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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.
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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
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Supplementary resources (9)

... 54 Hyperactivation of showing that bat ACE2s were less efficient overall than the hACE2 in relation to the susceptibility of SARS-CoV entry. 58 This lower efficiency of bat ACE2 (bACE2) compared with hACE2 may also occur with SARS-related CoVs (SARSr-CoVs). As highlighted by Guo et al., the bat R. sinicus carries SARSr-CoVs, and they demonstrated that the SARSr-CoV spike proteins had a higher binding affinity to hACE2 than to bACE2. ...
... On this scale, 37 bat species scored low or very low on the potential to be used as a receptor by SARS-CoV-2. In an earlier study,Hou et al. showed that the diversity of ACE2 among bats was greater than that observed in all known SARS-CoV susceptible mammals and that this genetic diversity stands in contrast to the homogenous ACE2 in humans.58 This diversity of ACE2 among bats is evident in the binding of SARS-CoV-2 to ACE2 within Rhinolophus bats. ...
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Why a systems analysis view of this pandemic? The current pandemic has inflicted almost unimaginable grief, sorrow, loss, and terror at a global scale. One of the great ironies with the COVID-19 pandemic, particularly early on, is counter intuitive. The speed at which specialized basic and clinical sciences described the details of the damage to humans in COVID-19 disease has been impressive. Equally, the development of vaccines in an amazingly short time interval has been extraordinary. However, what has been less well understood has been the fundamental elements that underpin the progression of COVID-19 in an individual and in populations. We have used systems analysis approaches with human physiology and pharmacology to explore the fundamental underpinnings of COVID-19 disease. Pharmacology powerfully captures the thermodynamic characteristics of molecular binding with an exogenous entity such as a virus and its consequences on the living processes well described by human physiology. Thus, we have documented the passage of SARS-CoV-2 from infection of a single cell to species jump, to tropism, variant emergence and widespread population infection. During the course of this review, the recurrent observation was the efficiency and simplicity of one critical function of this virus. The lethality of SARS-CoV-2 is due primarily to its ability to possess and use a variable surface for binding to a specific human target with high affinity. This binding liberates Gibbs free energy (GFE) such that it satisfies the criteria for thermodynamic spontaneity. Its binding is the prelude to human host cellular entry and replication by the appropriation of host cell constituent molecules that have been produced with a prior energy investment by the host cell. It is also a binding that permits viral tropism to lead to high levels of distribution across populations with newly formed virions. This thermodynamic spontaneity is repeated endlessly as infection of a single host cell spreads to bystander cells, to tissues, to humans in close proximity and then to global populations. The principal antagonism of this process comes from SARS-CoV-2 itself, with its relentless changing of its viral surface configuration, associated with the inevitable emergence of variants better configured to resist immune sequestration and importantly with a greater affinity for the host target and higher infectivity. The great value of this physiological and pharmacological perspective is that it reveals the fundamental thermodynamic underpinnings of SARS-CoV-2 infection.
... His February Nadesan 15 20 interview at NaturalNews synthesized elements from the Wuhan and Fort Detrick narratives, but attributed responsibility to an international assemblage of experts, primarily in the U.S., the U.K., Australia, Canada, and China. Boyle provided specific research citations for the gain-of-function engineering he claimed occurred across time (e.g., Hou et al., 2010). His evidence of engineering hinged especially on the unique properties of "furin-like cleavage site" that is identified in scientific research as distinguishing the novel coronavirus from SARS and MERS (e.g., see Coutard et al., 2020) and described U.S. outsourcing research to China due to the national ban. ...
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Experts, news media, and social media commentators struggled to make sense of SARS-CoV-2 January–May 2020 as disease caused by this virus, COVID-19, circulated the globe. This paper represents a longitudinal analysis of the primary narratives produced across expert, media, and social media sources to describe the virus, its phylogenetic origins, and biological effects. High expert uncertainty coupled with amplifying representations of risk across time drove collective sensemaking and conspiratorial narratives.
... However, this does not lead to any conclusion about the safety of the inoculations, since they were manipulated. 10. The mRNA inoculation trials gave the misleading 94-95% efficacy figures. ...
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The world has been on a lockdown since 2020, caused by the release of recombinant coronaviruses from Wuhan, China. This respiratory disease nicknamed COVID-19, along with the improper medical interventions and malpractices, have inflicted more casualties than an atomic bomb. Yet, individuals involved in the gain of function research and funding of the labs and those who were responsible for the release of these deadly viruses, have not been held accountable in any form to prevent a similar disaster. However, unjustly normal people are paying the price, by losing their loved ones, jobs, becoming depressed, morbid, restricted, harassed and the target of questionable medications. Surprisingly, the mandates are not fully based on pure science, but rather to the benefit of a minority. In this article, the less addressed issues that are either neglected or suppressed by media and policy makers, shall be reviewed and it will be revealed how real science revokes the advertised misinformation and disinformation about COVID-19.
... 99 ), which is used to model many interactions 100 . There is naturally high variation among the ACE2 receptors of bat species 105 , in addition to the high diversity of SARSr-CoVs 106 . New host infection and adaptation likely involves mutations in the viral spike protein, and potentially selection in an intermediate (bridge) host, to enable effective binding and use of human ACE2 and facilitate zoonotic spillover 104,107 . ...
Article
In the past two decades, three coronaviruses with ancestral origins in bats have emerged and caused widespread outbreaks in humans, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the first SARS epidemic in 2002–2003, the appreciation of bats as key hosts of zoonotic coronaviruses has advanced rapidly. More than 4,000 coronavirus sequences from 14 bat families have been identified, yet the true diversity of bat coronaviruses is probably much greater. Given that bats are the likely evolutionary source for several human coronaviruses, including strains that cause mild upper respiratory tract disease, their role in historic and future pandemics requires ongoing investigation. We review and integrate information on bat–coronavirus interactions at the molecular, tissue, host and population levels. We identify critical gaps in knowledge of bat coronaviruses, which relate to spillover and pandemic risk, including the pathways to zoonotic spillover, the infection dynamics within bat reservoir hosts, the role of prior adaptation in intermediate hosts for zoonotic transmission and the viral genotypes or traits that predict zoonotic capacity and pandemic potential. Filling these knowledge gaps may help prevent the next pandemic. Bats harbour a multitude of coronaviruses and owing to their diversity and wide distribution are prime reservoir hosts of emerging viruses. Ruiz-Aravena, McKee and colleagues analyse the currently available information on bat coronaviruses and discuss their role in recent and potential future spillovers.
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Pandemics originating from non-human animals highlight the need to understand how natural hosts have evolved in response to emerging human pathogens and which groups may be susceptible to infection and/or potential reservoirs to mitigate public health and conservation concerns. Multiple zoonotic coronaviruses, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV), SARS-CoV-2 and Middle Eastern respiratory syndrome-associated coronavirus (MERS-CoV), are hypothesized to have evolved in bats. We investigate angiotensin-converting enzyme 2 (ACE2), the host protein bound by SARS-CoV and SARS-CoV-2, and dipeptidyl-peptidase 4 (DPP4 or CD26), the host protein bound by MERS-CoV, in the largest bat datasets to date. Both the ACE2 and DPP4 genes are under strong selection pressure in bats, more so than in other mammals, and in residues that contact viruses. Additionally, mammalian groups vary in their similarity to humans in residues that contact SARS-CoV, SARS-CoV-2 and MERS-CoV, and increased similarity to humans in binding residues is broadly predictive of susceptibility to SARS-CoV-2. This work augments our understanding of the relationship between coronaviruses and mammals, particularly bats, provides taxonomically diverse data for studies of how host proteins are bound by coronaviruses and can inform surveillance, conservation and public health efforts.
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Emerging infectious diseases, especially if caused by bat-borne viruses, significantly impact public health and the global economy. There is an urgent need to understand the mechanism of interspecies transmission, particularly to humans. Viral genetics, host factors, including polymorphisms in the receptors, ecological, environmental, and population dynamics are major parameters to consider. Here, we describe the taxonomy, geographic distribution, and unique traits of bats associated with their importance as virus reservoirs. Then, we summarize the origin, intermediate hosts, and the current understanding of interspecies transmission of MERS-CoV, SARS-CoV, SARS-CoV-2, Nipah, Hendra, Ebola, Marburg virus and rotaviruses. Finally, the molecular interactions of viral surface proteins with host cell receptors are examined, and a comparison of these interactions in humans, intermediate hosts, and bats is conducted. This uncovers adaptive mutations in virus spike protein that facilitate cross-species transmission and risk factors associated with the emergence of novel viruses from bats.
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The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to deaths worldwide and decimation of the global economy. Despite its tremendous impact, the origin of SARS-CoV-2 has remained mysterious and controversial. The natural origin theory, although widely accepted, lacks substantial support. The alternative theory that the virus may have come from a research laboratory is, however, censored on peer-reviewed scientific journals. Nonetheless, SARS-CoV-2 shows biological characteristics that are inconsistent with a naturally occurring, zoonotic virus. In this report, the authors describe the genomic, structural, medical, and literature evidence, which, when considered together, strongly contradicts the natural origin theory. The evidence shows that SARS-CoV2 should be a laboratory product created by using bat coronaviruses ZC45 and/or ZXC21 as a template and/or backbone. Building upon the evidence, the authors further postulate a synthetic route for SARS-CoV-2, demonstrating that the lab-creation of this coronavirus is convenient and can be accomplished in approximately six months.
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Better methods to predict and prevent the emergence of zoonotic viruses could support future efforts to reduce the risk of epidemics. We propose a network science framework for understanding and predicting human and animal susceptibility to viral infections. Related approaches have so far helped to identify basic biological rules that govern cross-species transmission and structure the global virome. We highlight ways to make modelling both accurate and actionable, and discuss the barriers that prevent researchers from translating viral ecology into public health policies that could prevent future pandemics. A network science framework for understanding and predicting human and animal susceptibility to viral infections is proposed.
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The exact origin of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) responsible for unleashing the pandemic Corona Virus Disease (Covid-19) is still not established unambiguously. The intermediate and reservoir hosts of SARS-CoV-2 need to be identified with clarity and how the disease exploded into a pandemic, inevitability needs urgent scientific answers to contain and prevent future pandemics and crises. This perspective provides awareness of the peculiar features of SARS-CoV-2 and inspects the gaps in the natural zoonotic origin of the pandemic.
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The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Covid-19 pandemic has caused high morbidity and mortality rates worldwide. Virus entry into cells can be blocked using several strategies, including inhibition of protein-protein interactions (PPIs) between the viral spike glycoprotein and cellular receptors, as well as blocking of spike protein conformational changes that are required for cleavage/activation and fusogenicity. The spike-mediated viral attachment and entry into cells via fusion of the viral envelope with cellular membranes involve PPIs mediated by short peptide fragments exhibiting particular secondary structures. Thus, peptides that can inhibit these PPIs may be used as potential antiviral agents preventing virus entry and spread. This review is focused on peptides and peptidomimetics as PPI modulators and protease inhibitors against SARS-CoV-2. Abstract
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Macromolecular visualization is hampered by the fragmented set of available programs and the lack of cooperativity among them. The amount of visual information required for robust structural analysis is relatively dif®cult to generate and rarely allows further high-quality three-dimensional graphic rendering. Here, a modi®cation of MolScript [Kraulis (1991). J. Appl. Cryst. 24, 946±950] is presented which contains the capability of the original MolScript, the ability to carry out a majority of the options available in most other crystallographic visualization packages, as well as several new features of its own. POVScript+ (currently version 1.62) allows anisotropic displacement ellipsoid rendering (read in as a second-rank tensor from a PDB ®le), electron-density polygonization (in several formats derived from àmarching tetrahedra' approach), volumetric rendering of electron density and GRASP/MSMS surface-map input/output. Finally, POVRay output is supported (via a modi®ed version of PovScript) to generate high-quality renderings that are easily modi®ed for any of a number of purposes (e.g. animations or altered textures). POVScript+ provides a marked increase in the amount of structural and atomic detail possible, while still allowing a straightforward means of generating this information.
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In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses.
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The Chinese rufous horseshoe bat (Rhinolophus sinicus) has been suggested to carry the direct ancestor of severe acute respiratory syndrome (SARS) coronavirus (SCoV), and the diversity of SARS-like CoVs (SLCoV) within this Rhinolophus species is therefore worth investigating. Here, we demonstrate the remarkable diversity of SLCoVs in R. sinicus and identify a strain with the same pattern of phylogenetic incongruence (i.e. an indication of recombination) as reported previously in another SLCoV strain. Moreover, this strain possesses a distinctive 579 nt deletion in the nsp3 region that was also found in a human SCoV from the late-phase epidemic. Phylogenetic analysis of the Orf1 region suggested that the human SCoVs are phylogenetically closer to SLCoVs in R. sinicus than to SLCoVs in other Rhinolophus species. These findings reveal a closer evolutionary linkage between SCoV in humans and SLCoVs in R. sinicus, defining the scope of surveillance to search for the direct ancestor of human SCoVs.
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Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
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CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.
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A worldwide outbreak of severe acute respiratory syndrome (SARS) has been associated with exposures originating from a single ill health care worker from Guangdong Province, China. We conducted studies to identify the etiologic agent of this outbreak. We received clinical specimens from patients in seven countries and tested them, using virus-isolation techniques, electron-microscopical and histologic studies, and molecular and serologic assays, in an attempt to identify a wide range of potential pathogens. None of the previously described respiratory pathogens were consistently identified. However, a novel coronavirus was isolated from patients who met the case definition of SARS. Cytopathological features were noted in Vero E6 cells inoculated with a throat-swab specimen. Electron-microscopical examination revealed ultrastructural features characteristic of coronaviruses. Immunohistochemical and immunofluorescence staining revealed reactivity with group I coronavirus polyclonal antibodies. Consensus coronavirus primers designed to amplify a fragment of the polymerase gene by reverse transcription-polymerase chain reaction (RT-PCR) were used to obtain a sequence that clearly identified the isolate as a unique coronavirus only distantly related to previously sequenced coronaviruses. With specific diagnostic RT-PCR primers we identified several identical nucleotide sequences in 12 patients from several locations, a finding consistent with a point-source outbreak. Indirect fluorescence antibody tests and enzyme-linked immunosorbent assays made with the new isolate have been used to demonstrate a virus-specific serologic response. This virus may never before have circulated in the U.S. population. A novel coronavirus is associated with this outbreak, and the evidence indicates that this virus has an etiologic role in SARS. Because of the death of Dr. Carlo Urbani, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus.
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Using three different assays, we examined 103 serum samples collected from different civet farms and a market in China in June 2003 and January 2004. While civets on farms were largely free from SARS-CoV infection, approximately 80% of the animals from one animal market in Guangzhou contained significant levels of antibody to SARS-CoV, which suggests no widespread infection among civets resident on farms, and the infection of civets in the market might be associated with trading activities under the conditions of overcrowding and mixing of various animal species.
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An outbreak of severe acute respiratory syndrome (SARS) has been reported in Hong Kong. We investigated the viral cause and clinical presentation among 50 patients. We analysed case notes and microbiological findings for 50 patients with SARS, representing more than five separate epidemiologically linked transmission clusters. We defined the clinical presentation and risk factors associated with severe disease and investigated the causal agents by chest radiography and laboratory testing of nasopharyngeal aspirates and sera samples. We compared the laboratory findings with those submitted for microbiological investigation of other diseases from patients whose identity was masked. Patients' age ranged from 23 to 74 years. Fever, chills, myalgia, and cough were the most frequent complaints. When compared with chest radiographic changes, respiratory symptoms and auscultatory findings were disproportionally mild. Patients who were household contacts of other infected people and had older age, lymphopenia, and liver dysfunction were associated with severe disease. A virus belonging to the family Coronaviridae was isolated from two patients. By use of serological and reverse-transcriptase PCR specific for this virus, 45 of 50 patients with SARS, but no controls, had evidence of infection with this virus. A coronavirus was isolated from patients with SARS that might be the primary agent associated with this disease. Serological and molecular tests specific for the virus permitted a definitive laboratory diagnosis to be made and allowed further investigation to define whether other cofactors play a part in disease progression.