Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) BHK-21 cells were transfected with HA-tagged bat ACE2-expressing constructs and baited with His tagged-purified RBD proteins for bat sarbecoviruses. Cells were stained with anti-HA PE conjugated antibody (ACE2) and anti-His APC conjugated antibody (RBD). Live and singlet BHK-21 cells were gated as PE+ APC+ (ACE2+RBD+), relative to mock-transfected cells (pDISPLAY). (B) The SE% Dymax metric was used to calculate the percentage of ACE2+RBD+ cells, shown for all RBDs tested with our library of bat ACE2 and human ACE2. Representative datasets are shown for mock-transfected cells and raccoon dog ACE2 with SARS-CoV-2 RBD, with their respective SE Dymax % positive value noted. (C) Heatmap showing the binding of purified bat sarbecovirus proteins to ACE2s of human, M. lucifugus Rh. cornutus by ELISA (pink, EC 50 µg/ml) or flow cytometry (blue, % ACE2 + RBD + cells). Data is representative of a mean of 3 separate experiments. A cross in a box indicates this data was not obtained.

Article Snippet: BHK-21 cells were seeded in 6-well plates at 7.5 × 10 5 /well in DMEM-10% 1 day prior to transfection with 500 ng of different species, ACE2-expressing constructs or empty vector (pDISPLAY) ( S2 Table ) in OptiMEM (ThermoFisher Scientific) and TransIT-X2 (Mirus Bio) transfection reagent, according to the manufacturer’s recommendations.

Techniques: Transfection, Expressing, Construct, Purification, Staining, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Evolutionary history of bat sarbecovirus RBDs (NCBI reference shown for 110 amino acid sequences) was inferred by using the Maximum Likelihood method and Whelan and Goldman model. Initial tree(s) for the heuristic search were obtained automatically by Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances (225 total positions) estimated using the JTT model with discrete Gamma distribution (5 categories (+G, parameter = 0.3460)), and then selecting the topology with superior log likelihood value. Evolutionary analyses were conducted in MEGA 11. Of note, the HKU3-7 and HKU3-8, and the HKU3-1, 2, 3, 4, 5, 6, 7, 9, 19, 11 and 12 Spike sequences are identical, so the HKU3-8 and HKU3-1 sequences were used as representatives, respectively. Bat sarbecovirus RBDs cluster into clades: clade Ia (yellow), clade Ib (orange), clade II (blue), clade III (green), clade IV (purple) and clade V (red). Viruses selected for analysis in this study are highlighted in red. (B) World map highlighting the geographic location where sarbecoviruses investigated in this study were first isolated (coloured in blue), along with the distribution of the bat species these were isolated from ( https://www.iucnredlist.org/ ). (C) Structural images showing the binding of Rhinolophus affinis ACE2 with SARS-CoV-2 RBD (PDB 7XA7). Offset images highlight the conserved amino acid residues across the different bat ACE2s relative to Rh. affinis ACE2, and the conservation of bat sarbecovirus RBDs compared to SARS-CoV-2, screened in this study. The ACE2-RBD interacting residues are denoted. (D) Maximum likelihood phylogeny of the bat sarbecoviruses full length Spike and bat ACE2 amino acid sequences are shown. Bat sarbecovirus Spikes were used to generate lentiviral-based pseudotypes and used to infect BHK-21 cells overexpressing different bat ACE2s or human ACE2. A heatmap illustrating receptor usage is shown, representing the mean log RLU of 3-5 separate experiments. A vector only control (pDISPLAY) was included to demonstrate specificity and to set the background signal. The cognate bat receptor for each virus, where possible is highlighted with a red box. Absence of a red square is indicative of the lack of cognate receptor from our screen, either due to unavailability of ACE2 sequence, or because the original bat host is yet unknown (RhGB07: Rh. hipposideros ; SARS-CoV-2, SARS-CoV-1: unknown; BANAL-20-52: Rh. malayanus ; BANAL-20-236: Rh. marshalli ; RacCS203 – Rh. acuminatus ). (E) Sequence alignment of bat sarbecovirus RBDs at the ACE2-binding interface, with key amino acid residues denoted, using SARS-CoV-2 numbering. Regions of amino acid deletions between sequences are denoted in red. The corresponding human and Rh.affinis ACE2 residues that bind SARS-CoV-2 RBD are highlighted, with black indicating amino acid residues that are the same between human ACE2 and Rh. affinis ACE2, green indicating the same residues but a different amino acid, and the remainder highlighting unique binding residues for human ACE2 (blue) or Rh. affinis ACE2 (pink) only.

Article Snippet: BHK-21 cells were seeded in 6-well plates at 7.5 × 10 5 /well in DMEM-10% 1 day prior to transfection with 500 ng of different species, ACE2-expressing constructs or empty vector (pDISPLAY) ( S2 Table ) in OptiMEM (ThermoFisher Scientific) and TransIT-X2 (Mirus Bio) transfection reagent, according to the manufacturer’s recommendations.

Techniques: Isolation, Binding Assay, Plasmid Preparation, Control, Virus, Sequencing

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Heatmap showing virus entry by cell-cell fusion. HEK293T cells stably expressing half of a split GFP-Renilla (RLuc-GFP 1-7) luciferase reporter were transfected with bat sarbecovirus Spikes, and co-cultured with BHK-21 cells expressing the other half of the split reporter (RLuc-GFP 8-11), transfected with bat ACE2s. Productive cell-cell fusion results in reconstitution of the split reporter, from which luciferase signals can be measured. Data is shown as log (RLU) and is the average of three separate experiments. A pDISPLAY vector control was included to set the baseline value to account for background signals. The red boxes indicate the predicted cognate bat receptor for each bat sarbecovirus, where known. (B) XY scatter plots for each of the viruses assessed for their receptor usage profile, correlating the log (RLU) of pseudoparticle entry (X) against cell-cell fusion entry (Y), excluding clade II viruses that do not use ACE2 as their entry receptor (RfGB02: r= 0.8244, p<0.0001; RhGB07: r=0.9335, p<0.0001; Rc-o319: r=0.9467, p<0.0001; SARS-CoV-2: r = 0.6684, p=0.0024; BANAL-20-103: r=0.5308, p=0.0234; BANAL-20-236: r=0.6096, p=0.0072; BANAL-20-52: r=0.7343, p=0.0005; RaTG13: r=0.7719, p=0.0002; SARS-CoV-1: r=0.9005, p<0.0001; WIV-1: r=0.7724, p=0.0002; Rs4231: r=0.8259, p<0.0001). Each data point is labelled with the ACE2 corresponding ACE2 receptor and a Pearson’s correlation calculated with 95% confidence intervals. The Pearson r value and p value are both documented.

Article Snippet: BHK-21 cells were seeded in 6-well plates at 7.5 × 10 5 /well in DMEM-10% 1 day prior to transfection with 500 ng of different species, ACE2-expressing constructs or empty vector (pDISPLAY) ( S2 Table ) in OptiMEM (ThermoFisher Scientific) and TransIT-X2 (Mirus Bio) transfection reagent, according to the manufacturer’s recommendations.

Techniques: Virus, Stable Transfection, Expressing, Luciferase, Transfection, Cell Culture, Plasmid Preparation, Control

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Heatmap showing the receptor usage of bat sarbecovirus-pseudotyped viruses with mammalian ACE2s, including human, livestock/pets, rodents, potential intermediate hosts and monkeys. The mean log RLU from 3 separate experiments is shown and a pDISPLAY control to illustrate the background levels of entry. (B) Surface representation of human ACE2 coloured with mammalian ACE2 conservation (human ACE2: PDB 6M0J), with the outlined region denoting the RBD-interacting sites, with individual amino acids labelled. (C) WebLogo (University of California, Berkley, USA) plots summarising the amino acid divergence within the mammalian ACE2 sequences used in this screen. The single letter amino acid code is used with the vertical height presenting the relative frequency of that amino acid at each position. The positions at which ACE2 interacts with SARS-CoV-2 RBD are shown, N-terminal to C-terminal, with different colours representing amino acid conservation between the mammalian ACE2s examined in this study.

Article Snippet: BHK-21 cells were seeded in 6-well plates at 7.5 × 10 5 /well in DMEM-10% 1 day prior to transfection with 500 ng of different species, ACE2-expressing constructs or empty vector (pDISPLAY) ( S2 Table ) in OptiMEM (ThermoFisher Scientific) and TransIT-X2 (Mirus Bio) transfection reagent, according to the manufacturer’s recommendations.

Techniques: Control

Journal: bioRxiv

Article Title: Understanding the future risk of bat coronavirus spillover into humans – correlating sarbecovirus receptor usage, host range, and antigenicity

doi: 10.1101/2025.09.03.673949

Figure Lengend Snippet: (A) Infection of HEK293T cells stably expressing hACE2 with SARS-CoV-2, Rs4231 or chimeric pseudoparticles (Rs4231 + SARS-CoV-2 RBD, SARS-CoV-2 + Rs4231 RBD) Virus was titrated 5-fold on cells and Firefly luciferase signals measured; a negative control (non-enveloped, NE) was also included. (B) Pseudoparticle entry assay using SARS-CoV-2 and Rs4231 chimeras, on a subset of ACE2s that had striking entry profile in the initial screen (human, Rh. sinicus , Rh. shameli, Rh. macrotis, T. melanopogan, mouse, Syrian hamster, pig, raccoon dog and marmoset ACE2). The heatmap shows log(RLU) of psuedotype entry, with pDISPLAY included as a control to establish background luciferase signal.

Article Snippet: BHK-21 cells were seeded in 6-well plates at 7.5 × 10 5 /well in DMEM-10% 1 day prior to transfection with 500 ng of different species, ACE2-expressing constructs or empty vector (pDISPLAY) ( S2 Table ) in OptiMEM (ThermoFisher Scientific) and TransIT-X2 (Mirus Bio) transfection reagent, according to the manufacturer’s recommendations.

Techniques: Infection, Stable Transfection, Expressing, Virus, Luciferase, Negative Control, Control