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Serological Evidence of Bat SARS-Related Coronavirus Infection in Humans, China

Accepted Manuscript Posted Online
Virologica Sinica. DOI: 10.1007/s12250-017-4124-2
Received: 21 November 2017, Revised: 2 January 2018, Accepted: 8 January 2018
This article is protected by copyright. All rights reserved.
Serological evidence of bat SARS-related coronavirus infection in
humans, China
Running title: SARSr-CoV serological detection in human
Ning Wang1,2, Shi-Yue Li3, Xing-Lou Yang1, Hui-Min Huang3, Yu-Ji Zhang1, Hua Guo1,2, Chu-
Ming Luo1,2, Maureen Miller4, Guangjian Zhu4, Aleksei A. Chmura4, Emily Hagan4, Ji-Hua
Zhou5, Yun-Zhi Zhang5,6, Lin-Fa Wang7, Peter Daszak4, Zheng-Li Shi1
1. CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology,
Chinese Academy of Sciences, Wuhan 430071, China
2. University of Chinese Academy of Sciences, Beijing 100049, China
3. School of Health Sciences, Wuhan University, Wuhan 430071, China
4. EcoHealth Alliance, New York NY10001, USA
5. Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention, Yunnan Institute of
Endemic Diseases Control and Prevention, Dali 671000 China
6. School of Public Health, Dali University, Dali 671000, China
7. Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore 169857,
Zheng-Li Shi
CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese
Academy of Sciences, Wuhan 430071, China
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record.
Please cite this article as: Ning Wang, Shi-Yue Li, Xing-Lou Yang, Hui-Min Huang, Yu-Ji
Zhang, Hua Guo, Chu-Ming Luo, Maureen Miller, Guangjian Zhu, Aleksei A. Chmura, Emily
Hagan, Ji-Hua Zhou, Yun-Zhi Zhang, Lin-Fa Wang, Peter Daszak, Zheng-Li Shi. Serological
evidence of bat SARS-related coronavirus infection in humans, China. Virologica Sinica. DOI:
In our previous works, we have reported genetically diverse SARS-related coronaviruses
(SARSr-CoV) in a single bat cave, Yunnan province, China, and suggested that some SARSr-
CoVs may have high potential to infect humans without the necessity for an intermediate host.
In this report, we developed a specific ELISA based on the nucleocapsid protein of a SARSr-
CoV strain and detected its antibody in humans who are highly exposed to bat populations.
From 218 human serum samples, 6 were positive against the nucleocapsid protein by ELISA
and further confirmed by Western blot. For the first time, we demonstrated the SARSr-CoV had
spillover to humans, although did not cause clinical diseases.
KEYWORDS Bats, Coronavirus, SARS, SARS-related coronavirus, zoonoses, spillover
Dear Editor,
Severe acute respiratory syndrome coronavirus (SARS-CoV) was the causative agent of
the 20022003 SARS pandemic, which resulted in more than 8,000 human infections
worldwide with an approximately 10% fatality rate (Ksiazek et al. 2003; Peiris et al. 2004). The
virus infects both upper airway and alveolar epithelial cells, resulting in mild to severe lung
injury in humans (Peiris et al. 2003).
During investigation into the SARS epidemic, epidemiological evidence of a zoonotic
origin of SARS-CoV was identified (Xu et al. 2004). Isolation of SARS-related coronavirus
(SARSr-CoVs) from masked palm civets and detection of virus infection in humans working
at the wet market suggested that masked palm civets could serve as source of human infection
(Guan et al. 2003). Subsequent work has identified genetically diverse SARSr-CoVs in Chinese
horseshoe bats (Rhinolophus sinicus) in a county of Yunnan Province, China and provided
strong evidence that bats are the natural reservoir of SARS-CoV (Ge et al. 2013; Li et al. 2005;
Yang et al. 2016). Since then, diverse SARS-related coronaviruses (SARSr-CoVs) have been
detected and reported in bats in different regions globally (Hu et al. 2015). Importantly, SARSr-
CoVs which use the SARS-CoV receptor, angiotensin converting enzyme 2 (ACE2) have been
isolated (Ge et al. 2013). These results indicate that some SARSr-CoVs may have high potential
to infect human cells, without the necessity for an intermediate host. However, to date, no
evidence of direct transmission of SARSr-CoVs from bats to people has been reported.
In this study, we performed serological surveillance on residents who live in close
proximity to caves that are roost sites for bats carrying diverse SARSr-CoVs. In October 2015,
we collected serum samples from 218 residents in four villages in Jinning County, Yunnan
province, China (Figure 1A), located 1.16.0 km from two caves (Yanzi and Shitou). We have
been conducting longitudinal molecular surveillance of bats for CoVs in these caves since 2011
and they are inhabited by large numbers of bats including Rhinolophus spp., a major reservoir
of SARSr-CoVs. This region was not involved in the 20022003 SARS outbreaks and none of
the subjects exhibited any evident respiratory illness during sampling. Among those sampled,
139 are female and 79 male, median age of 48 (range 1280). Occupational data were available
for 208 (95.4%) participants: 85.3% farmers and 8.7% students. Most (81.2%) kept or owned
livestock or pet, and the majority (97.2%) had a history of exposure to or contact with livestock
or wild animals. Importantly, 20 (9.1%) participants have witnessed bats flying close to their
houses, and one had handled a bat corpse. As a control, we also collected 240 serum samples
from random blood donors in 2015 in Wuhan, Hubei Province more than 1,000 km away from
Jinning (Figure 1A) and where inhabitants have a much lower likelihood of contact with bats.
None of the donors had prior SARS infection or known contact with SARS patients.
His-tagged nucleocapsid protein (NP) of the following viruses were expressed and
purified in E.coli for this study: SARSr-CoV Rp3; human coronaviruses (HCoVs) HKU1,
OC43, 229E, NL63; Middle East Respiratory Syndrome Coronavirus (MERS-CoV); and Ebola
virus (EBOV). In addition, the receptor binding domain (RBD) of the spike protein (S) was also
produced in mammalian cells from SARS-CoV and bat SARSr-CoVs Rp3, WIV1, and SHC014
(Ge et al. 2013; Yang et al. 2016).
Polyclonal antibodies against each of the six NPs were prepared in rabbits as previously
published (He et al. 2006). Cross-reactivity was evaluated with ELISA and Western blot
(Supplementary Figure S1 S2). No significant cross-reactivity was detected among NPs and
their corresponding antibodies for Rp3, MERS-CoV, NL63, or 229E. Cross-reaction was
detected between OC43 and HKU1 as reported previously (Lehmann et al. 2008).
The Rp3 NP was chosen to develop a SARSr-CoV specific ELISA for serosurveillance.
Micro-titer plates were coated with 100 ng/well of recombinant Rp3 NP and incubated with
human sera in duplicates at a dilution of 1:20, followed by detection with HRP labeled goat
anti-human IgG antibodies (Proteintech, Wuhan, China) at a dilution of 1:20000. The 240
random serum samples collected in Wuhan and two SARS positive samples from Zhujiang
Hospital, Southern Medical University (kindly provided by Prof. Xiaoyan Che) were used to
set a cutoff value. We used the mean OD value of the 240 samples plus three standard deviations
to set the cutoff value at 0.41. A total of 6 positive samples were detected by ELISA (Figure
1B). The specificity of these positive samples was confirmed by Western blot with recombinant
Rp3 NP (Figure 1C) together with NP of NL63, MERS-CoV and EBOV. The degree of
reactivity in Western blot correlated well with the ELISA OD readings, providing further
confidence in the ELISA screening method. None of the sera reacted with NPs of either MERS-
CoV or EBOV. On the other hand, all 10 human sera (9 from Jinning and 1 from Wuhan),
regardless of their Rp3 NP reactivity, reacted strongly with the NL63 NP as expected due to
high prevalence of NL63 infection in humans worldwide (Abdul-Rasool and Fielding 2010).
We conducted a virus neutralization test for the six positive samples for the two SARSr-
CoVs, WIV1 and WIV16 (Ge et al. 2013; Yang et al. 2016). None of them were able to
neutralize either virus. These sera also failed to react in Western blot with any of the
recombinant RBD proteins from SARS-CoV or the three bat SARSr-CoVs (Rp3, WIV1, and
SHC014). We also performed the viral nucleic acid detection in the oral and fecal swab and
blood cells, none of them were positive.
The demography and travel history of the 6 positive individuals (4 male, 2 female) are as
follows. Two males (JN162, 45 yrs old, JN129, 51 yrs old) are from the Dafengkou village; two
males (JN117, 49 yrs old, JN059, 57 yrs old) from the Lvxi village; and two females (JN053,
JN041, both 55 yrs old), from the Tianjing village. In the 12 months prior to the sampling date,
JN041 was the only one who travelled outside of Yunnan, to Shenzhen, a city 1400 km away
from her home village (see Figure 1). JN053 and JN059 had travelled to another county 1.4 km
away from their village. JN162 had travelled to Kunming, the capital of Yunnan, 63 km away.
JN129 and JN117 had never left the village. It is worth to note that all of them have sighted
bats flying in their villages.
Our study provides the first serological evidence of likely human infection by bat SARSr-
CoVs or, potentially, related viruses. The lack of prior exposure to SARS patients by the people
surveyed, their lack of prior travel to areas heavily affected by SARS during the outbreak, and
the rapid decline of detectable antibodies to SARS-CoV in recovered patients within 23 years
after infection strongly suggests that positive serology obtained in this study is not due to prior
infection with SARS-CoV (Wu et al. 2007). The 2.7% seropositivity for the high risk group of
residents living in close proximity to bat colonies suggests that spillover is a relatively rare
event. During questioning, none of the 6 sero-positive subjects could recall any clinical
symptoms in the past 12 months, suggesting that their bat SARSr-CoV infection either occurred
before the time of sampling, or that infections was subclinical or caused only mild symptoms.
Our previous work based on cellular and humanized mouse infection studies suggest that these
viruses are less virulent than SARS-CoV (Ge et al. 2013; Menachery et al. 2016; Yang et al.
2016). Masked palm civets play a significant role as the intermediate host of SARS-CoV in the
20022003 outbreak (Guan et al. 2003). However, considering that these individuals have a
high chance of direct exposure to bat secretion in their villages, this study further support the
notion that some bat SARSr-CoVs are able to directly infect humans without intermediate hosts
as suggested by receptor entry and animal infection studies (Menachery et al. 2016).
The failure of these NP ELISA positive sera to either neutralize live virus or react with
RBD proteins in Western blot could be explained by at least two hypotheses. First, the immune
response to the bat SARSr-CoV S protein may be weaker than that to the NP protein or may
wane more rapidly, especially in subclinical infections, and hence its antibody level is too low
to be detected in our assay systems. Second, other bat SARSr-CoV variants may be circulating
in bats of these villages that have highly divergent S proteins that have not yet been detected in
our previous surveillance studies.
Coronaviruses are known to have a high mutation rate during replication and are prone to
recombination if different viruses infect the same individual (Knipe et al. 2013). From our
previous studies of bat SARSr-CoVs in the two caves near these villages, we have found
genetically highly diverse bat SARSr-CoVs and evidence of frequent coinfection of two or
more different SARSr-CoVs in the same bat (Ge et al. 2013). Our current study suggests that
our surveillance is not exhaustive, as one would have expected, and further more extensive
surveillance in this region is therefore warranted. It might also be prudent to combine
serological surveillance with molecular surveillance of bats in future, despite the technological
This study was jointly funded by the National Natural Science Foundation of China grant
(81290341) to ZLS; the National Institute of Allergy and Infectious Diseases of the National
Institutes of Health (Award Number R01AI110964) to PD and ZLS, United States Agency for
International Development (USAID) Emerging Pandemic Threats PREDICT project grant
(Cooperative Agreement no. AID-OAA-A-14-00102) to PD; and Singapore NRF-CRP grant
(NRF2012NRF-CRP001056) and CD-PHRG grant (CDPHRG/0006/2014) to LFW.
Conflict of Interest The authors declare that they have no conflict of interest.
Animal and Human Rights Statement This study was approved by the Wuhan Institute of
Virology Institutional Review Board (China) and by Hummingbird IRB (USA).
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Figure 1. SARSr-CoV serosurveillance. Map of Xiyang town, Jinning County, Yunnan
Province, China. Shown here is the location of the 4 villages (Tianjing, Dafengkou, Lvxi,
Lvxixin) around 2 bat caves (Yanzi Cave and Shitou Cave) chosen for this study (A). The map
of China is also shown in the inset indicating the location of Wuhan, where the negative control
sera were collected, in relation to Jinning, Shenzhen and the capital Beijing. Serological
reactivity of serum samples with recombinant SARSr-CoV NP protein. (B) ELISA test. The
dotted line represents the cutoff of the test. (C) Western blot analysis. Numbers on the left are
molecular masses in kDa.
Supplementary Figure S1. Two-way cross-reaction ELISA testing between 6 coronavirus NPs
and their corresponding rabbit polyclonal antibodies. The NP proteins (100 ng/well) were
coated in 96-well micro-plate and tested with polyclonal antibody against NPs of SARS-related
CoV Rp3 (PAbRp3), HCoV HKU1 (PAbHKU1), HCoV OC43 (PAbOC43), MERS-CoV (PAbMERS),
HCoV229E (PAb229E) and HCoV NL63 (PAbNL63), respectively. The serum was diluted at
1:16,000 or 1:64,000 (for PAb229E and PAbNL63). HRP labeled goat anti-rabbit IgG (1:20,000)
was used as secondary antibody and detected with TMB substrate. The horizontal line in the
diagram indicates cutoff value determined from negative rabbit sera collected before
Supplementary Figure S2. Two-way cross-reaction Western blotting between 6 coronavirus
NPs and their corresponding rabbit polyclonal antibodies. The NP proteins (100 ng) were run
on 12% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Roche Diagnostics
GmbH, Mannheim, Germany). The membrane was incubated with the different rabbit sera at
different dilutions indicated on the right (in brackets). Goat anti-rabbit IgG conjugated with AP
(Proteintech, Wuhan, China) were used for detection at a dilution of 1:2000. Influenza virus
H5N1 NP was used as negative control. Numbers at the left are molecular masses (in
... Since sarbecoviruses from bats and pangolins can enter human cells via an ACE2dependent mechanism [9], it is possible that these viruses can cross the species barrier and infect human hosts [10]. Numerous studies have provided serological evidence for the spread of bat CoV to human populations, especially in rural communities with frequent wildlife contact [7,11,12]. In addition, bat CoVs closely related to SARS-CoV-2 have recently been identified in rural areas in Southeast Asia, including Thailand and Cambodia [13,14]. ...
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... However, the role of civets in the emergence of SARS is uncertain, and other bat SARSr-CoVs can directly infect human cells, including airway epithelial cells, and thus have potential to spill over directly from bats to humans [14][15][16] . In support of this idea, serological evidence of prior infection with SARSr-CoVs was found in communities living near bat populations in China prior to the emergence of COVID-19, including in people who reported no contact with SARSr-CoV intermediate hosts 17,18 . Direct bat-to-human spillover events may occur more frequently than has been reported, but go unrecognized because they cause mild symptoms, cause symptoms similar to those of other infections, result in small case numbers, or lack sustained chains of human-to-human transmission. ...
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... Realistic pathways were identified for SARS-CoV-2 crossover from the bats in Yunnan and the princeps human cases in Wuhan [44]. Yunnan residents living close to the bat caves have ∼3% positivity rates for SARS-related coronaviruses (SARSr-CoV) [48]. While a considerable geographic gap exists between Yunnan and the locations of the princeps SARS-CoV-2 human cases, pointing to the difficulty in tracking the exact pathway of virus emergence, all current evidence supports SARS-CoV-2 emergence following zoonotic transmission [43]. ...
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... Recent history has seen regular deadly zoonotic coronavirus spillover events with the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 (1), Middle East respiratory syndrome (MERS) coronavirus in 2012 (2) and SARS-CoV-2 in December 2019 (3). SARS related coronaviruses are found in bats throughout Southeast Asia (4) and the serology of people living in proximity to a Rhinolophus spp bat colony suggests these zoonotic infections are not uncommon (5). Since the publication of the SARS-CoV-2 genome in January 2020 (3) there has been rapid development and deployment of vaccines for SARS-CoV-2 (6) and the clinical development of multiple SARS-CoV-2 spike specific neutralizing monoclonal antibodies (mAbs) from convalescent patients or animals, reviewed in (7). ...
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... In 2017, a team found all the genomic components of the SARS virus in two caves (Swallow Cave and Stone Cave) in Xiyang Yi Township, Jinning County, Yunnan Province, China, and pinpointed the source. They also Visited 218 villagers in Xiyang Yi Township, 81.2% raised or owned livestock or pets, and 9.1% witnessed bats flying near houses [14,[18][19][20]. ...
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Materials at nanometric dimensions offer novel abilities with different properties which cannot be observed with the same material in bulk form. Nanoscience typically refers to the study of nanomaterials, their properties, and related phenomena (Mulvaney, 2015). Nanotechnology refers to the moderation, advancement, and application of atomic or molecular structures at the base of one dimension in the nanoscale range (1–1000nm) to produce devices and products. These particles offer varying shapes, morphologies, compositions, dimensions, or surface characteristics (Fig. 1) (Li, Xiao, Chen, & Huang, 2021; Singh, Misra, Mohanty, & Sahoo, 2020). A diverse range of nanosized particles of both biological and abiological origin includes lipid nanoparticles, nanoemulsions, biodegradable polymers, dendrimers, carbon nanoparticles, exosomes, and viral coats for the potential protected delivery of vaccine components (Chintagunta, Krishna, & Nalluru, 2021; Nasrollahzadeh, Sajjadi, Soufi, Iravani, & Varma, 2020). Nanotechnology involved the manipulation of these materials and emerged as a powerful tool in multiple ways to support the fight against various emerging infections (Chintagunta et al., 2021). In the context of the current pandemic setoff by SARS COV-2, population immunization on a large scale is regarded as a foremost priority for the public health concern.
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Viral infections are inevitable, and since ancient times, there are an increasing number of records of such outbreaks affecting a million lives (Bauerfeind, von Graevenitz, Kimmig, et al., 2016). In 1918–20, the deadly viral outbreak (Spanish Flu) in human history affected two-third of the human population with a high mortality rate. In the 21st century, there has been a subsequent viral outbreak, including SARS in 2002 in China, MERS in the Middle East, NiPAH in India (2009), Ebola, and H1N1 (also new variants). The rise in such viral outbreaks affected human lives and posed challenges to the existing health-care system (Morens & Taubenberger, 2018; Tong, 2006). In the last two decades of the 21st century, more than ten viral outbreaks have been reported worldwide. More than 2.5 million deaths in the case of novel SARS-CoV-2 have been reported; however, the pandemic is not over yet. New strains of the novel SARS-CoV-2 are cautiously emerging in different geographical areas with varying infection and fatality rates (Junejo, Ozaslan, Safdar, et al., 2020). The coronavirus is most common viral infection to humans after H1N1 and its novel variants. All these viruses primarily target the lower and upper respiratory tract causing acute respiratory distress syndrome. The novel SARS-CoV-2 is the prime causative agent for the deadly COVID-19. Based on the recurrent viral outbreaks in the last two decades, it has been hypothesized that human-animal interaction is the prime cause for viral transmission from wild animals to humans (Rabaan et al., 2020).
Background: The SARS-CoV-2 rapid diagnostic test antibody (RDT-Ab) was most often used as an early detection tool for COVID-19 at the beginning of pandemic. Whereas the antibody response was formed in the second week after the onset of symptoms.Objective: To evaluate the diagnostic value of the SARS-CoV-2 RDT-Ab, including sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), and negative likelihood ratio (NLR), in patients at Diponegoro National Hospital, Semarang, Indonesia.Methods: Data subjects have been selected retrospectively using purposive sampling based on inclusion criteria (patients who had shortness of breath, pneumonia, suspected, possible, or confirmed COVID-19, and data on the results of the SARS-CoV-2 RDT-Ab IgM and/or IgG (Leccurate® SARS-CoV-2 Antibody Rapid Test Kit) with a valid RT-PCR as gold standard) and exclusion criteria (patients who only had one of either SARS-CoV-2 RDT-Ab or RT-PCR). Researchers analyzed the diagnostic value of SARS-CoV-2 RDT-Ab with RT-PCR which gave the possibility of true-positive, false-positive, true-negative, and false-negative results arranged in a 2x2 table. According to WHO, the diagnostic value is said to be good at least having a sensitivity value of 80% and specificity of 97%.Results: The diagnostic value of SARS-CoV-2 RDT-Ab with RT-PCR, which was evaluated from 1142 patients retrospectively, included IgM (Se 65.25%, Sp 89.51%, PPV 46.70%, NPV 94.81%, PLR 6.22, NLR 0.39), IgG (Se 58.16%, Sp 93.01%, PPV 53.95%, NPV 94.04%, PLR 8.32, NLR 0.45), IgM and IgG (Se 53.90%, Sp 94.21%, PPV 56.72%, NPV 93.55%, PLR 9.30, NLR 0.49), IgM and/or IgG (Se 69.50%, Sp 88.31%, PPV 45.58%, NPV 95.36%, PLR 5.95, NLR 0.35).Conclusion: SARS-CoV-2 RDT-Ab (Leccurate® SARS-CoV-2 Antibody Rapid Test Kit) is not ideal to be used as a rapid diagnostic test for COVID-19.Keywords: COVID-19, Rapid diagnostic test, RT-PCR, SARS-CoV-2 antibody
Epidemic risk assessment and response relies on rapid information sharing. Using examples from the past decade, we discuss the limitations of the present system for outbreak notifications, which suffers from ambiguous obligations, fragile incentives, and an overly narrow focus on human outbreaks. We examine existing international legal frameworks, and provide clarity on what a successful One Health approach to proposed international law reforms—including a pandemic treaty and amendments to the International Health Regulations—would require. In particular, we focus on how a treaty would provide opportunities to simultaneously expand reporting obligations, accelerate the sharing of scientific discoveries, and strengthen existing legal frameworks, all while addressing the most complex issues that global health governance currently faces.
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Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.
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Bats have been recognized as the natural reservoirs of a large variety of viruses. Special attention has been paid to bat coronaviruses as the two emerging coronaviruses which have caused unexpected human disease outbreaks in the 21st century, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), are suggested to be originated from bats. Various species of horseshoe bats in China have been found to harbor genetically diverse SARS-like coronaviruses. Some strains are highly similar to SARS-CoV even in the spike protein and are able to use the same receptor as SARS-CoV for cell entry. On the other hand, diverse coronaviruses phylogenetically related to MERS-CoV have been discovered worldwide in a wide range of bat species, some of which can be classified to the same coronavirus species as MERS-CoV. Coronaviruses genetically related to human coronavirus 229E and NL63 have been detected in bats as well. Moreover, intermediate hosts are believed to play an important role in the transmission and emergence of these coronaviruses from bats to humans. Understanding the bat origin of human coronaviruses is helpful for the prediction and prevention of another pandemic emergence in the future.
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Wereport the isolation and characterization of a novel bat coronavirus which is much closer to the severe acute respiratory syndrome coronavirus (SARS-CoV) in genomic sequence than others previously reported, particularly in its S gene. Cell entry and susceptibility studies indicated that this virus can use ACE2 as a receptor and infect animal and human cell lines. Our results provide further evidence of the bat origin of the SARS-CoV and highlight the likelihood of future bat coronavirus emergence in humans.
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The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.
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Even though coronavirus infection of humans is not normally associated with severe diseases, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome showed that highly pathogenic coronaviruses can enter the human population. Shortly thereafter, in Holland in 2004, another novel human coronavirus (HCoV-NL63) was isolated from a seven-month old infant suffering from respiratory symptoms. This virus has subsequently been identified in various countries, indicating a worldwide distribution. HCoV-NL63 has been shown to infect mainly children and the immunocommpromised, who presented with either mild upper respiratory symptoms (cough, fever and rhinorrhoea) or more serious lower respiratory tract involvement such as bronchiolitis and croup, which was observed mainly in younger children. In fact, HCoV-NL63 is the aetiological agent for up to 10% of all respiratory diseases. This review summarizes recent findings of human coronavirus HCoV-NL63 infections, including isolation and identification, phylogeny and taxonomy, genome structure and transcriptional regulation, transmission and pathogenesis, and detection and diagnosis.
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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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A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome (SARS). SCoV-like viruses were isolated from Himalayan palm civets found in a live-animal market in Guangdong, China. Evidence of virus infection was also detected in other animals (including a raccoon dog, Nyctereutes procyonoides) and in humans working at the same market. All the animal isolates retain a 29-nucleotide sequence that is not found in most human isolates. The detection of SCoV-like viruses in small, live wild mammals in a retail market indicates a route of interspecies transmission, although the natural reservoir is not known.
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An epidemic of severe acute respiratory syndrome (SARS) began in Foshan municipality, Guangdong Province, China, in November 2002. We studied SARS case reports through April 30, 2003, including data from case investigations and a case series analysis of index cases. A total of 1,454 clinically confirmed cases (and 55 deaths) occurred; the epidemic peak was in the first week of February 2003. Healthcare workers accounted for 24% of cases. Clinical signs and symptoms differed between children (<18 years) and older persons (> or =65 years). Several observations support the hypothesis of a wild animal origin for SARS. Cases apparently occurred independently in at least five different municipalities; early case-patients were more likely than later patients to report living near a produce market (odds ratio undefined; lower 95% confidence interval 2.39) but not near a farm; and 9 (39%) of 23 early patients, including 6 who lived or worked in Foshan, were food handlers with probable animal contact.
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.