A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence

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DOI: 10.1038/nm.3985
Cite this publication
Abstract
The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. Here we examine the disease potential of a SARS-like virus, SHC014-CoV, which is currently circulating in Chinese horseshoe bat populations. Using the SARS-CoV reverse genetics system, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse-adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild-type backbone can efficiently use multiple orthologs of the SARS receptor human angiotensin converting enzyme II (ACE2), replicate efficiently in primary human airway cells and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from infection with CoVs using the novel spike protein. On the basis of these findings, we synthetically re-derived an infectious full-length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Our work suggests a potential risk of SARS-CoV re-emergence from viruses currently circulating in bat populations.
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Sapna Devi, Anqi Li, Clare L V Westhorpe, Camden Y Lo, Latasha D Abeynaike, Sarah L Snelgrove, Pam Hall, Joshua D Ooi,
Christopher G Sobey, A Richard Kitching & Michael J Hickey
Nat. Med. 19, 107–112 (2013); published online 16 December 2012; corrected after print 12 August 2015
In the published article, in the Online Methods section, it is stated that the dose of DHE used is 20 mg/kg, when in fact DHE was administered at
2 mg/kg. The error has been corrected in the HTML and PDF versions of the article.
Corrigendum: PAR1 signaling regulates the retention and recruitment of
EPCR-expressing bone marrow hematopoietic stem cells
Shiri Gur-Cohen, Tomer Itkin, Sagarika Chakrabarty, Claudine Graf, Orit Kollet, Aya Ludin, Karin Golan, Alexander Kalinkovich,
Guy Ledergor, Eitan Wong, Elisabeth Niemeyer, Ziv Porat, Ayelet Erez, Irit Sagi, Charles T Esmon, Wolfram Ruf & Tsvee Lapidot
Nat. Med. 21, 1307–1317 (2015); published online 12 October 2015; corrected after print 18 November 2015
In the version of this article initially published, the first author’s name was incorrect. The error has been corrected in the HTML and PDF versions
of the article.
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repair following myocardial infarction
Mortimer Korf-Klingebiel, Marc R Reboll, Stefanie Klede, Torben Brod, Andreas Pich, Felix Polten, L Christian Napp, Johann Bauersachs,
Arnold Ganser, Eva Brinkmann, Ines Reimann, Tibor Kempf, Hans W Niessen, Jacques Mizrahi, Hans-Joachim Schönfeld,
Antonio Iglesias, Maria Bobadilla, Yong Wang & Kai C Wollert
Nat. Med. 21, 140–149 (2015); published online 12 January 2015; corrected after print 19 November 2015
In the version of this article initially published, the article number in reference 13 is incorrectly stated as ‘100ra190’ and should be ‘100ra90’. The
error has been corrected in the HTML and PDF versions of the article.
Corrigendum: A SARS-like cluster of circulating bat coronaviruses shows
potential for human emergence
Vineet D Menachery, Boyd L Yount Jr, Kari Debbink, Sudhakar Agnihothram, Lisa E Gralinski, Jessica A Plante, Rachel L Graham,
Trevor Scobey, Xing-Yi Ge, Eric F Donaldson, Scott H Randell, Antonio Lanzavecchia, Wayne A Marasco, Zhengli-Li Shi & Ralph S Baric
Nat. Med.; doi:10.1038/nm.3985; corrected 20 November 2015
In the version of this article initially published online, the authors omitted to acknowledge a funding source, USAID-EPT-PREDICT funding from
EcoHealth Alliance, to Z.-L.S. The error has been corrected for the print, PDF and HTML versions of this article.
Corrigendum: Long-term glycemic control using polymer-encapsulated
human stem cell–derived beta cells in immune-competent mice
Arturo J Vegas, Omid Veiseh, Mads Gürtler, Jeffrey R Millman, Felicia W Pagliuca, Andrew R Bader, Joshua C Doloff, Jie Li,
Michael Chen, Karsten Olejnik, Hok Hei Tam, Siddharth Jhunjhunwala, Erin Langan, Stephanie Aresta-Dasilva, Srujan Gandham,
James J McGarrigle, Matthew A Bochenek, Jennifer Hollister-Lock, Jose Oberholzer, Dale L Greiner, Gordon C Weir, Douglas A Melton,
Robert Langer & Daniel G Anderson
Nat. Med.; doi:10.1038/nm.4030; corrected online 18 February 2016
In the version of this article initially published online, the authors omitted acknowledgment recognizing the histology core of the Harvard Stem
Cell Institute and several individuals for their assistance. The error has been corrected for the print, PDF and HTML versions of this article.
446 VOLUME 22 | NUMBER 4 | APRIL 2016 NATURE MEDICINE
CORRIGENDA
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  • ... These discoveries, however, typically depend on the detection of viral genome or genome fragments using molecular approaches. Except for three strains isolated using interferon (IFN)-deficient Vero E6 or Vero cells 13,14 and one strain of genetically synthesized virus 15 , to date, most identified bat SARSr-CoVs have not been successfully isolated and cultivated. Despite many reports of bat primary cell cultures and immortalized bat cell lines 16 , as well as the commercially available bat cell lines in ATCC, isolation of SARSr-CoV using a bat cell line has never been documented. ...
    ... However, no direct evidence exists of SARSr-CoV infection in these bats, which might be owing to the nature of acute and self-limiting infection, seasonal fluctuation of virus growth in these bats 18 and the difficulty in accessing these animals in the wild. Thus, the absence of a readily accessible in vitro model that can faithfully represent native bat cells is a major obstacle to isolate and study bat viruses 16 , including bat SARSr-CoVs, which are predicted to possess high zoonotic potential 15 . ...
  • ... One shortcoming of these approaches is the inherent bias toward known viruses, specifically highly conserved genomic regions, which can be circumvented with HTS. Progenitor coronavirus strains related to SARS-CoV or SARS-CoV-2 may exist in a cohort of diverse viral variants in reservoirs [24], and this depth of information can be discovered via an unbiased approach like HTS. Genetic diversity of viruses within a single host reservoir may permit frequent transmission to incidental hosts, which becomes problematic when a genetic variant capable of infecting the new host population spills over [44]. ...
    ... Coronaviruses may emerge following random mutations permissive to infection of intermediate amplifying hosts, and/or recombination events that result in a large pool of variants that could infect humans, potentially without an intermediate host [24]. Due to the knowledge gap, with regard to the circulation of recombinant viruses, an understanding of the prevalence of unique, recombinant viruses will provide an advantage to predict the innate features of a virus with greater propensity for spillover. ...
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    Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.
  • ... These discoveries, however, typically depend on the detection of viral genome or genome fragments using molecular approaches. Except for three strains isolated using interferon (IFN)-deficient Vero E6 or Vero cells 13,14 and one strain of genetically synthesized virus 15 , to date, most identified bat SARSr-CoVs have not been successfully isolated and cultivated. Despite many reports of bat primary cell cultures and immortalized bat cell lines 16 , as well as the commercially available bat cell lines in ATCC, isolation of SARSr-CoV using a bat cell line has never been documented. ...
    ... However, no direct evidence exists of SARSr-CoV infection in these bats, which might be owing to the nature of acute and self-limiting infection, seasonal fluctuation of virus growth in these bats 18 and the difficulty in accessing these animals in the wild. Thus, the absence of a readily accessible in vitro model that can faithfully represent native bat cells is a major obstacle to isolate and study bat viruses 16 , including bat SARSr-CoVs, which are predicted to possess high zoonotic potential 15 . ...
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    A novel coronavirus—severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—emerged in humans in Wuhan, China, in December 2019 and has since disseminated globally1,2. As of April 16, 2020, the confirmed case count of coronavirus disease 2019 (COVID-19) had surpassed 2 million. Based on full-genome sequence analysis, SARS-CoV-2 shows high homology to SARS-related coronaviruses identified in horseshoe bats1,2. Here we show the establishment and characterization of expandable intestinal organoids derived from horseshoe bats of the Rhinolophus sinicus species that can recapitulate bat intestinal epithelium. These bat enteroids are fully susceptible to SARS-CoV-2 infection and sustain robust viral replication. Development of gastrointestinal symptoms in some patients with COVID-19 and detection of viral RNA in fecal specimens suggest that SARS-CoV-2 might cause enteric, in addition to respiratory, infection3,4. Here we demonstrate active replication of SARS-CoV-2 in human intestinal organoids and isolation of infectious virus from the stool specimen of a patient with diarrheal COVID-19. Collectively, we established the first expandable organoid culture system of bat intestinal epithelium and present evidence that SARS-CoV-2 can infect bat intestinal cells. The robust SARS-CoV-2 replication in human intestinal organoids suggests that the human intestinal tract might be a transmission route of SARS-CoV-2. Bat and human intestinal organoids can support replication of SARS-CoV-2, enabling further characterization of the virus lifecycle and investigation of potential mechanisms of enteric infection in COVID-19.
  • ... The receptor interaction site on S1 is considered the main target for efficient neutralization of cell infection and therefore a prime candidate for therapeutic antibody development [8][9][10][11][12] . Although the S protein of the SARS-CoV and SARS-CoV-2 share 77.5% identity, most of the mAbs isolated against SARS-CoV reportedly failed to cross-neutralize the novel virus [13][14][15] . ...
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    The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug, specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD, therefore they represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.
  • ... The cause was quickly identified as a novel CoV, which was eventually named SARS-CoV-2. Investigations of the epidemiological, clinical, laboratory and radiological characteristics, treatment, and outcomes of patients infected by SARS-CoV-2 demonstrated that the infection caused clusters of severe respiratory illness similar to SARS-CoV 6 . Like SARS-CoV, SARS-CoV-2 enters target cells through an endosomal pathway and also uses the same cell entry receptor, angiotensin-converting enzyme II (ACE2) 7 8 . ...
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    div> One of the most important SARS-CoV-2 protein targets for therapeutics is the 3C-like protease (main protease, Mpro). In our previous work1 we used the first Mpro crystal structure to become available, 6LU7. On February 4, 2020 Insilico Medicine released the first potential novel protease inhibitors designed using a de novo, AI-driven generative chemistry approach. Nearly 100 X-ray structures of Mpro co-crystallized both with covalent and non-covalent ligands have been published since then. Here we utilize the recently published 6W63 crystal structure of Mpro complexed with a non-covalent inhibitor and combined two approaches used in our previous study: ligand-based and crystal structure-based. We published 10 representative structures for potential development with 3D representation in PDB format and welcome medicinal chemists for broad discussion and generated output analysis. The molecules in SDF format and PDB-models for generated protein-ligand complexes are available here and at https://insilico.com/ncov-sprint/. Medicinal chemistry VR analysis was provided by Nanome team and the video of VR session is available at https://bit.ly/ncov-vr. </div
  • ... genes are closer to SARS-CoVs [60,62]. In a recent study based on available genomic sequences, 10 it was observed that SARS-CoV-2 (106 sequences) genome has much lower mutation rate and 11 genetic diversity than SARS-CoV (39 sequences), and in particular the spike protein-coding gene 12 is found relatively more conserved than other protein-encoding genes [63]. ...
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    SARS-CoV-2 is a newly emerging, highly transmissible, and pathogenic coronavirus in humans, which has caused global public health emergency and economic crisis. To date, millions of infections and thousands of deaths have been reported worldwide, and the numbers continue to rise. Currently, there is no specific drug or vaccine against this deadly virus; therefore, there is a pressing need to understand the mechanism through which this deadly virus enters the host cell. Viral entry into the host cell is a multistep process in which SARS-CoV-2 utilizes the receptor binding domain of the spike glycoprotein (S) to recognize ACE2 receptors on the human cells; this initiates the host cell entry by promoting the viral-host cell membrane fusion through large scale conformational changes in the S protein. Receptor recognition and fusion are critical and essential steps of viral infections and are key determinants of the viral host range and cross-species transmission. In this review, we summarize the current knowledge on the origin and evolution of SARS-CoV-2, roles of key viral factors and discuss the receptor recognition mechanisms of coronaviruses. We provide a comparative analysis of the SARS-CoV and SARS-CoV-2 S proteins, receptor-binding specificity, and discuss the differences in their antigenicity based on biophysical and structural characteristics. Finally, we dive into available medications, and the current COVID-19 treatment options, which will be beneficial for the scientific community as well as for the general public.
  • ... SARS-CoV-2 belongs to the β-coronavirus family and the associated severe acute respiratory syndrome (SARS), like the previous SARS-CoV and Middle East respiratory syndrome (MERS-CoV), it may cause life-threatening diseases [1][2][3][4][5][6]. Data from past epidemiological studies match with emerging observations, indicating a large, sex-dependent gap in disease infection and outcomes for SARS-CoV-2 disease (COVID- 19) as for previous SARS [7,8]. ...
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    In December 2019, a novel severe acute respiratory syndrome (SARS) from a new coronavirus (SARS-CoV-2) was recognized in the city of Wuhan, China. Rapidly, it became an epidemic in China and has now spread throughout the world reaching pandemic proportions. High mortality rates characterize SARS-CoV-2 disease (COVID-19), which mainly affects the elderly, causing unrestrained cytokines-storm and subsequent pulmonary shutdown, also suspected micro thromboembolism events. At the present time, no specific and dedicated treatments, nor approved vaccines, are available, though very promising data come from the use of anti-inflammatory, anti-malaria, and anti-coagulant drugs. In addition, it seems that males are more susceptible to SARS-CoV-2 than females, with males 65% more likely to die from the infection than females. Data from the World Health Organization (WHO) and Chinese scientists show that of all cases about 1.7% of women who contract the virus will die compared with 2.8% of men, and data from Hong Kong hospitals state that 32% of male and 15% of female COVID-19 patients required intensive care or died. On the other hand, the long-term fallout of coronavirus may be worse for women than for men due to social and psychosocial reasons. Regardless of sex- or gender-biased data obtained from WHO and those gathered from sometimes controversial scientific journals, some central points should be considered. Firstly, SARS-CoV-2 has a strong interaction with the human ACE2 receptor, which plays an essential role in cell entry together with transmembrane serine protease 2 (TMPRSS2); it is interesting to note that the ACE2 gene lays on the X-chromosome, thus allowing females to be potentially heterozygous and differently assorted compared to men who are definitely hemizygous. Secondly, the higher ACE2 expression rate in females, though controversial, might ascribe them the worst prognosis, in contrast with worldwide epidemiological data. Finally, several genes involved in inflammation are located on the X-chromosome, which also contains high number of immune-related genes responsible for innate and adaptive immune responses to infection. Other genes, out from the RAS-pathway, might directly or indirectly impact on the ACE1/ACE2 balance by influencing its main actors (e.g., ABO locus, SRY, SOX3, ADAM17). Unexpectedly, the higher levels of ACE2 or ACE1/ACE2 rebalancing might improve the outcome of COVID-19 in both sexes by reducing inflammation, thrombosis, and death. Moreover, X-heterozygous females might also activate a mosaic advantage and show more pronounced sex-related differences resulting in a sex dimorphism, further favoring them in counteracting the progression of the SARS-CoV-2 infection.
  • ... Among these proteins, the S glycoprotein plays crucial roles in viral entry and pathogenesis as its widely exposed structure forms large petal-shaped spikes on the surface of the virion [7]. Mutations in the spike glycoprotein can allow novel coronavirus strains to infect humans and spread pandemically [8]. Therefore, S gene encoding S glycoprotein has widely Vaccines 2020, 8, 220 2 of 8 been used for molecular analysis of coronaviruses due to the significant features of the S glycoprotein affecting the antigenicity and immunogenicity [2,4,[9][10][11][12][13]. ...
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    Vaccine development against SARS‐CoV‐2 has drawn attention around the globe due to the exploding pandemic. Although COVID‐19 is caused by a new coronavirus, SARS‐CoV‐2, previous research on other coronavirus vaccines, such as FIPV, SARS and MERS, has provided valuable information for the rapid development of COVID‐19 vaccine. However, important knowledge gaps remain – some are specific to SARS‐CoV‐2, others are fundamental to immunology and vaccinology. Here we discuss areas that need to be addressed for COVID‐19 vaccine development, and what can be learned from examples of vaccine development in the past. Since the beginning of the outbreak, the research progress on COVID‐19 has been remarkable. We are therefore optimistic about the rapid development of COVID‐19 vaccine. This article is protected by copyright. All rights reserved
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    Unlabelled: The receptor binding domain (RBD) of the spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) is a major target of protective immunity in vivo. Although a large number of neutralizing antibodies (nAbs) have been developed, it remains unclear if a single RBD-targeting nAb or two in combination can prevent neutralization escape and, if not, attenuate viral virulence in vivo. In this study, we used a large panel of human nAbs against an epitope that overlaps the interface between the RBD and its receptor, angiotensin-converting enzyme 2 (ACE2), to assess their cross-neutralization activities against a panel of human and zoonotic SARS-CoVs and neutralization escape mutants. We also investigated the neutralization escape profiles of these nAbs and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral strains, including neutralization escape viruses derived from other nAbs; however, no single nAb or combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani background) or increased virulence (GD03 background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single nAbs or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint. Importance: The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has resulted in severe human respiratory disease with high death rates. Their zoonotic origins highlight the likelihood of reemergence or further evolution into novel human coronavirus pathogens. Broadly neutralizing antibodies (nAbs) that prevent infection of related viruses represent an important immunostrategy for combating coronavirus infections; however, for this strategy to succeed, it is essential to uncover nAb-mediated escape pathways and to pioneer strategies that prevent escape. Here, we used SARS-CoV as a research model and examined the escape pathways of broad nAbs that target the receptor binding domain (RBD) of the virus. We found that neither single nAbs nor two nAbs in combination blocked escape. Our results suggest that targeting conserved regions with less plasticity and more structural constraint rather than the SARS-CoV RBD-like region(s) should have broader utility for antibody-based immunotherapy.
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    Unlabelled: Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats in China, Europe, and Africa, most have a genetic organization significantly distinct from human/civet SARS CoVs in the receptor-binding domain (RBD), which mediates receptor binding and determines the host spectrum, resulting in their failure to cause human infections and making them unlikely progenitors of human/civet SARS CoVs. Here, a viral metagenomic analysis of 268 bat rectal swabs collected from four counties in Yunnan Province has identified hundreds of sequences relating to alpha- and betacoronaviruses. Phylogenetic analysis based on a conserved region of the RNA-dependent RNA polymerase gene revealed that alphacoronaviruses had diversities with some obvious differences from those reported previously. Full genomic analysis of a new SARS-like CoV from Baoshan (LYRa11) showed that it was 29,805 nucleotides (nt) in length with 13 open reading frames (ORFs), sharing 91% nucleotide identity with human/civet SARS CoVs and the most recently reported SARS-like CoV Rs3367, while sharing 89% with other bat SARS-like CoVs. Notably, it showed the highest sequence identity with the S gene of SARS CoVs and Rs3367, especially in the RBD region. Antigenic analysis showed that the S1 domain of LYRa11 could be efficiently recognized by SARS-convalescent human serum, indicating that LYRa11 is a novel virus antigenically close to SARS CoV. Recombination analyses indicate that LYRa11 is likely a recombinant descended from parental lineages that had evolved into a number of bat SARS-like CoVs. Importance: Although many severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been discovered in bats worldwide, there are significant different genic structures, particularly in the S1 domain, which are responsible for host tropism determination, between bat SARS-like CoVs and human SARS CoVs, indicating that most reported bat SARS-like CoVs are not the progenitors of human SARS CoV. We have identified diverse alphacoronaviruses and a close relative (LYRa11) to SARS CoV in bats collected in Yunnan, China. Further analysis showed that alpha- and betacoronaviruses have different circulation and transmission dynamics in bat populations. Notably, full genomic sequencing and antigenic study demonstrated that LYRa11 is phylogenetically and antigenically closely related to SARS CoV. Recombination analyses indicate that LYRa11 is a recombinant from certain bat SARS-like CoVs circulating in Yunnan Province.
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    Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis.
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    Two novel coronaviruses have emerged in humans in the twenty-first century: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), both of which cause acute respiratory distress syndrome (ARDS) and are associated with high mortality rates. There are no clinically approved vaccines or antiviral drugs available for either of these infections; thus, the development of effective therapeutic and preventive strategies that can be readily applied to new emergent strains is a research priority. In this Review, we describe the emergence and identification of novel human coronaviruses over the past 10 years, discuss their key biological features, including tropism and receptor use, and summarize approaches for developing broadly effective vaccines.
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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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    The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. Using bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks using human lung epithelial cells infected with either wild type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression with approximately twice as many upregulated genes in the mutant virus samples by 48 hours post-infection, despite identical viral titers. Our data demonstrate that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4), which are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes, but revealed candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo.