Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry

Abstract

The discovery of SARS-like coronavirus in bats suggests that bats could be the natural reservoir of SARS-CoV. However, previous studies indicated the angiotensin-converting enzyme 2 (ACE2) protein, a known SARS-CoV receptor, from a horseshoe bat was unable to act as a functional receptor for SARS-CoV. Here, we extended our previous study to ACE2 molecules from seven additional bat species and tested their interactions with human SARS-CoV spike protein using both HIV-based pseudotype and live SARS-CoV infection assays. The results show that ACE2s of Myotis daubentoni and Rhinolophus sinicus support viral entry mediated by the SARS-CoV S protein, albeit with different efficiency in comparison to that of the human ACE2. Further, the alteration of several key residues either decreased or enhanced bat ACE2 receptor efficiency, as predicted from a structural modeling study of the different bat ACE2 molecules. These data suggest that M. daubentoni and R. sinicus are likely to be susceptible to SARS-CoV and may be candidates as the natural host of the SARS-CoV progenitor viruses. Furthermore, our current study also demonstrates that the genetic diversity of ACE2 among bats is greater than that observed among known SARS-CoV susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of SARS-CoV or its progenitor viruses. This calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of SARS-CoV.

Full text

ORIGINAL ARTICLE
Angiotensin-converting enzyme 2 (ACE2) proteins of different bat
species confer variable susceptibility to SARS-CoV entry
Yuxuan Hou •Cheng Peng •Meng Yu •Yan Li •
Zhenggang Han •Fang Li •Lin-Fa Wang •
Zhengli Shi
Received: 21 April 2010 / Accepted: 12 June 2010 / Published online: 22 June 2010
ÓSpringer-Verlag 2010
Abstract The discovery of SARS-like coronavirus in bats
suggests that bats could be the natural reservoir of SARS-
CoV. However, previous studies indicated the angiotensin-
converting enzyme 2 (ACE2) protein, a known SARS-CoV
receptor, from a horseshoe bat was unable to act as a func-
tional receptor for SARS-CoV. Here, we extended our
previous study to ACE2 molecules from seven additional bat
species and tested their interactions with human SARS-CoV
spike protein using both HIV-based pseudotype and live
SARS-CoV infection assays. The results show that ACE2s
of Myotis daubentoni and Rhinolophus sinicus support viral
entry mediated by the SARS-CoV S protein, albeit with
different efficiency in comparison to that of the human
ACE2. Further, the alteration of several key residues either
decreased or enhanced bat ACE2 receptor efficiency, as
predicted from a structural modeling study of the different
bat ACE2 molecules. These data suggest that M. daubentoni
and R. sinicus are likely to be susceptible to SARS-CoV and
may be candidates as the natural host of the SARS-CoV
progenitor viruses. Furthermore, our current study also
demonstrates that the genetic diversity of ACE2 among bats
is greater than that observed among known SARS-CoV
susceptible mammals, highlighting the possibility that there
are many more uncharacterized bat species that can act as a
reservoir of SARS-CoV or its progenitor viruses. This calls
for continuation and expansion of field surveillance studies
among different bat populations to eventually identify the
true natural reservoir of SARS-CoV.
Introduction
Severe acute respiratory syndrome coronavirus (SARS-
CoV) is the aetiological agent responsible for the SARS
outbreaks during 2002–2003, which had a huge global
impact on public health, travel and the world economy [4,
11]. The host range of SARS-CoV is largely determined by
the specific and high-affinity interactions between a defined
receptor-binding domain (RBD) on the SARS-CoV spike
protein and its host receptor, angiontensin-converting
enzyme 2 (ACE2) [6,7,9]. It has been hypothesized that
SARS-CoV was harbored in its natural reservoir, bats, and
was transmitted directly or indirectly from bats to palm
civets and then to humans [10]. However, although the
genetically related SARS-like coronavirus (SL-CoV) has
been identified in horseshoe bats of the genus Rhinolophus
[5,8,12,18], its spike protein was not able to use the
human ACE2 (hACE2) protein as a receptor [13]. Close
examination of the crystal structure of human SARS-CoV
RBD complexed with hACE2 suggests that truncations in
the receptor-binding motif (RBM) region of SL-CoV spike
Electronic supplementary material The online version of this
article (doi:10.1007/s00705-010-0729-6) contains supplementary
material, which is available to authorized users.
Y. Hou C. Peng Y. Li Z. Han Z. Shi (&)
State Key Laboratory of Virology, Wuhan Institute of Virology,
Chinese Academy of Sciences (CAS), Wuhan 430071, Hubei,
China
e-mail: zlshi@wh.iov.cn
M. Yu L.-F. Wang (&)
Australian Animal Health Laboratory, Commonwealth Scientific
and Industrial Research Organization Livestock Industries,
PO Bag 24, Geelong, VIC 3220, Australia
e-mail: linfa.wang@csiro.au
F. Li
Department of Pharmacology, University of Minnesota Medical
School, Minneapolis, MN 55455, USA
123
Arch Virol (2010) 155:1563–1569
DOI 10.1007/s00705-010-0729-6

protein abolish its hACE2-binding ability [7,10], and
hence the SL-CoV found recently in horseshoe bats is
unlikely to be the direct ancestor of human SARS-CoV.
Also, it has been shown that the human SARS-CoV spike
protein and its closely related civet SARS-CoV spike
protein were not able to use a horseshoe bat (R. pearsoni)
ACE2 as a receptor [13], highlighting a critical missing
link in the bat-to-civet/human transmission chain of SARS-
CoV.
There are at least three plausible scenarios to explain the
origin of SARS-CoV. First, some unknown intermediate
hosts were responsible for the adaptation and transmission
of SARS-CoV from bats to civets or humans. This is the
most popular theory of SARS-CoV transmission at the
present time [10]. Second, there is an SL-CoV with a very
close relationship to the outbreak SARS-CoV strains in a
non-bat animal host that is capable of direct transmission
from reservoir host to human or civet. Third, ACE2 from
yet to be identified bat species may function as an efficient
receptor, and these bats could be the direct reservoir of
human or civet SARS-CoV. Unraveling which scenario is
most likely to have occurred during the 2002–2003 SARS
epidemic is critical for our understanding of the dynamics
of the outbreak and will play a key role in helping us to
prevent future outbreaks. To this end, we have extended
our studies to include ACE2 molecules from different bat
species and examined their interaction with the human
SARS-CoV spike protein. Our results show that there is
great genetic diversity among bat ACE2 molecules, espe-
cially at the key residues known to be important for
interacting with the viral spike protein, and that ACE2s of
Myotis daubentoni and Rhinolophus sinicus from Hubei
province can support viral entry.
Materials and methods
Cell lines and antibodies
HeLa cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine
serum (Gibco, USA). Rabbit polyclonal antibodies against
ACE2 of R. pearsoni (RpACE2) were generated using
R. pearsoni ACE2 protein expressed in Escherichia coli
at the Wuhan Institute of Virology following standard
procedures.
Bat sample collection and identification
Bats were sampled from their natural habitats in Hubei,
Guangxi, Guizhou, Henan and Yunnan provinces in China
as described previously [8]. Bat identification was initially
determined in the field by morphology and later confirmed
in the laboratory by sequencing the mitochondrial cyto-
chrome b gene from samples of blood cells or rectal tissue
as described previously [1].
Bat ACE2 amplification and cloning
Total RNA was extracted from bat rectal tissue using
TRIzol Reagent (Invitrogen, USA) and treating with
RNase-free DNase I at 37°C for 30 min. First-strand cDNA
was synthesized from total RNA by reverse transcription
with random hexamers. Full-length bat ACE2 fragments
were amplified using the forward primer bAF2 (50-CTTGG
TACCATGTCAGGCTCTTYCTGG-30) and the reverse
primer bAR2 (50-CCGCTCGAGCTAAAAB[G/T/C]GA
V[G/A/C]GTCTGAACATCATC-30). The PCR mixture
(25 lL) contained 0.5 lL cDNA, 1.5 mM MgCl
2
and
0.2 lM of each primer, and the fragments were amplified
using the following parameters: 95°C for 5 min, 35 cycles
of 94°C for 30 s, 55°C for 45 s and 68°C for 3 min, with a
final elongation step at 68°C for 10 min. All bat ACE2s
were cloned into pCDNA3.1 with KpnI and XhoI, and this
was verified by sequencing.
Chimeric ACE2 construction
For samples in which full-length ACE2 amplification was
unsuccessful, the N-terminal region (1–1,170 bp) was
amplified using the forward primer bAF2 and the reverse
primer RMR (50-TTAGCTCCATTTCTTAGCAGGTAG
G-30). Chimeric ACE2 was constructed by combining the
N-terminal region of bat ACE2 with the C-terminal portion
of human ACE2 at the unique BamHI site (1,070–
1,075 bp). The chimera was subsequently cloned into
pCDNA3.1 with KpnI and XhoI and sequenced as above.
Construction of bat ACE2 mutants
ACE2 from M. daubentoni was chosen to generate a series
of ACE2 mutants using a QuikChange II Site-Directed
Mutagenesis Kit (Stratagene, USA). The altered amino
acid codon for each mutant is indicated as follows: I27T,
N31K, K35E, and H41Y. Mutants were confirmed by
sequencing.
Sequence analysis
All bat ACE2s were submitted to GenBank (EF569964,
GQ999931–GQ999938). Sequence alignment was per-
formed using ClustalX version 1.83 [15] and corrected
manually. A phylogenetic tree based on amino acid (aa)
sequences was constructed using the neighbor-joining (NJ)
method in MEGA version 4.1. [14].
1564 Y. Hou et al.
123

Analysis of ACE2 expression by western blotting
Lysates of HeLa cells expressing human ACE2 or bat
ACE2 were separated on a 4–10% SDS-PAGE gel, fol-
lowed by transfer to a polyvinylidene difluoride (PVDF)
membrane using a semi-dry protein transfer apparatus
(Bio-Rad, USA). The membrane was probed with a rabbit
polyclonal antibody against the bat ACE2 protein (1:200)
at room temperature for 1 h, followed by incubation with
alkaline-phosphatase-conjugated goat anti-rabbit IgG
(1:1,000) (Chemicon, Australia). The probed proteins were
visualized using NBT and BCIP color development (Pro-
mega, USA).
Pseudotype virus infection assays
An HIV-1-luciferase pseudotype virus carrying the SARS-
CoV BJ01 S protein, HIV/BJ01-S, was prepared as
described previously [13]. HeLa cells were seeded onto
96-well plates for 18 h and then transfected with 0.2 lg
recombinant plasmid containing bat or human ACE2 using
0.5 lL Lipofectamine 2000 (Invitrogen, USA) according to
the manufacturer’s protocol. At 24 h post-transfection,
30 lL medium containing HIV/BJ01-S was added to each
well. At 2–3 h postinfection, unadsorbed viruses were
removed, and fresh medium was added. The infection was
monitored by measuring luciferase activity, expressed from
the reporter gene carried by the pseudovirus, using a
luciferase assay kit (Promega, USA). Cells were lysed at
48 h postinfection by adding 20 lL lysis buffer provided
with the kit, and 10 lL of the resulting lysates were tested
for luciferase activity by the addition of 20 lL luciferase
substrate in a Turner Designs TD-20/20 luminometer. Each
infection experiment was conducted in triplicate, and all
experiments were repeated three times.
Live virus infection assays
Live SARS-CoV infection was carried out under BSL4
conditions at the Australian Animal Health Laboratory
(AAHL) as described previously [16,17]. Briefly, 48 h
after transfection, the time at which expression of the
ACE2 receptor on the HeLa cell surface is optimal,
2910
6
TCID
50
of virus was added to the cells for
infection. The cells were fixed 24 h later by treatment with
100% methanol for 10 min and washed five times with
PBST. The primary antibody, chicken anti-SARS-CoV S
(produced against the recombinant S protein expressed in
E. coli at AAHL), at a 1:500 dilution in 1% BSA/PBS, was
added and incubated with the cells for 1 h at room tem-
perature. An FITC anti-chicken conjugate (Chemicon,
Australia) at 1:1,000 in 1% BSA/PBS was added after
washing the cells five times and incubated with the cells for
1 h. Infection was monitored by immunofluorescent
microscopic analysis.
Results and discussion
Cloning and expression of ACE2 genes from different
bat species
ACE2 genes from seven bat species were amplified and
cloned (Fig. 1, sFig. 1). Full-length genes were obtained
from Rhinolophus ferrumequinum from Hubei province
(Rf-HB), R. macrotis from Hubei province (Rm-HB),
R. pearsoni from Guangxi (Rp-GX), R. pusillus from Hubei
province (Rpu-HB), R. sinicus from Guangxi province
(Rs-GX) and R. sinicus from Hubei province (Rs-HB). For
the following bat species, amplification of the full-length
coding region was not successful, and instead,the N-ter-
minal region was cloned in frame with the C-terminal
region of the human ACE2 gene to form a chimeric full-
length ACE molecule: R. pearsoni from Guizhou province
(Rp-GZ), Myotis daubentonii bat from Yunnan province
(Md-YN) and Hipposideros pratti bat from Henan province
(Hp-HN). The full-length sequences of bat ACE2 are
identical in size to that of hACE2 (805 aa in total).
Sequence comparison showed that bat ACE2s are closely
related to ACE2s of other mammals and have an aa
sequence identity of 80–82% to human and civet ACE2.
The aa identity of ACE2 from different bat families ranges
from 78 to 84%, and within the genus Rhinolophus, the
sequence identity increases to 89–98%. The major
sequence variation among bat ACE2s is located in the
N-terminal region, which has been identified in structural
studies as the SARS-CoV-binding region [6,7]. A phylo-
genetic tree was constructed based on the sequences of bat
ACE2 (sFig. 2) using the MEGA package [14].
All ACE2 genes were cloned into a eukaryotic expres-
sion vector and used to transfect HeLa cells. Western blot
analysis showed that all ACE2s were expressed efficiently
and at very similar levels and were recognized by a rabbit
anti-bat ACE2 antibody with an apparent molecular weight
of approximately 100–130 kDa (Fig. 2c).
Functionality of bat ACE2 as an SARS-CoV entry
receptor
To examine the susceptibility of different bat ACE2 mol-
ecules to SARs-CoV entry, the HIV/BJ01-S pseudovirus
system was used to infect HeLa cells transiently expressing
bat ACE2 or human ACE2 genes. Among the bat ACE2s,
only MdACE2 (MdACE2) and Rs-HB ACE2 demon-
strated significant pseudovirus infection, as deduced from
the significantly higher level of luciferase activity in
Bats as reservoir of SARS-CoV progenitor virus 1565
123

comparison to background activity in the negative control
(Fig. 2a). Although such assays are not to be viewed as an
absolute quantification of receptor activity, it is neverthe-
less worth noting that MdACE2-mediated infection seemed
to be more efficient than with Rs-HB ACE2. In the same
context, it is clear that the bat ACE2s were less efficient
overall than the human ACE2 in this particular assay sys-
tem. The biological significance of this observation
remains to be determined. The functionality of MdACE2
and Rs-HB ACE2 as SARS-CoV entry receptors was fur-
ther confirmed by infection with live virus. As shown in
Fig. 2b, both bat ACE2 proteins could clearly support
SARs-CoV infection. No attempt was made to quantify
infection efficiency in this study due to difficulties
encountered in conducting experiments under BSL4
conditions.
Structural modeling of bat ACE2 molecules
Homologous structural modeling of human SARS-CoV
RBD complexed with MdACE2 supports MdACE2 as a
receptor for human SARS-CoV S protein. The crystal
structure of human SARS-CoV RBD complexed with
hACE2 shows that two salt bridges at the SARS-CoV-
hACE2 interface, between hACE2 Lys31 and Glu35 and
between hACE2 Lys353 and hACE2 Glu38, are both
buried in a hydrophobic environment and contribute criti-
cally to the SARS-CoV-hACE2 interactions (Fig. 3a, c)
[7]. Disturbance of the formation of either of these salt
bridges weakens SARS-CoV-hACE2 binding. The Lys31-
Glu35 salt bridge at the SARS-CoV-hACE2 interface
becomes an Asn31-Lys35 hydrogen bond at the SARS-
CoV-Md-YNACE2 interface (Fig. 3b), which possibly
weakens virus-receptor binding but still is largely com-
patible with the virus-receptor interface. Thr27 on hACE2
supports the Lys31-Gu35 salt bridge through hydrophobic
interactions with Tyr475 (Fig. 3a); Ile27 on MdACE2
supports the Asn31-Lys35 hydrogen bond more efficiently
than Thr27 through tighter hydrophobic interactions with
Tyr475 (Fig. 3b). Moreover, Tyr41 on hACE2 supports the
Lys353-Glu38 salt bridge (Fig. 3c); His41 on MdACE2
supports the same salt bridge less efficiently than Tyr41
(Fig. 3d). Overall, MdACE2 is an efficient receptor for
SARS-CoV, despite the fact that its receptor activity is
lower than that of hACE2.
Compared with MdACE2, Rs-HB ACE2 contains
Glu31 and Glu35, which are not compatible with each
other due to their same negative charges, which disfavor
Fig. 1 Sequence alignment of
SARS-CoV binding regions of
ACE2s from 9 bats, civet and
human. The GenBank accession
numbers of bat, civet and
human ACE2 are as follows:
human (NM021804), civet
(AY881174), Rf-HB
(GQ999931),
Rm-HB (GQ999932), Rs-GX
(GQ999933), Rp-GX
(EF569964), Hp-HN
(GQ999934),
Rp-GZ (GQ999935), Rs-HB
(GQ999936), Md-
YN(GQ999937) and Rpu-HB
(GQ999938). The alignment
was generated using ClustalX
v1.83. In black are single, fully
conserved residues. In gray are
strongly conserved residues. In
light gray are weakly conserved
residues. Asterisks indicate
residues that interact directly
with the receptor-binding
domain of the SARS-CoV S
protein
1566 Y. Hou et al.
123

virus-receptor binding. However, Rs-HB ACE2 also con-
tains Thr27 and Tyr41, both of which support SARS-CoV
entry by contributing favorably to the hydrophobic inter-
actions at the virus-receptor interface. Thus, Rs-HB is a
low-efficiency receptor for SARS-CoV. All of the other bat
ACE2 molecules contain combinations of the aforemen-
tioned key residues that are completely incompatible with
virus–receptor interactions. More specifically, they either
contain same-charged residues at the 31 and 35 positions,
which repel each other, or contain His41 and Lys27, which
Fig. 2 Testing of the ability of bat ACE2 proteins to mediate
pseudovirus HIV/BJ01-S and live SARS- CoV infection. aHeLa cells
transfected with plasmids encoding bat and human ACE2s were
infected with pseudovirus HIV/BJ01-S. Infectivity was determined by
measuring the activity of reporter luciferase as described in ‘‘Mate-
rials and methods’’. HeLa cells transfected with pcDNA3.1 and
human ACE2 were used as the negative and positive controls,
respectively. All tests were performed in triplicate, and the experi-
ments were repeated three times. The error bar represents the
calculated standard deviation. I27T, N31K, K35E, and H41Y are
mutants of MdACE2 that were made using a QuikChange II Site-
Directed Mutagenesis Kit. bSARS-CoV live virus infection using the
ACE2s from bats as described in ‘‘Materials and methods’’. HeLa
cells transfected with pcDNA3.1 and human ACE2 were used as the
negative and positive controls, respectively. cExpression of bat or
human ACE2. Lysates from HeLa cells transfected with plasmid
expressing human or bat ACE2 were analyzed by western blot. Rabbit
anti-bat ACE2 polyclonal antibody (upper panel)orb-actin mono-
clonal antibody (lower panel) was used as the primary antibody. Lane
1vector pcDNA3.1 control; lanes 2–10 bat ACE2 from samples
Rf-HB, Rm-HB, Rpu-HB, Hp-HN, Rp-HB, Rp-GZ, Rs-GX, Rs-HB
and Md-YN; lanes 11–14 Md-YN ACE2 mutant I27T, N31K, K35E
and H41Y; lane 15 human ACE2. The abbreviations of bat species are
given in the main text
Bats as reservoir of SARS-CoV progenitor virus 1567
123

disfavor SARS-CoV binding (Fig. 1). In particular, Lys27
on some of these bat ACE2 molecules is incompatible with
certain hydrophobic residues, such as Leu443 and Phe460,
on SARS-CoV RBD (Fig. 3a, b). Therefore, these bat
ACE2 molecules are not receptors for SARS-CoV.
Site-directed mutagenesis analysis
To confirm the above homologous structural analysis, we
carried out site-directed mutagenesis on MdACE2. Our
results show that mutations E31K, K35E, and I27T all
dramatically decrease the receptor activity of MdACE2,
whereas mutation H41Y greatly increases its receptor
activity (Fig. 2a). Therefore, our mutagenesis data further
confirmed that key residues in ACE2 determine the
receptor activity of MdACE2.
Our finding that M. daubentoni and R. sinicus could
support SARS-CoV infection has important implications in
relation to the origin of SARS-CoV. Since all lines of
investigation have indicated that ACE2-binding affinity is
among the important determinants for SARS-CoV host
range, our data would suggest that M. Daubentonii and
R. sinicus have the potential to serve as the direct reservoirs
for human SARS-CoV or its highly related civet SARS-
CoV. To further investigate the potential of M. Dauben-
tonii and R. sinicus as reservoirs for SARS-CoV, more
efforts will have to be directed toward widening the sur-
veillance of bats in these families and in different geo-
graphical locations.
Another important finding of our current study is the
great genetic diversity of bat ACE2 proteins, which is in
contrast to the genetically homogenous hACE2 [10].
Sequence variations of bat ACE2, especially in positions
that are critical to SARS-CoV binding, such as residues 27,
31, 35, and 41, suggest that, in addition to the Md-YN and
Rs-HB ACE2s, there may be many other bats with an
ACE2 protein that makes them susceptible to SARS-CoV
entry. This again highlights the need for more field sur-
veillance and molecular characterization of different bat
ACE2 proteins until the true reservoir host of SARS-CoV
is identified and its spillover mechanisms and transmission
pathways are fully characterized.
Acknowledgments This work was jointly funded by the State Key
Program for Basic Research Grants (2005CB523004, 2010CB530100)
from the Chinese Ministry of Science, Technology and the Knowledge
Innovation Program Key Project administered by the Chinese Academy
of Sciences (KSCX1-YW-R-07) to Z.S. and the CSIRO CEO Science
Leader Award to L.-F.W. We thank Gary Crameri and Jennifer Barr for
help with live virus infection studies.
Fig. 3 Homologous structural
modeling of SARS-CoV and
Md-YN ACE2 (MdACE2)
interactions. aCritical salt
bridge between hACE2 Lys31
and Glu35 and the hydrophobic
residues surrounding it, based
on the experimentally
determined crystal structure of
SARS-CoV RBD complexed
with hACE2 (PDB 2AJF).
bHomologous structural
modeling of the hydrogen bond
between MdACE2 Asn31 and
Lys35. The modeling was done
in the program O [3]. cCritical
salt bridge between hACE2
Lys353 and Glu38 and the
hydrophobic residues
surrounding it, based on the
structure of SARS-CoV RBD
complexed with hACE2.
dHomologous structural
modeling of the salt bridge
between MdaACE2 Lys353 and
SARS-CoV Glu38 and the
hydrophobic residues
surrounding it. Structural
illustrations were prepared
using the program Povscript [2]
1568 Y. Hou et al.
123

References
1. Cui J, Han N, Streicker D, Li G, Tang X, Shi Z, Hu Z, Zhao G,
Fontanet A, Guan Y, Wang L, Jones G, Field HE, Daszak P,
Zhang S (2007) Evolutionary relationships between bat coro-
naviruses and their hosts. Emerg Infect Dis 13:1526–1532
2. Fenn TD, Ringe D, Petsko GA (2003) POVScript?: a program
for model and data visualization using persistence of vision ray-
tracing. J Appl Crystallogr 36:944–947
3. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved
methods for buildingprotein models in electron density maps and the
location of errors in these models. Acta Crystallogr A 47:110–119
4. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery
S, Tong S, Urbani C, Comer JA, Lim W, Rollin PE, Dowell SF,
Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD,
Rota P, Fields B, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc
JW, Bellini WJ, Anderson LJ (2003) A novel coronavirus asso-
ciated with severe acute respiratory syndrome. N Engl J Med
348:1953–1966
5. Lau SK, Woo PC, Li KS, Huang Y, Tsoi HW, Wong BH, Wong
SS, Leung SY, Chan KH, Yuen KY (2005) Severe acute respi-
ratory syndrome coronavirus-like virus in Chinese horseshoe
bats. Proc Natl Acad Sci USA 102:14040–14045
6. Li F, Li W, Farzan M, Harrison SC (2005) Structure of SARS
coronavirus spike receptor-binding domain complexed with
receptor. Science 309:1864–1868
7. Li F (2008) Structural analysis of major species barriers between
humans and palm civets for severe acute respiratory syndrome
coronavirus infections. J Virol 82:6984–6991
8. Li W, Shi Z, Yu M, Ren W, Smith C, Epstein JH, Wang H,
Crameri G, Hu Z, Zhang H, Zhang J, McEachern J, Field H,
Daszak P, Eaton BT, Zhang S, Wang LF (2005) Bats are natural
reservoirs of SARS-like coronaviruses. Science 310:676–679
9. Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, Luo S, Wong SK,
Huang IC, Xu K, Vasilieva N, Murakami A, He Y, Marasco WA,
Guan Y, Choe H, Farzan M (2005) Receptor and viral determi-
nants of SARS-coronavirus adaptation to human ACE2. EMBO J
24:1634–1643
10. Li W, Wong SK, Li F, Kuhn JH, Huang IC, Choe H, Farzan M
(2006) Animal origins of the severe acute respiratory syndrome
coronavirus: insight from ACE2-S-protein interactions. J Virol
80:4211–4219
11. Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY (2003) Coronavirus
as a possible cause of severe acute respiratory syndrome. Lancet
361:1319–1325
12. Ren W, Li W, Yu M, Hao P, Zhang Y, Zhou P, Zhang S, Zhao G,
Zhong Y, Wang S, Wang LF, Shi Z (2006) Full-length genome
sequences of two SARS-like coronaviruses in horseshoe bats and
genetic variation analysis. J Gen Virol 87:3355–3359
13. Ren W, Qu X, Li W, Han Z, Yu M, Zhou P, Zhang SY, Wang LF,
Deng H, Shi Z (2008) Difference in receptor usage between
severe acute respiratory syndrome (SARS) coronavirus and
SARS-like coronavirus of bat origin. J Virol 82:1899–1907
14. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0.
Mol Biol Evol 24:1596–1599
15. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins
DG (1997) The CLUSTAL_X windows interface: flexible strat-
egies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25:4876–4882
16. Tu C, Crameri G, Kong X, Chen J, Sun Y, Yu M, Xiang H, Xia
X, Liu S, Ren T, Yu Y, Eaton BT, Xuan H, Wang LF (2004)
Antibodies to SARS coronavirus in civets. Emerg Infect Dis
10:2244–2248
17. Yu M, Stevens V, Berry JD, Crameri G, McEachern J, Tu C, Shi
Z, Liang G, Weingartl H, Cardosa J, Eaton BT, Wang LF (2008)
Determination and application of immunodominant regions of
SARS coronavirus spike and nucleocapsid proteins recognized by
sera from different animal species. J Immunol Methods 331:1–12
18. Yuan J, Hon CC, Li Y, Wang D, Xu G, Zhang H, Zhou P, Poon
LL, Lam TT, Leung FC, Shi Z (2010) Intraspecies diversity of
SARS-like coronaviruses in Rhinolophus sinicus and its impli-
cations for the origin of SARS coronaviruses in humans. J Gen
Virol 91:1058–1062
Bats as reservoir of SARS-CoV progenitor virus 1569
123