Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Detection of SARS-CoV-2 infected cells using high-content imaging under control conditions. Fluorescent signals: green (anti-SARS-CoV NP mAb), blue (Hoechst). (B) Representative flow cytometry analysis plots showing NP+ cells. (C) Measurement of fluorescent intensity in NP+ cells. (D) Percentage of infected MSCs. (E) The supernatants of infected MSCs were collected and titrated on Vero-E6/TMPRSS2 cells to measure infectious viral particle production using the focus-forming assay. (F) After RNA extraction from infected MSCs, the expression of IDO1 was examined using real-time PCR. The graph displays the expression of IDO1 normalized to GAPDH , with the mock group serving as the control. Data are represented as means ± SEM from three independent biological replicates. For , the Delta condition was available from only two independent donors and was therefore presented descriptively and excluded from the statistical analysis. Statistical comparisons among the remaining groups were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. One-way ANOVA showed a significant overall difference among groups, F(2, 6) = 6.794, P = 0.0287. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Infection, Imaging, Control, Flow Cytometry, Analysis, Focus Forming Assay, RNA Extraction, Expressing, Real-time Polymerase Chain Reaction

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Representative western blot depicting the expression of NRP1 in AD-MSCs (No siRNA), cells transfected with non-targeting siRNA (siControl), or siNRP1 for 24 hours. β-actin was used as a loading control. (B) Graph displaying the quantification of NRP1 normalized to β-actin (n = 3). This experiment represents three independent biological replications. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Transfected cells were visualized using high-content imaging. Fluorescent signals: green for anti-NRP1 mAb, blue for Hoechst. (D) Percentage of NRP1-expressing cells normalized to cells transfected with siControl. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was conducted using a paired t-test. Next, siRNA-transfected cells were infected with different variants of SARS-CoV-2. (E) SARS-CoV-2-infected cells were detected using high-content imaging. Fluorescent signals: green for anti-SARS-CoV NP mAb, blue for Hoechst. (F) The fluorescent intensity of NP+ cells was measured. All data represent three independent experiments. Statistical analysis among each group of variant was performed using an unpaired t-test. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Western Blot, Expressing, Transfection, Control, Analysis, Imaging, Infection, Variant Assay

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Fluorescence intensity of NP-positive UCB-CD34 + HSPCs after infection with WT or Omicron variants of SARS-CoV-2. (B) Infectious viral particle production was determined by the focus-forming assay on Vero-E6/TMPRSS2 cells. (C) HSPC phenotype after infected with SARS-CoV-2 was investigated via the expression of primitive markers through flow cytometry. (D) The representative of HSC phenotype after infected with SARS-CoV-2. (E) Fluorescent intensity of NP-positive UCB-CD34 + HSPCs after infected with heat-inactivated WT and Omicron variant of SARS-CoV-2 compared to active virus. (F) The representative of HSC phenotype after infected with heat-inactivated WT and Omicron compared to active virus. All data are represented as mean ± SEM from three independent biological replicates. Statistical comparisons among Mock, WT, and Omicron in each group were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. For , the unpaired t-test were performed among each group by comparing between Mock vs. WT, Mock vs. Omicron, WT vs. heat-inactivated WT, and Omicron vs. heat-inactivated Omicron. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Fluorescence, Infection, Focus Forming Assay, Expressing, Flow Cytometry, Variant Assay, Virus

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Representative CFU colonies generated from human UCB-CD34 + HSPCs after exposure to WT or Omicron SARS-CoV-2 compared to Mock. (B) Quantification of burst-forming unit–erythroid (BFU-E), colony-forming unit-granulocyte/macrophage (CFU-GM), and total CFU output (C) Frequency of early erythroid progenitors (CD34 − CD36 ⁺ CD71 ⁺ CD235 − ) following in vitro erythroid differentiation of SARS-CoV-2-exposed CD34 ⁺ cells. (D) CFU output (BFU-E, CFU-GM, and total) in the virus inhibition experiment comparing active versus heat-inactivated virus. (E) Frequency of early erythroid progenitors (CD34 − CD36 ⁺ CD71 ⁺ CD235 − ) in the virus inhibition experiment. The results represent the means and SEM of three independent experiments. For and , the unpaired t-test were performed among each group by comparing of total CFUs, BFU-E, and CFU-GM between Mock vs. WT, Mock vs. Omicron, WT vs. heat-inactivated WT, and Omicron vs. heat-inactivated Omicron. For and , statistical comparisons among Mock, WT, and Omicron in each group were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: To stain the viral NP, a primary rabbit mAb antibody specific for SARS-CoV NP (Sino Biological Inc.) was used at a dilution ratio of 1:1000.

Techniques: Generated, In Vitro, Virus, Inhibition

Journal: iScience

Article Title: A multimodal in situ framework for spatial characterization applied on SARS-CoV-2 live-attenuated vaccines in the nasal mucosa

doi: 10.1016/j.isci.2026.117730

Figure Lengend Snippet: Viral protein distribution and target cell tropism in the nasal mucosa at 2 dpi (A) SARS-NP positive areas in the olfactory epithelium were comparable between WT and OTS-206 inoculated hamsters and significantly larger than in OTS-228. (B) In the respiratory epithelium, larger SARS-NP positive areas were observed after OTS-206 than after WT inoculation. (C–E) Representative sections illustrate multifocal to diffuse SARS-NP labeling in epithelial cells of the olfactory epithelium (levels 2–3) and respiratory epithelium (levels 1–2; predominantly ciliated epithelial cells) following (C) WT and (D) OTS-206 inoculation. In contrast, (E) OTS-228 antigen was restricted to single cells or oligofocal clusters of few epithelial cells (overview, scale bars, 1 mm; detail, scale bars, 100 μm). (F) In the lamina propria of the olfactory mucosa, abundant viral antigen was detected after WT inoculation, whereas only minimal antigen was observed after OTS-206 and OTS-228. (G) WT led to prominent infection of numerous Bowman’s gland epithelial cells (black arrow) and only individual virus-positive glandular cells following OTS inoculation (scale bars, 100 μm). (H) The vomeronasal organ (black dotted contour) was affected in all WT-infected hamsters, but not in OTS-inoculated hamsters (scale bars, 100 μm). (I) SARS-NP antigen (black arrow) was detected in the cytoplasm of macrophages of the nasal-associated lymphoid tissue in all groups (scale bars, 100 μm). (J–L) Representative immunofluorescence images (63× magnification) of the olfactory epithelium identified (J) sustentacular cells (CK18, green) as the main target cell (SARS-NP, red) for all groups. (K) Few SARS-NP positive olfactory sensory neurons (Tuj1, cyan; magnified image, white dotted contour) were detected, while axon bundles in the lamina propria (overview image, white dotted contour) remained negative following OTS inoculation. (L) SARS-NP positive Bowman’s glands (CK18, green; white dotted contour). Kruskal-Wallis test followed by Dunn’s correction was performed (A, B, F). ∗ p < 0.05, ns = not significant. Mean with standard deviation is shown. SARS-NP: SARS-CoV nucleocapsid protein; CK18: cytokeratin peptide 18; Tuj1: neuron-specific class III beta-tubulin. n = 5 hamsters per group; 2 dpi; nasal levels 1, 2, and 3, with a total of 7 regions of interest, were analyzed.

Article Snippet: SARS-NP (1:3000, Rockland, 200–401-A50) IHC was performed as described above.

Techniques: Olfactory, Labeling, Infection, Virus, Tissue, Immunofluorescence, Standard Deviation

Journal: iScience

Article Title: A multimodal in situ framework for spatial characterization applied on SARS-CoV-2 live-attenuated vaccines in the nasal mucosa

doi: 10.1016/j.isci.2026.117730

Figure Lengend Snippet: Host gene responses in the olfactory epithelium at 2 (WT, OTS-206, OTS-228) and 5 dpi (OTS-228 only). RNA in situ hybridization of interferon beta 1 (IFN β1), retinoic acid inducible gene I (RIG I), myxovirus resistance protein 1 (Mx1), and tumor necrosis factor alpha (TNF α) combined with SARS-NP and Mx1 antigen detection (A) WT: SARS-NP (red) positive sustentacular cells (SCs) showed mainly moderate to abundant nuclear IFN-β1 RNA (purple), few to moderate RIG-I (green), and few Mx1 (yellow) RNA signals. Non-infected olfactory sensory neurons (OSNs) exhibited few to moderate cytoplasmic RIG-I and Mx1 RNA. (B) OTS-206: SARS-NP positive SCs showed moderate to abundant cytoplasmic IFN-β1 RNA. Virus antigen positive SCs and negative OSNs exhibited moderate RIG-I and Mx1 RNA. (C and D) OTS-228: At 2 dpi (C), RNA expression of all targets was below the detection limit. At 5 dpi (D), IFN-β1 remained undetectable, whereas few to moderate cytoplasmic RIG-I and Mx1 RNA were detected in both SARS-NP positive and negative SCs. (E–H) TNF-α RNA (cyan) was present at moderate to abundant levels at 2 dpi after WT and OTS-206 inoculation and restricted to SARS-NP positive SCs, showing (E) nuclear aggregation in WT and (F) predominantly cytoplasmic distribution in OTS-206. (G and H) OTS-228: TNF-α RNA was undetectable at 2 dpi (G), but at 5 dpi (H), few cytoplasmic signals were present in individual SARS-NP positive SCs. (I–L) Mx1 RNA and antigen. (I) WT antigen positive SCs showed absent or few Mx1 RNA (yellow) signals and minimal protein (blue), whereas non-infected SCs exhibited low to moderate levels of both. (J) OTS-206 antigen positive and- negative SCs with moderate to abundant Mx1 RNA and antigen. (K and L) OTS-228 antigen positive cells with minimal Mx1 protein at 2 dpi (K), and moderate to abundant Mx1 RNA and protein at 5 dpi (L). Dotted contours indicate SCs and OSNs in magnified images. SARS-NP: SARS-CoV nucleocapsid protein. n = 2 hamsters per group; 2 (all groups) and 5 dpi (OTS-228 only); 6 images (63× magnification) per animal taken from regions of interest were evaluated for location (nuclear, cytoplasmic, both) and abundance of RNA signals (few, moderate, abundant).

Article Snippet: SARS-NP (1:3000, Rockland, 200–401-A50) IHC was performed as described above.

Techniques: Olfactory, RNA In Situ Hybridization, Infection, Virus, RNA Expression

Journal: iScience

Article Title: A multimodal in situ framework for spatial characterization applied on SARS-CoV-2 live-attenuated vaccines in the nasal mucosa

doi: 10.1016/j.isci.2026.117730

Figure Lengend Snippet:

Article Snippet: SARS-NP (1:3000, Rockland, 200–401-A50) IHC was performed as described above.

Techniques: Plasmid Preparation, Virus, Indirect ELISA, Recombinant, RNAscope, Multiplex Assay, Software, Negative Control, Confocal, Laser-Scanning Microscopy

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Detection of SARS-CoV-2 infected cells using high-content imaging under control conditions. Fluorescent signals: green (anti-SARS-CoV NP mAb), blue (Hoechst). (B) Representative flow cytometry analysis plots showing NP+ cells. (C) Measurement of fluorescent intensity in NP+ cells. (D) Percentage of infected MSCs. (E) The supernatants of infected MSCs were collected and titrated on Vero-E6/TMPRSS2 cells to measure infectious viral particle production using the focus-forming assay. (F) After RNA extraction from infected MSCs, the expression of IDO1 was examined using real-time PCR. The graph displays the expression of IDO1 normalized to GAPDH , with the mock group serving as the control. Data are represented as means ± SEM from three independent biological replicates. For , the Delta condition was available from only two independent donors and was therefore presented descriptively and excluded from the statistical analysis. Statistical comparisons among the remaining groups were performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. One-way ANOVA showed a significant overall difference among groups, F(2, 6) = 6.794, P = 0.0287. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: After that, the plate was stained with 1:2500 of rabbit anti-SARS-CoV NP (Sino Biological Inc.) and then anti-rabbit HRP (DAKO) was used as secondary antibody.

Techniques: Infection, Imaging, Control, Flow Cytometry, Analysis, Focus Forming Assay, RNA Extraction, Expressing, Real-time Polymerase Chain Reaction

Journal: PLOS One

Article Title: Omicron SARS-CoV-2 shows NRP1-linked susceptibility in adipose-derived MSCs and alters primitive cord blood CD34⁺ HSPCs

doi: 10.1371/journal.pone.0360439

Figure Lengend Snippet: (A) Representative western blot depicting the expression of NRP1 in AD-MSCs (No siRNA), cells transfected with non-targeting siRNA (siControl), or siNRP1 for 24 hours. β-actin was used as a loading control. (B) Graph displaying the quantification of NRP1 normalized to β-actin (n = 3). This experiment represents three independent biological replications. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. (C) Transfected cells were visualized using high-content imaging. Fluorescent signals: green for anti-NRP1 mAb, blue for Hoechst. (D) Percentage of NRP1-expressing cells normalized to cells transfected with siControl. Data are presented as mean ± SEM from three independent experiments. Statistical analysis was conducted using a paired t-test. Next, siRNA-transfected cells were infected with different variants of SARS-CoV-2. (E) SARS-CoV-2-infected cells were detected using high-content imaging. Fluorescent signals: green for anti-SARS-CoV NP mAb, blue for Hoechst. (F) The fluorescent intensity of NP+ cells was measured. All data represent three independent experiments. Statistical analysis among each group of variant was performed using an unpaired t-test. * P < 0.05, ** P < 0.01, *** P = 0.0001, **** P < 0.0001.

Article Snippet: After that, the plate was stained with 1:2500 of rabbit anti-SARS-CoV NP (Sino Biological Inc.) and then anti-rabbit HRP (DAKO) was used as secondary antibody.

Techniques: Western Blot, Expressing, Transfection, Control, Analysis, Imaging, Infection, Variant Assay