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      The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins

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          Abstract

          SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, leading to the Coronavirus Disease 2019 (COVID-19) pandemic that continues to cause significant global mortality in human populations. Given its sequence similarity to SARS-CoV, as well as related coronaviruses circulating in bats, SARS-CoV-2 is thought to have originated in Chiroptera species in China. However, whether the virus spread directly to humans or through an intermediate host is currently unclear, as is the potential for this virus to infect companion animals, livestock, and wildlife that could act as viral reservoirs. Using a combination of surrogate entry assays and live virus, we demonstrate that, in addition to human angiotensin-converting enzyme 2 (ACE2), the Spike glycoprotein of SARS-CoV-2 has a broad host tropism for mammalian ACE2 receptors, despite divergence in the amino acids at the Spike receptor binding site on these proteins. Of the 22 different hosts we investigated, ACE2 proteins from dog, cat, and cattle were the most permissive to SARS-CoV-2, while bat and bird ACE2 proteins were the least efficiently used receptors. The absence of a significant tropism for any of the 3 genetically distinct bat ACE2 proteins we examined indicates that SARS-CoV-2 receptor usage likely shifted during zoonotic transmission from bats into people, possibly in an intermediate reservoir. Comparison of SARS-CoV-2 receptor usage to the related coronaviruses SARS-CoV and RaTG13 identified distinct tropisms, with the 2 human viruses being more closely aligned. Finally, using bioinformatics, structural data, and targeted mutagenesis, we identified amino acid residues within the Spike–ACE2 interface, which may have played a pivotal role in the emergence of SARS-CoV-2 in humans. The apparently broad tropism of SARS-CoV-2 at the point of viral entry confirms the potential risk of infection to a wide range of companion animals, livestock, and wildlife.

          Abstract

          A study using a combination of surrogate entry assays and live virus suggests that SARS-CoV-2 may have a broad host-range, revealing that the virus's spike protein can use a broad range of host ACE2 receptors to enter cells and that the sequence of this protein might have changed during the zoonotic jump into humans.

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          A pneumonia outbreak associated with a new coronavirus of probable bat origin

          Since the outbreak of severe acute respiratory syndrome (SARS) 18 years ago, a large number of SARS-related coronaviruses (SARSr-CoVs) have been discovered in their natural reservoir host, bats 1–4 . Previous studies have shown that some bat SARSr-CoVs have the potential to infect humans 5–7 . Here we report the identification and characterization of a new coronavirus (2019-nCoV), which caused an epidemic of acute respiratory syndrome in humans in Wuhan, China. The epidemic, which started on 12 December 2019, had caused 2,794 laboratory-confirmed infections including 80 deaths by 26 January 2020. Full-length genome sequences were obtained from five patients at an early stage of the outbreak. The sequences are almost identical and share 79.6% sequence identity to SARS-CoV. Furthermore, we show that 2019-nCoV is 96% identical at the whole-genome level to a bat coronavirus. Pairwise protein sequence analysis of seven conserved non-structural proteins domains show that this virus belongs to the species of SARSr-CoV. In addition, 2019-nCoV virus isolated from the bronchoalveolar lavage fluid of a critically ill patient could be neutralized by sera from several patients. Notably, we confirmed that 2019-nCoV uses the same cell entry receptor—angiotensin converting enzyme II (ACE2)—as SARS-CoV.
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            SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

            Summary The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
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              MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.

              We present the latest version of the Molecular Evolutionary Genetics Analysis (Mega) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, Mega has been optimized for use on 64-bit computing systems for analyzing larger datasets. Researchers can now explore and analyze tens of thousands of sequences in Mega The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit Mega is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OS X. The command line Mega is available as native applications for Windows, Linux, and Mac OS X. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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                Author and article information

                Contributors
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: InvestigationRole: MethodologyRole: Project administrationRole: Writing – original draftRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: Investigation
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: Conceptualization
                Role: ConceptualizationRole: Writing – review & editing
                Role: Investigation
                Role: Investigation
                Role: ConceptualizationRole: InvestigationRole: MethodologyRole: VisualizationRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Academic Editor
                Journal
                PLoS Biol
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, CA USA )
                1544-9173
                1545-7885
                21 December 2020
                December 2020
                21 December 2020
                : 18
                : 12
                : e3001016
                Affiliations
                [1 ] The Pirbright Institute, Woking, Surrey, United Kingdom
                [2 ] Department of Microbial Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom
                [3 ] Department of Pathology, University of Cambridge, Cambridge, United Kingdom
                University of Wisconsin-Madison, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                https://orcid.org/0000-0002-4450-5911
                https://orcid.org/0000-0003-4735-2065
                https://orcid.org/0000-0002-8307-507X
                https://orcid.org/0000-0003-1869-6176
                https://orcid.org/0000-0002-6509-3354
                https://orcid.org/0000-0002-7437-1723
                https://orcid.org/0000-0002-1395-1775
                https://orcid.org/0000-0003-0120-7196
                https://orcid.org/0000-0002-4100-9269
                https://orcid.org/0000-0002-2213-3248
                https://orcid.org/0000-0002-4012-1283
                https://orcid.org/0000-0002-6300-109X
                https://orcid.org/0000-0003-4547-4034
                https://orcid.org/0000-0002-5640-2266
                Article
                PBIOLOGY-D-20-02408
                10.1371/journal.pbio.3001016
                7751883
                33347434
                fec05da5-db68-4315-9c5a-3c85336db572
                © 2020 Conceicao et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 August 2020
                : 17 November 2020
                Page count
                Figures: 6, Tables: 0, Pages: 27
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100000265, Medical Research Council;
                Award ID: MR/P021735/1
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BB/R019843/1
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BBS/E/I/00007034
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BBS/E/I/00007030
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000268, Biotechnology and Biological Sciences Research Council;
                Award ID: BBS/E/I/00007039
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100006041, Innovate UK;
                Award ID: 971555
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: 098406/Z/12/B
                Award Recipient :
                This work was supported by the following grants to DB: a UK Research and Innovation (UKRI; www.ukri.org) Medical Research Council (MRC) New Investigator Research Grant (MR/P021735/1), a UKRI Biotechnology and Biological Sciences Research Council (BBSRC, www.ukri.org) project grant (BB/R019843/1) and Institute Strategic Programme Grant (ISPG) to The Pirbright Institute (BBS/E/I/00007034, BBS/E/I/00007030 and BBS/E/I/00007039) and an Innovate UK Department for Health and Social Care project (SBRI Vaccines for Global Epidemics – 795 Clinical; Contract 971555 ‘A Nipah vaccine to eliminate porcine reservoirs and safeguard human health’). SCG is a Sir Henry Dale Fellow, jointly funded by the Wellcome Trust and the Royal Society (098406/Z/12/B). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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