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

Journal: Nature Communications

Article Title: Optimizing a human monoclonal antibody for better neutralization of SARS-CoV-2

doi: 10.1038/s41467-025-61472-z

Figure Lengend Snippet: a Top view of the RBD inner face in complex with the 19-77 antibody. 19-77 heavy chain and light chain are shown in marine and light blue, respectively. The residues in the RBD are colored by the sequence entropy in circulating SARS-CoV-2 variants. The blue and cyan boundaries show the footprints of 19-77 and human ACE2 (hACE2), respectively. b Structure modeling of how A475V on the RBD affects 19-77 neutralization. The clashes are shown as red plates. c – e Comparison of residues 455 and 456 on RBD and P100 HC in D614G ( c ), EG.5.1 ( d ), and HK.3 ( e ) structures. The van der Waals clashes are shown as green plates.

Article Snippet: To make human ACE2-Fc (hACE2) protein, pcDNA3-sACE2-WT(732)-IgG1 (Addgene plasmid #154104, gift of Erik Procko) plasmid was transfected into Expi293 cells using 1 mg/mL polyethyleneimine (PEI) at a ratio of 1:3, and the supernatants were collected after 5 days. hACE2 was purified from the cell supernatant by using rProtein A Sepharose (GE).

Techniques: Sequencing, Neutralization, Comparison