Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Diagram of the RBD substitutions that distinguish Omicron BA.2.86 from Wuhan-Hu-1 (top), Omicron KP.3.1.1 from BA.2.86 (middle), and Omicron LP.8.1 from BA.2.86 (bottom). Dashed lines show propagation of BA.2.86 changes to KP.3.1.1 and LP.8.1, and italicized mutation in LP.8.1 (H445R) indicates a secondary substitution at a site that previously changed from Wuhan-Hu-1 to BA.2.86. Wuhan-Hu-1 reference spike numbering is used throughout the manuscript. ( B-D ) Quality control of the KP.3.1.1 and LP.8.1 RBD site-saturation mutagenesis library as assessed by PacBio sequencing, illustrating the distribution of number of amino acid mutations per barcoded variant (B), the average number of mutations of each type across library variants (C), and the distribution of mutations across sites in the RBD over all variants (D). ( E, F ) Representative FACS gates used to sort RBD + singlet cells for ACE2 binding (E) and singlet cells for RBD expression (F), which is followed by high-throughput sequencing of cells sorted into each bin. ( G, H ) Correlation in per-mutant deep mutational scanning measurements between independently barcoded replicate libraries for ACE2-binding affinity (G) and RBD expression (H) experiments. Red dash indicates the 1:1 line.

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Mutagenesis, Control, PacBio Sequencing, Variant Assay, Binding Assay, Expressing, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Heatmap illustrating the impacts of all mutations in the KP.3.1.1 and LP.8.1 RBD on ACE2-binding affinity as determined from FACS-seq experiments with yeast-displayed RBD mutant libraries. ACE2 contact residues (black squares, bottom) defined as RBD residues with non-hydrogen atoms <5Å from ACE2 in the BA.2.86 RBD structure (PDB 8QSQ ). Raw data available in Supplemental Data 1. ( B ) Deep mutational scanning data from (A) mapped to the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ ), illustrating the average effect of mutations at a site (left), and the maximal effect of any mutation at a site (right). Sites of interest are labeled. ACE2 (key motifs only) is shown as transparent gray cartoon. ( C ) Heatmap illustrating the impacts of all mutations in the KP.3.1.1 and LP.8.1 RBD on yeast-surface expression levels, a proxy for folding and expression efficiency. Raw data available in Supplemental Data 1. An interactive version of these heatmaps alongside previously assayed SARS-CoV-2 variant RBDs is available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-KP3-LP8/RBD-heatmaps/ .

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Binding Assay, Mutagenesis, Labeling, Expressing, Variant Assay

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Correlation between KP.3.1.1 mutant effects on ACE2 binding in the pseudovirus whole-spike DMS study of Dadonaite et al. (change in potency of pseudoviral neutralization via soluble ACE2 protein) versus the yeast-display RBD DMS study presented here (change in binding affinity from FACS-seq titration). Correlations are separated based on residue distance from the ACE2 interface in the ACE2-bound BA.2.86 RBD structure (PDB 8QSQ ). Mutations whose effect on spike-mediated pseudoviral entry was < -2 units per the assay of Dadonaite et al. were not included because it is difficult to reliably measure soluble-ACE2-inhibition of entry when entry is poor. ( B ) Structural context of residues F374 and H505 in the spike trimer (PDB 9ELH ). Center, overall top-down view of the spike trimer with two RBDs in the “down” and one in the “up” conformation. Left, zoom-in on the down-down interface of RBD packing; right, zoom-in on the up-down interface of RBD packing.

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Mutagenesis, Binding Assay, Neutralization, Titration, Residue, Inhibition

Journal: bioRxiv

Article Title: Deep mutational scanning of recent SARS-CoV-2 variants highlights changing amino acid preferences within epistatic hotspot residues

doi: 10.64898/2026.03.11.711006

Figure Lengend Snippet: ( A ) Epistatic shift in the effects of mutations on ACE2 binding at each RBD position as measured in the Wuhan-Hu-1 (previously reported in ), KP.3.1.1 or LP.8.1 background compared to those previously measured in Omicron BA.2.86 (previously reported in ). ( B ) Mutation-level plots of epistatic shifts between BA.2.86 and KP.3.1.1 or LP.8.1 at sites of interest. Each scatterplot shows the measured ACE2-binding affinity of each amino acid (plotting character, – indicates deletion character) in the BA.2.86 versus KP.3.1.1 or LP.8.1 backgrounds. Red dashed lines mark the respective wildtype RBD affinities on each axis, and the gray dashed line indicates the additive (non-epistatic) expectation. Interactive plots enabling the comparison of all SARS-CoV-2 variants and scatterplots for all RBD sites are available at https://tstarrlab.github.io/SARS-CoV-2-RBD_DMS_Omicron-KP3-LP8/epistatic-shifts/ .

Article Snippet: Induced cells were washed with PBS-BSA (BSA 0.2 mg/L), split into 16-OD*mL aliquots, and incubated with biotinylated monomeric human ACE2 protein (ACROBiosystems AC2-H82E8) across a concentration range from 10 -6 to 10 -13 M at 1-log intervals, plus a 0 M sample.

Techniques: Binding Assay, Mutagenesis, Comparison

Journal: npj Viruses

Article Title: K5 polysaccharides inhibit SARS-CoV-2 infection by preventing spike-proteolytic priming

doi: 10.1038/s44298-025-00163-4

Figure Lengend Snippet: VeroE6 cells or A549 ACE2+ cells were treated with increasing concentrations of heparin, K5, K5OSH, and K5NOSH. a , b The cells remained viable in the presence of heparin and the K5 compounds as evaluated by measuring the ATP levels. VeroE6 or A549 ACE2+ cells were infected with the B.1 c , d or Omicron BA.1 e , f isolates in the presence or the absence of increasing concentrations of heparin, K5, K5OSH, and K5NOSH. Infection was reduced in a concentration-dependent manner as shown by the percentage of plaque reduction compared to SARS-CoV-2 alone. Data are presented as the mean value ± standard error of three independent replicates. * P < 0.05; ** P < 0.005.

Article Snippet: Reagents and materials were used as received, unless otherwise mentioned, and were purchased from the following: Human recombinant SARS-CoV-2 Wuhan-Hu-1 spike His-Tag protein and RBD from Sino Biological (#40592-V08B); ACE2 from Acrobiosystem (#AC2-H52H8); Bovine Serum Albumin (BSA) from Merck (#810037); Human recombinant furin from OriGene Technologies Inc. (#TP304279M); Conventional heparin (13.6 kDa - purity ≥95%) from a commercial batch of unfractionated sodium heparin from Laboratori Derivati Organici S.p.A. (#9041-08-1).

Techniques: Infection, Concentration Assay

Journal: Journal of Virology

Article Title: Increased pathogenicity and transmission of SARS-CoV-2 Omicron XBB.1.9 subvariants, including HK.3 and EG.5.1, relative to BA.2

doi: 10.1128/jvi.01342-25

Figure Lengend Snippet: Evolution, prevalence, and replicative kinetics of XBB.1.9 subvariants. ( A ) Evolutionary origins of the XBB.1.9 sublineages, including XBB.1.9.1, EG.5.1, and HK.3. Synonymous mutations in nucleotides and amino acid mutations are shown in bold and bold-italic font, respectively. ( B ) Prevalence of XBB.1.9.1 (blue), EG.5.1 (green), and HK.3 (orange) in China (CHN), the United States (USA), Europe (EUP), and the Republic of Korea (ROK) for 14 months from January 2023 (2023.01) to March 2024 (2024.03). ( C and D ). Replicative kinetics of BA.2 (dark red), XBB.1.9.1 (blue), EG.5.1 (green), and HK.3 (orange) in terms of viral titers (upper panel) and viral loads (lower panel) in Vero E6, Vero E6 TMPRSS2+ , HeLa hACE2+ , Huh-7, and Caco2 cells. Cells were infected at an MOI of 0.01. The significance of the differences in replication between BA.2 and XBB.1.9.1, EG.5.1, or HK.3 is indicated above the lines by the asterisks in colors corresponding to the individual viruses. The significance of the differences in replication between HK.3 and XBB.1.9.1 or EG.5.1 is indicated by gray or black asterisks below the lines. A detection reference (from a weakly positive sample, CT = 27.0) is represented by dashed lines. ( E ) Viability of HK.3-infected cells. Significance of viability differences between Vero E6 and Vero E6 TMPRSS2+ cells is revealed. ( F ) Relative RNA expressions of TMPRSS2 (left) and ACE2 (right). Significance of the differences in TMPRSS2 expression between Vero E6 and other cells and in ACE2 expression between HeLa hACE2+ cells and others is indicated. ( G ) Ratio of viral titers (upper panel) and viral loads (lower panel) in Vero E6 cells with high versus low TMPRSS2 expression. ( H ) Replicative kinetics of two HK.3 isolates. Statistical analyses were conducted using Student’s t -test. Significances: P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***). Viral titer reflects the number of infectious viral particles (TCID 50 /mL), whereas viral load represents RNA replication levels (copy number of genomic RNA).

Article Snippet: The hACE2 protein with an Fc tag (Acro Biosystems) was immobilized onto the sample flow cell of the sensor chip.

Techniques: Infection, Expressing

Journal: Journal of Virology

Article Title: Increased pathogenicity and transmission of SARS-CoV-2 Omicron XBB.1.9 subvariants, including HK.3 and EG.5.1, relative to BA.2

doi: 10.1128/jvi.01342-25

Figure Lengend Snippet: Characteristics of the spikes of XBB.1.9 subvariants. ( A ) Spike-mediated infection determined by pseudovirus assays. XBB.1-S of the XBB.1 lineage and XBB.1-P of the XBB.1.9 lineage were included. The infection efficiency of BA.2 has been set to 1 to show relative infectivity. ( B ) Spike-mediated cell‒cell fusion based on luciferase activity. BA.2 (dark red), XBB.1-P (blue), EG.5.1 (green), and HK.3 (orange) are indicated by solid lines. D614G (pink), XBB.1-S (purple), and a negative control (N.C. in gray) are indicated by dotted lines. The significance of the differences between XBB.1 variants and BA.2 is indicated in colors corresponding to the individual XBB variants, which are placed within black rectangles by the asterisks, respectively. ( C ) Spike-mediated syncytia formation (scale bar: 400 µm). ( D ) The proteolytic processing of spike protein was analyzed in authentic SARS-CoV-2 virions propagated in Vero E6 TMPRSS2+ cells, including the ancestral strain (BJ05P14), Delta, and Omicron subvariants (BA.2, XBB.1.9.1, EG.5.1, and HK.3). Relative spike protein expression levels of BA.2, XBB.1.9.1, EG.5.1, and HK.3 virions were determined (a representative result) with an exposure time of 1 ms (left). The ratio of S2 subunit bands to full-length S protein (S2/S) was quantified (three biological replicates) using ImageJ/Fiji software (right). The ratio of BA.2 has been set to 1. ( E ) Purification of XBB.1-S, XBB.1-P, EG.5.1, and HK.3 spikes. ( F ) Comparison of the binding affinities of the XBB.1 spikes to hACE2. SPR characterization of the spike includes XBB.1-S, XBB.1-P, EG.5.1, and HK.3 interacting with hACE2. The dissociation constant is revealed above the lines. Sensorgrams depict different concentrations of ligands. Statistical analyses were conducted using Student’s t -test. Significances: P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

Article Snippet: The hACE2 protein with an Fc tag (Acro Biosystems) was immobilized onto the sample flow cell of the sensor chip.

Techniques: Infection, Luciferase, Activity Assay, Negative Control, Expressing, Software, Purification, Comparison, Binding Assay

Journal: Journal of Virology

Article Title: Increased pathogenicity and transmission of SARS-CoV-2 Omicron XBB.1.9 subvariants, including HK.3 and EG.5.1, relative to BA.2

doi: 10.1128/jvi.01342-25

Figure Lengend Snippet: Competitive fitness of XBB.1.9.1 and EG.5.1/HK.3 in wild-type hamsters. ( A ) Relative infection tropism of spikes. The infectivity ratio of ghACE2 to hACE2 is determined as tropism. ( B ) Flow chart of competitive fitness. ( C and D ) A mixture of XBB.1.9.1 and EG.5.1 ( C ) or HK.3 ( D ) at viral titer ratios of 1:1 (upper panel) or 1:3 (lower panel) was inoculated into hamsters. The RNA proportion of XBB.1.9.1 in the mixture was shown by numbers in the bars. Firstly, the RNA proportion of XBB.1.9.1 in initial inoculum was 80.8% or 54.5% ( C ) and 94.9% or 90.1% ( D ) which was displayed on the right of the initial proportion (yellow number). Secondly, the RNA proportion of XBB.1.9.1 in tissue samples (3 DPI) was shown in the bars (white number) below the horizontal of each figure grouping. The area in the bar means the RNA proportions of XBB.1.9.1 (blue) and EG.5.1 (green) or HK.3 (orange). Tissue samples are the lung and turbinate: lung (left) and turbinate (right).

Article Snippet: The hACE2 protein with an Fc tag (Acro Biosystems) was immobilized onto the sample flow cell of the sensor chip.

Techniques: Infection

Journal: Journal of Virology

Article Title: Increased pathogenicity and transmission of SARS-CoV-2 Omicron XBB.1.9 subvariants, including HK.3 and EG.5.1, relative to BA.2

doi: 10.1128/jvi.01342-25

Figure Lengend Snippet: In vivo virological characteristics of XBB.1.9 subvariants in K18-hACE2 hamsters. K18-hACE2 hamsters were intranasally inoculated with BA.2, XBB.1.9.1, EG.5.1, or HK.3. Four hamsters per group were used to measure the various parameters ( A, B, and C ). Four hamsters per group were euthanized at 3 DPI and used for data collection ( D–H ). The data (in A to E) of the mock, BA.2, XBB.1.9.1, EG.5.1, and HK.3 groups are shown in gray, red, blue, green, and orange, respectively (as shown in panel A ). ( A ) Body weights of the infected hamsters. Significant differences between the mock group and each infected group are revealed above the lines using asterisks in the colors corresponding to the respective infected group. ( B ) Percentage survival of the infected hamsters. Survival differences between multiple XBB.1.9 variants and BA.2 were analyzed using a Log-rank (Mantel-Cox) test with significance displayed in colors corresponding to the individual XBB.1.9 variant. ( C ) Viral loads in the nasal lavages of hamsters. The viral load baseline is indicated by dotted gray lines. ( D and E ) Radar chart of pathology ( D ) and pathology scores ( E ) of the infected lungs of hACE2 hamsters. The average of BA.2 (dark red), XBB.1.9.1 (blue), EG.5.1 (green), and HK.3 (orange) infected hamster (of 3–4 individuals) was indicated. ( F ) H&E staining and IHC images of the lungs of the infected hamsters. The lungs of two infected individuals in each group, namely, repetition 1 (REP1) and repetition 2 (REP2), are shown. The time point of tissue samples corresponds to 3 DPI. The scale bar represents 100 µm. ( G and H ) Viral titers ( G ) and viral loads ( H ) in the lungs (dark red) or turbinates (gray) of the infected hamsters. Statistical analyses were conducted using Student’s t -test. Significances: P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***).

Article Snippet: The hACE2 protein with an Fc tag (Acro Biosystems) was immobilized onto the sample flow cell of the sensor chip.

Techniques: In Vivo, Infection, Variant Assay, Staining