruses in bats.
J. Virol. 84, 3889–3897 (2010).
15. De Souza Luna, L. K. et al. Generic detection of coronaviruses and
differentiation at the prototype strain level by reverse transcription-PCR and
nonfluorescent low-density microarray. J. Clin. Microbiol. 45, 1049–1052 (2007).
16. Cui, J. et al. Evolutionary relationships between bat coronaviruses and their hosts.
Emerg. Infect. Dis. 13, 1526–1532 (2007).
17. Yuan, J. et al. Intraspecies diversity of SARS-like coronaviruses in Rhinolophus
sinicus andits implications for the origin of SARS coronaviruses in humans. J. Gen.
Virol. 91, 1058–1062 (2010).
18. Ren, W. et al. Full-length genome sequences of two SARS-like coronaviruses in
horseshoebats and genetic variationanalysis. J. Gen. Virol.87 , 3355–3359 (2006).
19. Martin, D. P. et al. RDP3: a flexible and fast computer program for analyzing
recombination. Bioinformatics 26, 2462–2463 (2010).
20. Wu, K., Peng, G., Wilken, M., Geraghty, R. J. & Li, F. Mechanisms of host receptor
adaptation by severe acute respiratory syndrome coronavirus. J. Biol. Chem. 287,
8904–8911 (2012).
21. Li, W. et al. Receptor and viral determinants of SARS-coronavirus adaptation to
human ACE2. EMBO J. 24, 1634–1643 (2005).
22. Lau, S. K. et al. Ecoepidemiology and complete genome comparison
of different strains of severe acute respiratory syndrome-related Rhinolophus bat
coronavirusin China reveal bats as a reservoir for acute, self-limitinginfection that
allows recombination events. J. Virol. 84, 2808–2819 (2010).
23. Lau, S. K. et al. Coexistence of different genotypes in the same
bat and serologicalcharacterization of Rousettus bat coronavirusHKU9 belonging
to a novel Betacoronavirus subgroup. J. Virol. 84, 11385–11394 (2010).
24. Song, H. D. et al. Cross-host evolution of severe ac ute respiratory syndrome coronavirus
in palm civet and human. Proc. Natl Acad. Sci. USA 102, 2430–2435 (2005).
25. Memish, Z. A. et al. Middle East respiratory syndrome coronavirus in bats, Saudi
Arabia. Emerg. Infect. Dis. 19, 11 (2013).
26. Chan, J. F. et al. Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-
EMC) the beginning of anotherSARS-like pandemic? J. Infect.65, 477–489 (2012).
27. Ithete, N. L. et al. Close relative of human Middle East respiratory syndrome
coronavirus in bat, South Africa. Emerg. Infect. Dis. 19, 1697–1699 (2013).
28. Morse, S. S. et al. Prediction and prevention of the nextpandemic zoonosis. Lancet
380, 1956–1965 (2012).
29. Hou, Y. et al. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat
species confer variable susceptibility to SARS-CoV entry. Arch. Virol. 155,
1563–1569 (2010).
Acknowledgements We acknowledge financial support from the State Key Program
for Basic Research (2011CB504701 and 2010CB530100), National Natural Science
Foundation of China (81290341 and 31321001), Scientific and technological basis
special project (2013FY113500), CSIRO OCE Science Leaders Award, National
Institute of Allergy and Infectious Diseases (NIAID) award number R01AI079231, a
National Institutes of Health (NIH)/National Science Foundation (NSF) ‘Ecology and
Evolution of Infectious Diseases’ award from the NIH Fogarty International Center
(R01TW005869), an award from the NIH Fogarty International Center supported by
International Influenza Funds from the Office of the Secretary of the Department of
Health and Human Services (R56TW009502), and United States Agency for
International Development (USAID) Emerging Pandemic Threats PREDICT. The
contents are the responsibility of the authorsand do not necessarily reflect the viewsof
NIAID, NIH, NSF, USAID or the United States Government. We thank X. Che from
ZhujiangHospital,Southern MedicalUniversity, forproviding humanSARS patient sera.
Author Contributions Z.-L.S. and P.D. designedand coordinated the study.X.-Y.G., J.-L.
L. and X.-L.Y. conducted majority of experiments and contributed equally to the study.
A.A.C., B.H., W.Z., C.P., Y.-J.Z., C.-M.L., B.T., N.W. and Y.Z. conducted parts of the
experiments and analyses. J.H.E., J.K.M. and S.-Y.Z. coordinated the field study. X.-Y.G.,
J.-L.L., X.-L.Y., B.T. and G.-J.Z. collected the samples. G.C. and L.-F.W. designed and
supervised part of the experiments. All authors contributed to the interpretations and
conclusions presented. Z.-L.S. and X-Y.G. wrote the manuscript with significant
contributions from P.D. and L-F.W. and input from all authors.
Author Information Sequences of three bat SL-CoV genomes, bat SL-CoV RBD and
R. sinicus ACE2 genes have been deposited in GenBank under accession numbers
KC881005–KC881007 (genomes from SL-CoV RsSHC014, Rs3367 and W1V1,
respectively), KC880984–KC881003 (bat SL-CoV RBD genes) and KC881004
(R. sinicus ACE2), respectively. Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version ofthe paper. Correspondence
and requests for materials should be addressed to P.D.
(daszak@ecohealthalliance.org) or Z.-L.S. (zlshi@wh.iov.cn).
A549
RSKT
V
ero E6
PK-15
DAPI Cy3 Merged
Time after
infection (h)
0 12 24 48
0 12 24 48
0 12 24 48
0 12 24 48
22 µm
1 × 10
3
1 × 10
4
1 × 10
5
TCID50 ml–1
1 × 10
3
1 × 10
5
1 × 10
7
1 × 10
9
TCID50 ml–1
1 × 10
3
1 × 10
4
1 × 10
5
TCID50 ml–1
1 × 10
3
1 × 10
4
1 × 10
5
TCID50 ml–1
Figure 4
|
Analysis of host range of SL-CoV-WIV1 determined by
immunofluorescence assay and real-time PCR. Virus infection in A549,
RSKT, Vero E6 and PK-15 cells. Virus replication was detected as described for
Fig. 3. The columns (from left to right) show staining of nuclei (blue), virus
replication (red), merged double-stained images and real-time PCR results,
respectively. n53; error bars represent s.d.
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4 | NATURE | VOL 000 | 00 MONTH 2013
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METHODS
Sampling. Bats were trapped in their natural habitat as described previously
5
.
Throat and faecal swab samples were collected in viral transport medium (VTM)
composed of Hank’s balanced salt solution, pH7.4, containing BSA (1%), ampho-
tericin (15 mgml
21
), penicillin G (100 U ml
21
) and streptomycin (50 mgml
21
). To
collect fresh faecal samples, clean plastic sheets measuring 2.0 by 2.0 m were placed
under knownbat roosting sites at about18:00 h each evening.Relatively fresh faecal
samples werecollected from sheets at approximately05:30–06:00 the next morning
and placed in VTM. Samples were transported to the laboratory and stored at
280 uC until use. All animals trapped for this study were released back to their
habitat after sample collection. All sampling processes were performed by veter-
inarians with approval from Animal Ethics Committee of the Wuhan Institute of
Virology (WIVH05210201) and EcoHealth Alliance under an inter-institutional
agreement with University of California, Davis (UC Davis protocol no. 16048).
RNA extraction, PCR and sequencing. RNA was extracted from 140 mlofswab
or faecal sampleswith a Viral RNA Mini Kit (Qiagen)following the manufacturer’s
instructions. RNA was eluted in 60 ml RNAse-free buffer (buffer AVE, Qiagen),
then aliquoted and stored at 280 uC. One-step RT–PCR (Invitrogen) was used to
detect coronavirus sequences as described previously
15
. First round PCR was con-
ducted in a 25-ml reaction mix containing 12.5 mlPCR23reaction mix buffer,
10 pmol of each primer, 2.5 mM MgSO4, 20 U RNase inhibitor, 1ml SuperScript
III/ Platinum Taq Enzyme Mix and 5 ml RNA. Amplification of the RdRP-gene frag-
ment was performed as follows: 50 uC for 30 min, 94 uC for 2 min, followed by 40
cycles consisting of 94 uC for 15 s, 62 uC for 15 s, 68 uC for 40 s, and a final exten-
sion of 68 uC for 5 min. Second round PCR was conducted in a 25-ml reaction mix
containing 2.5 ml PCR reaction buffer, 5 pmol of each primer, 50mM MgCl
2
,
0.5 mM dNTP, 0.1 ml Platinum Taq Enzyme (Invitrogen) and 1 ml first round
PCR product. The amplification of RdRP-gene fragment was performed as fol-
lows: 94 uC for 5 min followed by 35 cycles consisting of 94 uC for 30 s, 52 uC for
30 s, 72 uC for 40 s, and a final extension of 72 uC for 5 min.
To amplify the RBD region, one-step RT–PCR was performed with primers
designed based on availableSARS-CoV or bat SL-CoVs (first round PCR primers;
F, forward; R, reverse: CoVS931F-59-VWGADGTTGTKAGRTTYCCT-39and
CoVS1909R-59-TAARACAVCCWGCYTGWGT-39; second PCR primers: CoVS
951F-59-TGTKAGRTTYCCTAAYATTAC-39and CoVS1805R-59-ACATCYTG
ATANARAACAGC-39).First-round PCR was conducted in a 25-ml reaction mix
as described above except primers specific for the S gene were used. The ampli-
fication of the RBDregion of the S gene was performed as follows:50 uC for 30 min,
94 uC for 2 min, followed by 35 cycles consisting of 94 uC for 15 s, 43 uC for 15 s,
68 uC for 90 s, and a final extension of 68 uC for 5 min. Second-round PCR was
conducted in a 25-ml reaction mix containing2.5 ml PCR reaction buffer, 5 pmol of
each primer, 50 mM MgCl
2
,0.5mMdNTP,0.1mlPlatinumTaqEnzyme(Invitrogen)
and 1 ml first round PCR product. Amplification was performed as follows: 94 uC
for 5 min followed by 40 cycles consisting of 94 uC for 30 s, 41 uC for 30 s, 72 uC for
60 s, and a final extension of 72 uC for 5 min.
PCR products were gel purified and cloned into pGEM-T Easy Vector (Promega).
At least four independent clones were sequenced to obtain a consensus sequence
for each of the amplified regions.
Sequencing full-length genomes. Degenerate coronavirus primers were designed
based on all available SARS-CoV and bat SL-CoV sequences in GenBank and specific
primers were designedfrom genome sequences generated from previousrounds of
sequencing in this study (primer sequences will be provided upon request). All
PCRs were conducted usingthe One-Step RT–PCR kit (Invitrogen). The 59and 39
genomic ends were determined using the 59or 39RACE kit (Roche), respectively.
PCR products were gel purified and sequenced directly or following cloning into
pGEM-T EasyVector (Promega). At least four independentclones were sequenced
to obtain a consensus sequence for each of the amplified regions and each region
was sequenced at least twice.
Sequence analysis and databank accession numbers. Routine sequence manage-
ment and analysis was carried out using DNAStar or Geneious. Sequence align-
ment and editing was conducted usingClustalW, BioEdit or GeneDoc. Maximum
Likelihood phylogenetic trees based on the protein sequences were constructed
using a Poisson model with bootstrapvalues determined by 1,000 replicates in the
MEGA5 software package.
Sequences obtained in this study have been deposited in GenBank as follows
(accession numbersgiven in parenthesis): full-length genome sequence of SL-CoV
RsSHC014 and Rs3367 (KC881005, KC881006); full-length sequence of WIV1 S
(KC881007); RBD (KC880984-KC881003); ACE2 (KC8810040). SARS-CoV
sequences used in this study: human SARS-CoV strains Tor2 (AY274119), BJ01
(AY278488), GZ02 (AY390556) and civet SARS-CoV strain SZ3 (AY304486). Bat
coronavirus sequencesused in this study: Rs672 (FJ588686), Rp3 (DQ071615), Rf1
(DQ412042), Rm1 (DQ412043), HKU3-1 (DQ022305), BM48-31 (NC_014470),
HKU9-1 (NC_009021), HKU4 (NC_009019),HKU5 (NC_009020), HKU8 (DQ249228),
HKU2 (EF203067), BtCoV512(NC_009657),1A (NC_010437). Othercoronavirus
sequences used in thisstudy: HCoV-229E (AF304460), HCoV-OC43(AY391777),
HCoV-NL63 (AY567487), HKU1 (NC_006577),EMC (JX869059), FIPV (NC_002306),
PRCV (DQ811787), BWCoV (NC_010646), MHV (AY700211), IBV (AY851295).
Amplification, cloning and expression of the bat ACE2 gene. Construction of
expression clones for human and civet ACE2 in pcDNA3.1 has been described
previously
29
. Bat ACE2 was amplified from a R. sinicus (sample no. 3357). In brief,
total RNA was extractedfrom bat rectal tissue using the RNeasy Mini Kit (Qiagen).
First-strandcomplementary DNA wassynthesizedfrom total RNAby reverse trans-
cription with random hexamers. Full-length bat ACE2 fragments were amplified
using forward primerbAF2 and reverse primer bAR2 (ref. 29).The ACE2 gene was
cloned into pCDNA3.1 with KpnI and XhoI, and verified by sequencing. Purified
ACE2 plasmids were transfected to HeLa cells. After 24 h, lysates of HeLa cells
expressing human, civet, or bat ACE2 were confirmed by western blot or immu-
nofluorescence assay.
Western blot analysis. Lysates of cells or filtered supernatants containing pseu-
doviruses were separated by SDS–PAGE, followed by transfer to a nitrocellulose
membrane (Millipore). For detection of S protein, the membrane was incubated
with rabbit anti-Rp3 S fragment (amino acids 561–666) polyantibodies (1:200),
and the bound antibodies were detected by alkaline phosphatase (AP)-conjugated
goat anti-rabbit IgG (1:1,000). For detection of HIV-1 p24 in supernatants,mono-
clonal antibody against HIVp24 (p24 MAb) was used as the primary antibody at a
dilution of 1:1,000, followed by incubation with AP-conjugated goat anti-mouse IgG
at the same dilution. To detect the expression of ACE2 in HeLa cells, goat antibody
againstthe humanACE2 ectodomain (1:500)was used as thefirst antibody, followed
by incubation with horseradish peroxidase-conjugated donkey anti-goat IgG (1:1,000).
Virus isolation. Vero E6 cell monolayers were maintained in DMEM supplemen-
ted with 10% FCS. PCR-positive samples (in 200ml buffer) were gradient centri-
fuged at 3,000–12,000g, and supernatant were diluted 1:10 in DMEM beforebeing
added to Vero E6 cells.After incubation at 37 uC for 1 h, inocula were removedand
replaced with fresh DMEMwith 2% FCS. Cells were incubated at 37 uC for 3 days
and checked daily for cytopathic effect. Double-dose triple antibiotics penicillin/
streptomycin/amphotericin (Gibco) were includedin all tissue culturemedia (peni-
cillin 200 IU ml
21
, streptomycin 0.2 mg ml
21
, amphotericin 0.5 mgml
21
). Three
blind passages were carriedout for each sample. Aftereach passage, both theculture
supernatant and cell pellet were examined for presence of virus by RT–PCR using
primerstargeting the RdRP or S gene.Virions in supernatant (10 ml) were collected
and fixed using 0.1% formaldehyde for 4 h, then concentrated by ultracentrifuga-
tion through a 20%sucrose cushion (5 ml) at 80,000gfor 90 min using a Ty90 rotor
(Beckman). The pelleted viral particles were suspended in 100 ml PBS, stained with
2% phosphotungstic acid (pH 7.0) and examined using a Tecnai transmission
electron microscope (FEI) at 200 kV.
Virus infectivity detected by immunofluorescence assay. Cell lines used for this
study and their cultureconditions are summarized in ExtendedData Table 5. Virus
titre was determined in Vero E6 cells by cytopathic effect (CPE) counts. Cell lines
fromdifferentorigins and HeLacells expressingACE2 from human,civet or Chinese
horseshoe bat were grown on coverslipsin 24-well plates (Corning) incubated with
bat SL-CoV-WIV1 at a multiplicity of infection 510 for 1h. The inoculum was
removed and washed twice with PBS and supplemented with medium. HeLa cells
without ACE2 expression and Vero E6 cells were used as negative and positive
controls, respectively. At 24 h after infection, cells werewashed with PBS and fixed
with 4% formaldehyde in PBS (pH 7.4) for 20 min at 4 uC. ACE2 expression was
detected using goat anti-human ACE2 immunoglobulin (R&D Systems) followed
by FITC-labelled donkey anti-goat immunoglobulin (PTGLab). Virus replication
was detected using rabbit antibody against the SL-CoV Rp3 nucleocapsid protein
followedby Cy3-conjugatedmouse anti-rabbitIgG. Nuclei were stainedwith DAPI.
Staining patterns were examined using a FV1200 confocal microscope (Olympus).
Virus infectivity detected by real-time RT–PCR. Vero E6, A549, PK15, RSKT
and HeLa cells with orwithout expression of ACE2 of different originswere inocu-
lated with 0.1 TCID
50
WIV-1 and incubated for 1 h at 37 uC. After removing the
inoculum, the cells were cultured with medium containing 1% FBS. Supernatants
were collected at 0, 12, 24 and 48 h. RNA from 140 ml of each supernatant was
extracted with the ViralRNA Mini Kit (Qiagen) following manufacturer’sinstruc-
tions and eluted in 60 ml buffer AVE (Qiagen). RNA was quantified on the ABI
StepOne system, with the TaqMan AgPath-ID One-Step RT–PCR Kit (Applied
Biosystems) in a 25 ml reaction mix containing 4mlRNA,13RT–PCR enzyme
mix, 1 3RT–PCR buffer, 40 pmol forward primer (59-GTGGTGGTGACGGCA
AAATG-39), 40pmol reverse primer (59-AAGTGAAGCTTCTGGGCCAG-39)
and 12 pmol probe (59-FAM-AAAGAGCTCAGCCCCAGATG-BHQ1-39). Ampli-
fication parameters were 10 min at 50 uC, 10 min at 95 uC and 50 cycles of 15 s at 95 uC
and 20 s at 60 uC. RNA dilutions from purified WIV-1 stock were used as a standard.
Serum neutralization test. SARS patient sera wereinactivated at 56 uC for 30 min
and then used for virus neutralization testing. Sera were diluted starting with 1:10
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and then serially twofold diluted in 96-well cell plates to 1:40. Each 100ml serum
dilution was mixed with 100 ml viral supernatant containing 100 TCID5
0
of WIV1
and incubated at 37 uC for 1 h. The mixture wasadded in triplicate wells of 96-well
cell plates with plated monolayers of Vero E6 cells and further incubated at 37uC
for 2 days. Serum from a healthy blood donor was used as a negative control in
each experiment. CPE was observed using an inverted microscope 2days after
inoculation. The neutralizing antibody titre was read as the highest dilution of
serum which completely suppressed CPE in infected wells. The neutralization test
was repeated twice.
Recombination analysis. Full-length genomic sequences of SL-CoV Rs3367 or
RsSHC014 were aligned with those of selected SARS-CoVs and bat SL-CoVs using
Clustal X. The aligned sequences were preliminarily scanned for recombination
events using Recombination Detection Program(RDP) 4.0 (ref. 19). The potential
recombination events suggested by RDP owing to their strong Pvalues (,10–20)
were investigated further by similarity plot and bootscan analyses implemented in
Simplot 3.5.1. Phylogenetic origin of the major and minor parental regions of
Rs3367 or RsSHC014 were constructed from the concatenated sequences of the
essential ORFs of the majorand minor parental regions of selected SARS-CoVand
SL-CoVs. Two genome regions between three estimated breakpoints (20,827–
26,553 and 26,554–28,685) were aligned independently using ClustalX and gene-
rated two alignments of 5,727base pairs and 2,133 base pairs. The two alignments
were used to construct maximum likelihood trees to better infer the fragment
parents. All nucleotide numberings in this study are based on Rs3367 genome
position.
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Extended Data Figure 1
|
Sequence alignment of CoV S protein RBD.
SARS-CoV S protein (amino acids 310–520) is aligned with homologous
regions of bat SL-CoVs using ClustalW. The newly discovered bat SL-CoVs are
indicated with a bold vertical line on the left. The key amino acid residues
involved in the interaction with human ACE2 are numbered on the top of the
aligned sequences.
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Extended Data Figure 2
|
Alignment of CoV S protein S1 sequences.
Alignment of S1 sequences (amino acids 1–660) of the two novel bat SL-CoV S
proteins with those of previously reported bat SL-CoVs and human and
civet SARS-CoVs. The newly discovered bat SL-CoVs are boxed in red.
SARS-CoV GZ02, BJ01 and Tor2 were isolated from patients in the early,
middle and late phase, respectively, of the SARS outbreak in 2003. SARS-CoV
SZ3 was identified from P. larvata in 2003 collected in Guangdong, China.
SL-CoV Rp3, Rs 672 and HKU3-1 were identified from R. sinicus collected in
Guangxi, Guizhou and Hong Kong, China, respectively. Rf1 and Rm1 were
identified from R. ferrumequinum and R. macrotis, respectively, collected in
Hubei Province, China. Bat SARS-related CoV BM48-31 was identified from
R. blasii collected in Bulgaria.
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Extended Data Figure 3
|
Complete RdRP sequence phylogeny.
Phylogenetic tree of bat SL-CoVs and SARS-CoVs on the basis of complete
RdRP sequences (2,796 nucleotides). Bat SL-CoVs RsSHC014 and Rs3367 are
highlighted by filled circles. Three established coronaivirus genera,
Alphacoronavirus,Betacoronavirus and Gammacoronavirus are marked as a,b
and c, respectively. Four CoV groups in the genus Betacoronavirus are
indicated as A, B, C and D, respectively. MHV, murine hepatitis virus;
PHEV, porcine haemagglutinating encephalomyelitis virus; PRCV, porcine
respiratory coronavirus; FIPV, feline infectious peritonitis virus; IBV,
infectious bronchitis coronavirus; BW, beluga whale coronavirus.
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Extended Data Figure 4
|
Sequence phylogeny of the complete S protein of
SL-CoVs and SARS-CoV. Phylogenetic tree of bat SL-CoVs and SARS-CoVs
on the basis of complete S protein sequences (1,256 amino acids).
Bat SL-CoVs RsSHC014 and Rs3367 are highlighted by filled circles. Bat CoV
HKU9 was used as an outgroup.
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Extended Data Figure 5
|
Detection of potential recombination events.
a,b, Similarity plot (a) and bootscan analysis (b) detected three recombination
breakpoints in the bat SL-CoV Rs3367 or SHC014 genome. The three
breakpoints were located at the ORF1b (nt 20,827), M (nucleotides 26,553) and
N (nucleotides 28,685) genes, respectively. Both analyses were performed with
an F84 distance model, a window size of 1,500base pairs and a step size of
300 base pairs.
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Extended Data Table 1
|
Summary of sampling detail and CoV prevalence
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Extended Data Table 2
|
Genomic sequence identities of bat SL-CoVs with SARS-CoVs
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Extended Data Table 3
|
Genomic annotation and comparison of bat SL-CoV Rs3367 with human/civet SARS-CoVs and other bat SL-CoVs
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Extended Data Table 4
|
Genomic annotation and comparison of bat SL-CoV RsSHC014 with human/civet SARS-CoVs and other bat SL-CoVs
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Extended Data Table 5
|
Cell lines used for virus isolation and susceptibility tests
*Infectivity was determined by the presence of viral antigen detected by immunofluorescence assay.
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