We would like to thank the following for assistance in establishing the SARS-CoV pseudotype systems: Ed Wright (Viral Pseudotype Unit, University of Sussex), Nigel Temperton and Simon Scott (Viral Pseudotype Unit, University of Kent), Brian Willett (University of Glasgow Centre for Virus Research), Emma Bentley and Giada Mattiuzzo (National Institute for Biological Standards and Control [NIBSC]) and Michael Letko (National Institute of Allergy and Infectious Diseases). polyethyleneimine; PBS T, Cells were fixed and stained at 48 hpi. Immunoglobulins, also known as antibodies, are membrane-bound or secreted glycoproteins produced by B lymphocytes. The data in each row are normalised to the signal seen for human ACE2 (top), with results representing the mean percentage calculated from 3 separate experiments performed on different days. No, Is the Subject Area "Chickens" applicable to this article? 4. Multiple Sequence Alignment objects¶. We thus hypothesised that SARS-CoV-2 could use the ACE2 receptor to infect a range of nonhuman, non-bat hosts. A vector-only control (pDISPLAY) was added to demonstrate specificity. https://doi.org/10.1371/journal.pbio.3001016.g003. However, concurrent sequence analysis identified a number of variable residues within the RBD, which interact directly with ACE2 (Fig 4A, inset panel). (B) Expression of TMPRSS2 alone does not support SARS-CoV-2 entry pseudotypes. Fixed cells were resuspended in PBS before being analysed using the MACSQuant Analyzer 10 (Miltenyi Biotech), and the percentage of PE-positive cells was calculated by comparison with unstained and stained mock-transfected samples. This can potentially be explained by the terminal hydroxyl group of Y505, which makes a hydrogen bond with ACE2 E37, and by the aromatic region of the tyrosine side chain that forms hydrophobic interactions with the alkyl region of the K353 side chain (Fig 6). This research has identified vertebrate species where cell entry is most efficient, allowing prioritisation of in vivo challenge studies to assess disease susceptibility. coronavirus disease 2019; COOT, In both instances, the corresponding vector controls, pcDNA3.1 and pDISPLAY, were separately transfected for comparison. For the receptors where we had previously seen high levels of cell–cell fusion (hamster, pig, and rabbit), we observed robust viral replication (Fig 3C). 12/12/2018 RDP and Fungene Pipelines are back online now! (A) SARS-CoV-2 pseudotype entry was assayed in BHK-21 transfected cells overexpressing ACE2 from the indicated species. Is the Subject Area "SARS CoV 2" applicable to this article? The issues causing long delays in RDP and Fungene Pipelines in the past week have been resolved. The other substitutions between bat ACE2 proteins and other mammals are likely to be benign. Investigation, The identification, isolation, and sequencing of SARS-CoV-2 progenitors in animal reservoirs or intermediate hosts could provide important information to explain how this virus emerged in human populations. We ran BEAST using sample collection dates with the Hasegawa-Kishino-Yano mutation model, 30 with the strict clock mode. 10/04/2020 RDP Taxonomy Updated Now using RDP taxonomy 18. Indeed, our mutational analysis of SARS-CoV-2 and RaTG13 Spike confirmed that changes within this region have a significant impact on human ACE2 interactions, changes which are implicated in the emergence of SARS-CoV-2 in the human population. The β-coronavirus SARS Coronavirus 2 (SARS-CoV-2) emerged in late 2019, causing a large epidemic of respiratory disease in the Hubei Province of China, centred in the city of Wuhan [1]. Supernatant samples were harvested at 48 hpi for titration by TCID-50. 0. receptor binding domain; RLU, https://doi.org/10.1371/journal.pbio.3001016.s001. Cell lines representing a broad range of animal species were used to determine the host range/tropism of SARS-CoV-2 (S1 Table) (Cell Culture Central Services Unit, The Pirbright Institute, UK). To quantify firefly luciferase, media was replaced with 50 μL Bright-Glo substrate (Promega, USA), diluted 1:2 with serum-free, phenol red-free DMEM, incubated in the dark for 2 min and read on a Glomax Multi+ Detection System (Promega). Except for L79I, which is similarly substituted in pangolin and pig ACE2, all of these substitutions are likely to reduce Spike RBD binding. The mean RLU data from 3 independent experiments is plotted with the human ACE2 value highlighted in red. In particular, differences in receptor usage between closely related host species or viruses provides an opportunity to pinpoint amino acid substitutions at the interaction interface that inhibit Spike protein binding and fusion, and ultimately determine host range. Blots were washed in PBS-T and incubated with anti-mouse secondary antibody conjugated with horseradish peroxidase (Cell Signalling, USA) at 1:1,000 in PBS-T for 1 h at room temperature. Contributed equally to this work with: ACE2, angiotensin-converting enzyme 2; HA, human influenza hemagglutinin tag; hACE2, human ACE2; hAPN, human aminopeptidase N; hDPP4, human dipeptidyl peptidase 4; NE, non-enveloped; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2. https://doi.org/10.1371/journal.pbio.3001016.s002. Please provide at least 2 tree elements, or select a file to upload. Writing – review & editing, Roles This variation was also evident when aligning the 23 ACE2 sequences included in our study, which identified a number of highly variable residues within the overlapping SARS-CoV and SARS-CoV-2 binding sites, including Q24, D30, K31, H34, L79, and G354 (Fig 1D). MEGA is an integrated tool for conducting automatic and manual sequence alignment, inferring phylogenetic trees, mining web-based databases, estimating rates of molecular evolution, and testing evolutionary hypotheses. RNA was first transcribed using SuperScript II Reverse Transcriptase (Thermo Fisher Scientific), with oligo dT primers and 50 ng of input RNA in each reaction. The specificity of the SARS-CoV-2 and SARS-CoV assays were further confirmed by comparing hACE2-mediated fusion to human aminopeptidase N (hAPN) or dipeptidyl peptidase 4 (hDPP4) fusion, the coronavirus group I and MERS-CoV receptors, respectively. For SARS-CoV-2, a bat origin is supported by the 2013 identification of a related coronavirus RaTG13 from Rhinolophus affinis (intermediate horseshoe bat), which is 96% identical at the genome level to SARS-CoV-2 [1]. This appeared to be a mis-annotation in the genome as the 3′ end showed very high identity to the collectrin gene. Codon-optimised SARS-CoV, SARS-CoV-2, and RaTG13 Spike sequences were synthesised and cloned into pcDNA3.1+ (BioBasic) (S3 Table). 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. (G) Flow cytometry was performed to examine surface expression of each ACE2 protein on non-permeabilised cells. (A–D) Pseudotype and cell–cell fusion assays were established for SARS-CoV-2 (A and B) and SARS-CoV (C and D) using multiple internal controls. We also acknowledge the support of Nadine Lewis (BioBasic) for help with gene synthesis as well as The Pirbright Institute’s Flow Cytometry Facility and The Pirbright Institute Cell Servicing Unit. In particular, the F449Y, L486F, Y493Q, D501N, and H505Y mutations to RaTG13 Spike markedly increased human ACE2 receptor usage. https://doi.org/10.1371/journal.pbio.3001016.s006. During the SARS-CoV epidemic, where civets were identified as the intermediate reservoir of infection, a shifting pattern of increasing and decreasing ACE2 usage was observed in individual isolates of SARS-CoV taken from civets and humans (although they shared approximately 99% similarity to each other), providing evidence for adaptation to individual host receptors [13,20] with a particular focus on differential adaptation to human ACE2 residues K31 (T31 in civets) and K353. Live and singlet BHK-21 were gated as PE-positive, relative to mock-transfected cells (top panel). The cell–cell fusion activity of individual point mutants, as well as a full chimaera containing all 8 mutations (chimaera), was then examined using human ACE2 expressing target cells with activity normalised to the fusion seen with the wildtype (WT) viral glycoprotein of (C) SARS-CoV-2 or (D) RaTG13.