Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Kinetic analysis of B07, B09, and B10 VHHs on hACE2-Fc by BioLayer Interferometry (BLI) using various concentrations of VHHs (as indicated on the figure). AHC biosensors were used to immobilize hACE2-Fc. K D of each VHHs is indicated. One representative experiment out of two. b Epitope mapping. sACE2-Fc (5 µg/mL) was immobilized onto the AHC biosensors. After a baseline step, a first VHH was applied (VHH1; 5 µg/mL). The sensor was dipped in a mixture of VHH1 and the competitor VHH2 at the same concentration (5 µg/mL). An anti-IgM specific VHH was used as control (ctl). (Top) Immobilized sACE2 pre-bound to B07 (VHH1) and VHH2 being B09, B10, or IgM (ctl). (Middle) Immobilized sACE2 pre-bound to B09 (VHH1) and VHH2 being B07, B10, or IgM (ctl). (Bottom) Immobilized sACE2 pre-bound to B10 (VHH1) and VHH2 being B07, B09, or IgM (ctl). Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Concentration Assay, Control

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: Cells were incubated with the different VHHs (10 µg/mL), stained with an anti-myc antibody and a AF488-conjugated anti-mouse antibody, before being analyzed by flow cytometry. a B07, B09, B10 binding efficacy on HEK293-hACE2-expressing cells. Data are means ± SD of three independent experiments. Parental HEK293 cells were used as control. b B07, B09, B10 binding efficacy on cells expressing endogenous hACE2 (IGROV-1). Anti-IgE VHH was used as a control (ctl). Data are means ± SD, n = 6 (ctl, B07) or 3 (B09, B10) independent experiments. c Fluorescence diagram overlays: B07, B09, B10 efficacy on cells expressing exogenous (HEK293-hACE2) or endogenous hACE2 (IGROV-1). Background (blue) corresponds to the fluorescence intensity obtained on parental cells (HEK293) or using a VHH control (anti-IgE VHH) (IGROV-1). Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Incubation, Staining, Flow Cytometry, Binding Assay, Expressing, Control, Fluorescence

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: hACE2 activity in the presence of VHHs B07, B09, B10 or absence (-) was assayed using the SensoLyte 390 ACE2 Activity Assay Kit, which measures fluorogenic peptide cleavage. a Soluble hACE2 (sACE2) activity in the presence of 10 µM VHHs. An ACE2 inhibitor, provided with the kit, was used as control (DX600, 1 µM). Data are means ± SD of three independent experiments. Paired T test two-sided: ns, nonsignificant; ** p = 0.0053. b VHHs B07, B09, B10 were used at different doses (one representative experiment out of three performed in duplicates. Mean values of the duplicate). The mock control (buffer) was arbitrarily positioned at 10 -14 . c The ACE2 inhibitor DX600 was used at different doses. Data are means ± SD of seven independent experiments. The mock control (buffer) was arbitrarily positioned at 10 -14 . d Cell surface hACE2 activity in the presence of saturating concentration of VHHs ( > 20 µM) or 1 µM DX600. Data are means ± SD of four independent experiments. Paired T test two-sided: ns, nonsignificant; ** p = 0.0070. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Activity Assay, Control, Concentration Assay

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a S-fuse assay. Inhibition by B07, B09, B10 VHHs after infection of S-Fuse cells (U2OS-hACE2 GFP1-10 and GFP11) with different SARS-CoV-2 variants (Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5, XBB1.16.1, EG.5.1.3, BA.2.86.1). Data are mean of two independent experiments. The dashed line indicates the limit of detection. An anti-IgM (or IgE) specific VHH was used as a control (ctl). IC50 values are indicated in the table. b Inhibition of 5 µg/mL spike binding to hACE2-expressing HEK293 cells by increasing concentrations of VHHs B07, B09 or B10. HEK293-hACE2 cells pretreated or not with the VHHs and incubated with soluble spike (S) protein (ancestral spike) were stained with an anti-S antibody. Results were normalized for nonspecific (0%) and specific binding in the absence of inhibitor (100%). Experiments were fitted to a one-site competitive binding model. Data are mean ± SD of three (B07, B09) independent experiments or mean of two independent experiments (B10). c Displacement of S (ancestral spike) binding by B07 (left) or B09 (right) for various concentrations of S (5 µg/mL, 8 µg/mL, 10 µg/mL). Experiments were carried out as in ( b ) (one representative experiment out of two - 10 µg/mL, or three - 5 µg/mL, 8 µg/mL). d Relationship between the observed IC50 values for B07 or B09 displacement of S binding and initial concentration of S (ancestral spike). Results represent means ± SD of three independent experiments (5 µg/mL, 8 µg/mL) or means of two independent experiments (10 µg/mL). Linear regression analysis: R B07 = 0.9005; R B09 = 0.7896. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Inhibition, Infection, Control, Binding Assay, Expressing, Incubation, Staining, Concentration Assay

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Crystal structure of hACE2 (light blue) in complex with B07 (purple) and B10 (yellow) (PDB: 9R19). b Surface representation of the complex between hACE2 (light blue) and B07 (purple) rotated to 90 ° C from a . For clarity B10 was not displayed. c Open-book representation and footprints (in white) of B07 on hACE2 (left) and hACE2 on B07 (right). The buried surfaces (BSA) are indicated under each surface. Residues of hACE2 in red correspond to those for which alanine substitutions compromise B07 VHH binding to hACE2 mutants ( f ). d B07 complementarity determining regions (CDRs) colored in green (CDR1), blue (CDR2), and red (CDR3). hACE2 footprint on B07 is represented. e Hydrogen bonds and salt bridges (yellow dashed lines) between hACE2 and B07. f Impact of hACE2 alanine substitution on B07 binding. Cells were transfected with myc tagged or untagged hACE2 alanine mutants, incubated with an anti-myc antibody or B07 (10 µg/mL), respectively and stained with a mouse anti-myc antibody and a AF647-conjugated anti-mouse antibody before being analyzed by flow cytometry. Data are mean ± SD of four (anti-myc) or three (B07) independent experiments. Source Data are provided as a Source Data file. g Electrostatic potential mapped on the surface of the structure of hACE2 and B07. h Superimposition of the binding area of B07 (purple) and SARS-CoV-2 RBD (PDB: 6M0J, green) on hACE2 (light blue). i Footprints of the site of interaction of B07 and RBD on hACE2. The contours are colored in purple (B07) or in green (RBD). The common area is striped.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Binding Assay, Transfection, Incubation, Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Kinetic analysis of VHH B07-Fc on hACE2-His by BioLayer Interferometry (BLI) using various concentrations of VHH B07-Fc (indicated on the figure). HIS2 biosensors were used to immobilize 1 µg/mL of hACE2-His. b VHH B07-Fc binding on cells expressing exogenous (HEK293-hACE2) or endogenous hACE2 (IGROV-1). Experiments were performed as in Fig. but using 0.1 µg/mL of VHH B07-Fc. Data are mean ± SD of six (HEK293) or four (IGROV-1) independent experiments. c Fluorescence diagram overlays as performed in Fig. . d hACE2, phalloidin (F-actin) and DAPI staining on primary human nasal epithelial cells (hNECs). Representative immunofluorescence staining (out of 4 independent experiments) of hACE2 (Red: B07-Fc staining) in combination with phalloidin (Cyan) and DAPI (Blue). Scale bars: ctl-Fc, 10 µm; B07-Fc, 5 µm. e Enzymatic activity of soluble hACE2 (sACE2) in the presence of VHH B07-Fc or absence using the SensoLyte 390 ACE2 Activity Assay Kit, as performed in Fig. . DX600 was an inhibitor control of the kit (one representative experiment out of two; mean values of duplicates). f Cell surface hACE2 activity in the presence of saturating concentration of VHHs (10 µM) or 1 µM DX600. Data are means ± SD of three independent experiments. Paired T test two-sided: ns, nonsignificant; ** p = 0.0070. g Inhibition of fusion (S-Fuse assay) by B07-Fc after infection of S-Fuse cells with different SARS-CoV-2 variants: Delta B.1.617.2, BA.1, BQ.1.1, XBB.1.5, XBB.1.16.1, EG.5.1.3, BA.2.86.1, JN.1.1, KP.3.3. The dashed line indicates the limit of detection. Data are mean of two independent experiments, except for BQ1.1 (three experiments). h Inhibition of infection quantified by counting SARS-CoV-2 nucleoprotein (N) positive cells. IGROV-1 cells were pre-incubated 1 h with serial dilutions of B07-Fc and infected with the indicated variants at 3 × 10 -2 infectious units per cell. Cells were stained with a pan-coronavirus anti-N antibody at day 1 pi. The percentage of inhibition is represented. One representative experiment performed in duplicates. i − k Impact of ACE2 genetic variation on VHH B07-Fc binding. i , j Cells were transfected with different ACE2 constructs ( i ) hACE2 polymorphism; ( j ) human, hamster, or mouse ACE2, incubated with B07-Fc (0.1 µg/mL or different doses) and stained with an AF647-conjugated anti-human antibody before being analyzed by flow cytometry. i Data are mean ± SD of independent experiments: n = 5 (S19P; I21T; K26R; T27A; E35K) or n = 4 (E37K; K68E; M82I, P84T) or n = 3 (H34N). j Data are mean ± SD of 5 (human ACE2) or 3 (hamster and mouse ACE2) independent experiments. k Kinetic analysis of B07-Fc on hamster ACE2-His by BioLayer Interferometry (BLI) using various concentrations of VHH B07-Fc (indicated on the figure). HIS2 biosensors were used to immobilize 2 µg/mL of His-ACE2. K D App is indicated. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Binding Assay, Expressing, Fluorescence, Staining, Immunofluorescence, Activity Assay, Control, Concentration Assay, Inhibition, Infection, Incubation, Transfection, Construct, Flow Cytometry

Journal: Nature Communications

Article Title: Targeting ACE2 with a camelid antibody inhibits SARS-CoV-2 binding and has protective effects in vivo

doi: 10.1038/s41467-025-65144-w

Figure Lengend Snippet: a Schematic diagram showing the experimental design of B07-Fc prophylaxis in XBB.1.5 infected K18-hACE2 mice. Animals received intranasally (i.n.) 7 mg/kg VHH-B07-Fc (B07-Fc) or 7 mg/kg VHH-Fc ctl (ctl-Fc). Twenty-four hours later, they were infected with 10 5 PFU XBB.1.5. SARS-CoV-2 intranasally (i.n.). Three days post-infection, different tissues were collected for analysis. b Genomic RNA load measured by RT-qPCR of SARS-CoV-2 in lung. c SARS-CoV-2 infectious particles measured by conventional plaque assays. d Tubulin, phalloidin (F-actin), SARS-CoV-2 nucleocapsid (N) (viral replication) and DAPI staining on nasal respiratory epithelium, olfactory epithelium, and lung bronchioli extracted from K18-hACE2 uninfected mice, and animals that received VHH-Fc (Ctl-Fc or B07-Fc). Representative immunofluorescence staining of Tubulin (Green) in combination with phalloidin (Cyan), SARS-CoV-2 N protein (Red), and DAPI (Blue). This was performed on nasal, olfactory, and pulmonary epithelium of two VHH ctl-treated mice and four B07-Fc treated mice. Scale bars: 10 µm. e Relationship between viral genomic RNA load measured in the lung by RT-qPCR and B07-Fc concentration quantified in the lung by ELISA. f Schematic diagram showing the experimental design of B07-Fc prophylaxis in D614G infected K18-hACE2 mice. Animals received intranasally (i.n.) 5 mg/kg VHH-B07-Fc (B07-Fc) or 5 mg/kg VHH-Fc ctl (ctl-Fc). Twenty-four hours later, they were infected with 10 4 PFU D614G SARS-CoV-2 i.n. Mice were clinically monitored between days 3 to 6. Mice were euthanized on day 6 and lungs were collected for analysis. g Body weight measured over time post-infection in ctl-Fc ( n = 5) and B07-Fc-treated groups. Mice of the B07-Fc group were split into VHH_high ( n = 4) and VHH_low ( n = 6) according to the B07-Fc concentration measured in the lung on day 6 p.i. (higher or lower than 200 ng/g of lung, respectively). Data are presented as mean values +/- SD. Linear mixed model two sided (no adjustment for multiple comparisons): ns, nonsignificant (p = 0.26); ** p = 0.00378; *** p = 0.000396. h Relationship between body weight loss and B07-Fc concentration quantified in the lung by ELISA on day 6 p.i. Source Data are provided as a Source Data file.

Article Snippet: The construct human ACE2 (hACE2) was obtained by substitution of the sequence encoding mCardinal from mCardinal-C1 plasmid (addgene, #54799) with hACE2 from the pLenti6-attB-hACE2-BSD .

Techniques: Infection, Quantitative RT-PCR, Staining, Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay

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: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

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: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

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: Expression of tagged ACE2 proteins was carried out in lysates from BHK-21 cells transfected with ACE2 constructs and probed for HA expression by Western blotting. Sarbecovirus Spikes were used to generate lentivral-based pseudotypes, and the viral supernatant purified by ultra-centrifugation under a 20% sucrose cushion. Purified viruses were probed with an anti-FLAG antibody (except RhGB07 and RfGB02, which are untagged), an anti-Spike antibody (S, full-length Spike; S1/S2, cleaved variant) and the lentivirus internal control, anti-p24 antibody.

Article Snippet: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: Expressing, Transfection, Construct, Western Blot, Purification, Centrifugation, Variant Assay, 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 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: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

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) left: CryoEM density of the trimer spike coloured by the protomer chains the density originates from. Glycans are highlighted in teal. Center: the trimeric spike viewed from the top rendered in cartoon representation. Right: the isolated RhGB07 RBD represented as cartoon in grey with two glycans of interest shown. (B) The C-alpha trace of the RBD of RhGB07 rendered as balls-and-sticks superimposed with the RBD of SARS-CoV-1 and SARS-CoV-2. The insets show a zoomed-in view of two regions of the RBD that show the greatest changes in the position of the C-alpha of the RBD. The region containing the ins1-loop and delta4-loop coincides with putative ACE2-interacting residues based on mapping the equivalent residues of SARS-CoV-1 and SARS-CoV-2. The ACE2 interacting residues were determined by superimposing SARS-CoV-1 and RhGB07 RBD onto the SARS-CoV-2 RBD – ACE2 (PDB: 6M0J) structure and identifying interacting residues. Highlighted residues of RhGB07 show the putative interacting residues that differ in identity to SARS-CoV-2.

Article Snippet: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: Isolation

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) Sequence conservation of selected sarbecoviruses were mapped on to the RhGB07 spike. Areas least conserved are in red and most conserved are blue. The least conserved regions are at the top of the RBD, the NTD and close to a putative polybasic site. (B) The C-alpha trace of the RBD of RhGB07 rendered as balls-and-sticks superimposed with the RBD of PRD-0038 apo or PRD-0039 bound to R. Alcyone ACE2. The insets show a zoomed-in view of two regions of the RBD that show the greatest changes in the position of the C-alpha of the RBD. The region containing the ins1-loop and delta4-loop coincides with putative ACE2-interacting residues based on mapping the equivalent residues.

Article Snippet: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: 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 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: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

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) Schematic of the emergence of SARS-CoV-2 variants depicting the divergence of the B lineage variants, including those variants of concern that grew to global dominance (B.1, Alpha, Beta, Delta, Omicron [black lines]), and the Omicron subvariants [red lines], some of which emerged via recombination events (dotted lines). Heatmap showing the bat and mammalian ACE2 usage of SARS-CoV-2 variants with (B) the B.1, Alpha, Beta, Delta and BA.1 variants, or (E) BA.2 and its subvariants BA.4/5, XBB, XBB1.5, BA.2.86. Data are shown as the mean fold change in pseudotype entry (RLU) of 3 separate experiments, relative to human ACE2. (C) Structure of SARS-CoV-2 BA.1 (PDB 7XO7) with mutations listed compared to B.1. Zoomed images of the Spike protein for B.1 (PDB 6M0J) and BA.1 (PDB 7WBP), highlighting key residues at positions 417, 484, 493 and 501 and the resultant interactions with human ACE2. (D) Specific amino acids within the B.1 RBD were mutagenised to make the RBD more BA.1-like (K417N, E484A, Q493R or N501Y) (top panel), or within BA.1 RBD to make the RBD more B.1-like (N417K, A484E, R493Q or Y501N) (bottom panel), either by themselves or in combination. Entry of these mutants was tested with Rh. cornutus, Rh. pusillus , Rh. ferrumequinum , and L. Lyra ACE2s. Data is shown as log fold-change in pseudotype entry, with green bars indicating an increase in virus entry and blue bars a decrease in entry, relative to B.1 (top panel) or BA.1 (bottom panel), highlighted also in .

Article Snippet: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: Virus

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) Table outlining the different convalescent plasma/serum used to investigate the antigenicity of bat sarbecoviruses. These include samples from individuals following infection with the wildtype (WT), Alpha, Gamma, Delta, or Omicron BA.1 variants, either confirmed by PCR or assumed based on epidemiological data. The sample set also includes the WHO International Standards provided by MRHA (NIBSC), pooled vaccinee plasma (low and high titre) and negative plasma, pooled from individuals pre-2019. Information about the COVID-19 standards and reagents can be found here: WHO/BS/2022.2427: Establishment of the 2nd WHO International Standard for anti-SARS-CoV-2 immunoglobulin and Reference Panel for antibodies to SARS-CoV-2 variants of concern . (B) Heatmap showing the log (IC 50 ) values of neutralisation (the dilution at which there was a 50% reduction in luciferase activity) of SARS-CoV-2, BANAL-20-52, BANAL-20-103, BANAL-20-236, Rs4231, WIV-1, RhGB07, Rc-o319 and BA.1 viruses in the presence of the MHRA (NIBSC) reference reagents. HEK293T cells stably expressing human ACE2 were used for all neutralisation assays, apart from RhGB07 and Rc-o319, where HEK293T cells overexpressing Rh. alcyone or Rh. cornutus were used, respectively (indicated by an asterisk). (C) Violin plots show collated IC 50 values for the neutralisation of these viruses with each sera set, with the median, upper and lower quartiles (red lines) and limit of detection (black dotted line) shown. A one-way ANOVA with Dunnet’s multiple comparison was performed, comparing to SARS-CoV-2, with significance values indicated (* = p<0.05, ** = p<0.005, ***= p<0.0005, ns = non-significant).

Article Snippet: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: Clinical Proteomics, Infection, Luciferase, Activity Assay, Stable Transfection, Expressing, Comparison

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: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: Infection, Stable Transfection, Expressing, Virus, Luciferase, Negative Control, 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) Dissociation constant (K D (M), 2-4 replicates ± sd) of mAb binding kinetics calculated by SPR (bars) and ELISA EC 50 (µg/ml, 2 replicates ± sd) values (broken lines with filled symbols) for a subset of mAbs and sarbecovirus RBDs. For K D , no bar indicates no binding by SPR. For EC 50 , lower limit of detection: <0.08µg/ml, upper limit of detection: >5µg/ml. (B) Neutralisation (IC 50 ) titres shown for each mAb against bat sarbecovirus pseudotyped viruses (µg/ml), with limits of detection indicated (lower limit: <0.08µg/ml; upper limit: >50µg/ml for IC 50 ). HEK293T cells stably expressing human ACE2 were used for all neutralisation assays. apart from RhGB07 and Rc-o319, where HEK293T cells overexpressing Rh. alcyone or Rh. cornutus were used, respectively. (C) RhGB07 trimer and RBD, with antibody competition groups highlighted, and are coloured with the same colouring as the SARS-CoV-2 trimer in . Regions of divergence in binding epitopes in RhGB07 compared to SARS-CoV-2 are depicted, with amino acid residues denotes. Colours in all graphs/structures indicate mAb binding competition groups (Group 1 = purple, Group 2 = orange, Group 3 = red, Group 3.5 = blue, Group 4 = green). EC 50 , K D and IC 50 values be found in .

Article Snippet: Mammalian ACE2 constructs were subcloned into pDISPLAY with an N-terminal signal peptide (the murine Ig k-chain leader sequence) and HA-tag (BioBasic, Canada; Twist Bioscience, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: SARS-CoV-2 Nsp2 recruits GIGYF2 near viral replication sites and supports viral protein production

doi: 10.1093/nar/gkaf674

Figure Lengend Snippet: Critical role of Nsp2 in the early phase of SARS-CoV-2 life cycle. ( A ) Schematic of wild-type (WT) and Nsp2-deleted (ΔNsp2) SARS-CoV-2 generation. SARS-CoV-2 DNA fragments were used to generate the DNA encoding the full-length gRNA. For the ΔNsp2 virus generation, the Nsp2 sequence is removed from the first fragment. The proteolytic cleavage sites are indicated with arrowheads. The ligated DNAs were then transcribed in vitro . The full-length RNAs were subsequently electroporated into N-expressing Vero (Vero-N) cells to produce viruses. These viruses are amplified in Nsp2-expressing Vero (Vero-Nsp2) cells. ( B ) Expression of viral proteins, Nsp1, 2, and 3, in Vero cells infected with WT or ΔNsp2 SARS-CoV-2. ( C ) Schematic of viral MOI measurement using parental and Vero-Nsp2 cells. The WT and ΔNsp2 SARS-CoV-2 virus titers were measured by plaque assay using WT and Nsp2-expressing Vero cells. Data are shown as mean ± standard deviation (SD) ( n = 4). One-sided Student’s t -test. ( D ) Relative RNA levels of Nsp12 and N following WT or ΔNsp2 SARS-CoV-2 infection. Calu-3 cells were infected with the indicated virus (MOI = 0.05). The RNAs were extracted at 24 hpi and subjected to RT-qPCR. Data are shown as mean ± SD ( n = 3). One-sided paired t -test. ( E , F ) Time-course measurement of relative RNA levels of N and Nsp12 following WT or ΔNsp2 SARS-CoV-2 infection. Calu-3 cells (E) or 293T-ACE2 cells (F) were infected with the indicated virus (MOI = 1). The RNAs were extracted at the indicated time point post-infection and subjected to RT-qPCR. Data are shown as mean ± SD ( n = 3). * P < .05, ** P < .01, One-sided Student’s t -test.

Article Snippet: ACE2-overexpressing HEK293T cells were generated by transducing ACE2 constructs (Addgene, 161758) into HEK293T cells (Clontech, 632180).

Techniques: Virus, Sequencing, In Vitro, Expressing, Amplification, Infection, Plaque Assay, Standard Deviation, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: SARS-CoV-2 Nsp2 recruits GIGYF2 near viral replication sites and supports viral protein production

doi: 10.1093/nar/gkaf674

Figure Lengend Snippet: GIGYF2 is a functionally important binding partner of SARS-CoV-2 Nsp2. ( A ) Schematic of Nsp2 interactome study in Calu-3 cells infected with SARS-CoV-2 (MOI = 0.05). Cells were harvested at 48 hpi following formaldehyde crosslinking. Immunoprecipitation (IP) was carried out using (i) an anti-Nsp2 antibody in both uninfected and infected cells and (ii) anti-Nsp2 and IgG antibodies in infected cells. ( B ) Protein–protein interaction network of Nsp2 with host proteins. Proteins exhibiting enrichment under both conditions were classified as Nsp2 interactors. The edges width indicates the degree of enrichment in anti-Nsp2 immunoprecipitation between infected and uninfected cells. ER localization data were curated from Protein Atlas. ( C ) UpSet plot illustrating the overlap of SARS-CoV-2 Nsp2 host protein interactome from our fCLIP–MS with the Nsp2 AP-MS data and pro-viral candidates from CRISPR KO screens. Each bar represents the number of proteins overlapping between the indicated datasets. ( D ) The protein sequence similarity of Nsp2 in various human coronaviruses with SARS-CoV-2 Nsp2. ( E ) Physical interaction between GIGYF2 and Nsp2 proteins of various human coronaviruses. HEK293T cells were transfected with untagged (-), N-terminal tagged (N), or C-terminal tagged (C) Nsp2 proteins and harvested 48 h after transfection. IP was performed in the presence of RNase A overnight using the indicated antibodies. ( F ) Relative RNA levels of Nsp12 and N in GIGYF2 knockdown Calu-3 cells following SARS-CoV-2 infection (MOI = 0.05). RNAs were extracted at 24 hpi for RT-qPCR. Data are shown as mean ± SD ( n = 3). One-sided paired t -test. ( G , H ) Time-course measurement of relative RNA levels of N and Nsp12 in parental and GIGYF2 KO cells following SARS-CoV-2 infection. Parental and GIGYF2 KO Calu-3 cells (G) or 293T-ACE2 cells (H) were infected with SARS-CoV-2 (MOI = 1). Data are shown as mean ± SD ( n = 3). * P < .05, ** P < .01, one-sided Student’s t -test. ( I ) Protein–protein interaction network of GIGYF2 with associated host proteins. IP was performed with anti-GIGYF2 antibodyor IgG, followed by LC-MS/MS ( n = 2). Proteins with enrichment (anti-GIGYF2/IgG) equal to or greater than 50 were represented as core interactors. The width of the edges is proportional to enrichment. Host proteins associated with ribosome quality control (RQC) are highlighted. ( J ) Relative RNA levels of Nsp12 and N following depletion of GIGYF2 cofactors in SARS-CoV-2-infected cells. Calu-3 cells were transfected with siRNA targeting either ZNF598 or DDX6 for 48 h and then infected with SARS-CoV-2 (MOI = 0.05). RNAs were extracted at 24 hpi for RT-qPCR. Data are shown as mean ± SD ( n = 3). One-sided paired t -test.

Article Snippet: ACE2-overexpressing HEK293T cells were generated by transducing ACE2 constructs (Addgene, 161758) into HEK293T cells (Clontech, 632180).

Techniques: Binding Assay, Infection, Cross-linking Immunoprecipitation, Immunoprecipitation, Protein-Protein interactions, CRISPR, Sequencing, Transfection, Knockdown, Quantitative RT-PCR, Liquid Chromatography with Mass Spectroscopy, Control

Journal: Nucleic Acids Research

Article Title: SARS-CoV-2 Nsp2 recruits GIGYF2 near viral replication sites and supports viral protein production

doi: 10.1093/nar/gkaf674

Figure Lengend Snippet: SARS-CoV-2 Nsp2 recruits GIGYF2 to the vicinity of viral replication organelle. ( A ) Time-dependent subcellular localization of GIGYF2 upon SARS-CoV-2 infection. A549-ACE2 cells were infected with SARS-CoV-2 (MOI = 10). Immunofluorescence staining of GIGYF2 (green) was performed at the indicated time points, along with DAPI staining (blue). ( B ) Subcellular localization of GIGYF2 and Nsp2 following infection of SARS-CoV-2 expressing FLAG-Nsp2. The upper panel depicts WT and FLAG-Nsp2 constructs, with proteolytic cleavage sites indicated by arrowheads. A549-ACE2 were infected with SARS-CoV-2 expressing FLAG-Nsp2 (MOI = 10). Immunofluorescence staining of GIGYF2 (green) and FLAG (red) was performed at 6 hpi, along with DAPI staining (blue). Overlap was quantified by Pearson’s colocalization coefficient ( n = 11 images per sample). One-sided Student’s t -test. The bottom panel displays the fluorescence signal intensity profile across a marked line within the magnified regions, indicating the relative distribution of GIGYF2 and Nsp2. ( C ) Subcellular localization of GIGYF2 and dsRNA following WT or ΔNsp2 SARS-CoV-2 infection. The upper panel depicts WT and ΔNsp2 constructs, with proteolytic cleavage sites indicated by arrowheads. A549-ACE2 cells were infected with either WT or ΔNsp2 SARS-CoV-2 virus (MOI = 1 for WT and 10 for ΔNsp2). Immunofluorescence staining of GIGYF2 (green) and dsRNA (red) was performed at 24 hpi, along with DAPI staining (blue). Overlap was quantified by Pearson’s colocalization coefficient ( n = 11 images for WT and n = 10 for ΔNsp2). One-sided Student’s t -test. The bottom panel displays the fluorescence signal intensity profile across a marked line within the magnified regions, indicating the relative distribution of GIGYF2 and dsRNA. ( D ) Subcellular localization of Nsp2 and dsRNA following SARS-CoV-2 infection in GIGYF2 KO cells. Parental and GIGYF2 KO 293T-ACE2 cells were infected with SARS-CoV-2 (MOI = 1). Immunofluorescence staining of GIGYF2 (green) and dsRNA (red) was performed at 24 hpi, along with DAPI staining (blue). ( E ) Subcellular localization of ZNF598 and Nsp2 following infection of SARS-CoV-2 expressing FLAG-Nsp2. A549-ACE2 cells were infected with SARS-CoV-2 expressing FLAG-Nsp2 (MOI = 0.22). Immunofluorescence staining of ZNF598 (green) and FLAG (red) was performed at 24 hpi, along with DAPI staining (blue). ( F ) Subcellular localization of ZNF598 and dsRNA following ΔNsp2 SARS-CoV-2 infection. A549-ACE2 cells were infected with ΔNsp2 SARS-CoV-2 (MOI = 5). Immunofluorescence staining of ZNF598 (green) and dsRNA (red) was performed at 24 hpi, along with DAPI staining (blue).

Article Snippet: ACE2-overexpressing HEK293T cells were generated by transducing ACE2 constructs (Addgene, 161758) into HEK293T cells (Clontech, 632180).

Techniques: Infection, Immunofluorescence, Staining, Expressing, Construct, Fluorescence, Virus

Journal: Nucleic Acids Research

Article Title: SARS-CoV-2 Nsp2 recruits GIGYF2 near viral replication sites and supports viral protein production

doi: 10.1093/nar/gkaf674

Figure Lengend Snippet: GIGYF2 interacts with viral RNA and ensures viral protein expression. ( A ) IFN-β measurement using ELISA. Parental and GIGYF2 KO variants of Calu-3 and 293T-ACE2 cells were treated with 1 μg/ml poly(I:C) for 6 h. ELISA was performed to quantify IFN-β levels. Data are shown as mean ± SD ( n = 3). One-sided Student’s t -test. ( B ) A scatter plot representing the comparison of gene expression levels in Calu-3 cells at 5 hpi with ΔNsp2 ( y -axis) and WT ( x -axis) SARS-CoV-2 viruses (MOI = 0.05). The mean of normalized read counts in each condition is represented. Interferon-stimulated genes (ISGs) are marked in green. n = 2 for each condition. ( C ) Schematic of GIGYF2 fCLIP-seq after SARS-CoV-2 infection. Calu-3 cells were crosslinked with formaldehyde and subjected to RNase A treatment, followed by immunoprecipitation using IgG or anti-GIGYF2 antibody. ( D ) Enrichment of GIGYF2 fCLIP-seq reads across the SARS-CoV-2 genome (bin size = 100 nt, shift = 100 nt). Different genomic regions are color-coded, with regions showing significant peak enrichment highlighted in red. The line and shadow indicate the mean enrichment (anti-GIGYF2/IgG) and the standard deviation, respectively ( n = 2). ( E ) Volcano plot showing the enrichment of GIGYF2 fCLIP-seq peaks in the viral genome. ( F ) Zoom-in view of selected GIGYF2 fCLIP-seq peaks in the viral genome (bin size = 50 nt, shift = 1 nt). The line and shadow indicate the mean enrichment (anti-GIGYF2/IgG) and the standard deviation, respectively ( n = 2). ( G ) Schematic representation of a dual promoter vector designed for independent expression of GIGYF2 binding targets and green fluorescent protein (GFP). (H,I) Expression changes of GIGYF2 targets, M ( H ) and Orf6-FLAG ( I ), upon GIGYF2 depletion. 293T-ACE2 parental and GIGYF2 KO cells were transfected with the dual promoter vectors expressing the indicated GIGYF2 targets, with or without Nsp2. The original vector (pmiR-GLO dual luciferase) was used as negative control (NC). The band intensities of the GIGYF2 targets were measured using ImageJ software and normalized to GFP. Data are shown as mean ± SD ( n = 3). * P < .05, one-sided paired t -test. ( J ) Relative mRNA expression of GIGYF2 targets (M, Orf6-FLAG) upon GIGYF2 depletion. 293T-ACE2 parental and GIGYF2 KO cells were transfected with the dual promoter reporter plasmids and subjected to RT-qPCR. Data are shown as mean ± SD ( n = 3). One-sided paired t -test.

Article Snippet: ACE2-overexpressing HEK293T cells were generated by transducing ACE2 constructs (Addgene, 161758) into HEK293T cells (Clontech, 632180).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Gene Expression, Infection, Immunoprecipitation, Standard Deviation, Plasmid Preparation, Binding Assay, Transfection, Luciferase, Negative Control, Software, Quantitative RT-PCR