Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Characterization and anti-ZIKV activity of CBNK-EVs. (a) Isolation of CBNK cells by fluorescence-activated cell sorting. (b) Collection of CBNK-EVs and NTA for size distribution and concentration. (c) Morphological observation of CBNK-EVs by transmission electron microscopy, scale bar:100 nm. (d) WB analysis of specific markers for CBNK-EVs. (e) Anti-ZIKV activity of CBNK-EVs assessed by immunofluorescence. Vero-E6 cells were seeded in 12-well plates at 1 × 10 5 cells per well one day prior to experiments. Cells were pretreated with varying volumes of CBNK-EVs (stock concentration: 3 × 10 11 particles/mL) for 1 h to achieve the indicated final particle counts, followed by infection with ZIKV (MOI = 1) for 24 h in the presence of fresh medium containing the same EVs. ZIKV-infected cells were quantified by counting E protein-positive cells in three random fields by two independent investigators. Scale bar, 40 μm. (f) Statistical analysis of the data presented in (e) panel. (g) Vero-E6 cells were treated as in (e) and infected with ZIKV (MOI = 1) for 24 h. (h) ZIKV E and NS1 RNA levels were detected by quantitative RT-PCR. Data were normalized to GAPDH, log10-transformed, and presented as fold change compared to the virus control group (set to 1). Negative values indicate inhibition of viral replication. (i) Plaque reduction assay assessing the antiviral activity of CBNK-EVs against ZIKV. (j) Statistical analysis of the percentage of plaque reduction. (k) ZIKV NS5 protein levels in (g) were analyzed by Western blot. Data in panels (f, h, j and k) are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Vero E6, BHK21, 293T and HTR-8/Svneo cells were purchased from ATCC.

Techniques: Activity Assay, Isolation, Fluorescence, FACS, Concentration Assay, Transmission Assay, Electron Microscopy, Immunofluorescence, Infection, Quantitative RT-PCR, Transformation Assay, Virus, Control, Inhibition, Western Blot

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Functional components and mechanisms of CBNK-EVs in combating ZIKV infection. (a) Schematic of three treatment modalities: 1) pre-treatment of cells with CBNK-EVs, 2) pre-treatment of virus with CBNK-EVs, and 3) co-incubation of CBNK-EVs with ZIKV. (b) Vero-E6 cells were seeded at 1 × 10 5 cells per well in 12-well plates and subjected to the treatments outlined in (a), followed by infection with ZIKV (MOI = 1) for 1 h. After 48 h, ZIKV E and ZIKV NS5 protein levels were analyzed by Western blot (left). Densitometric analysis of the protein bands is shown (right, n = 3). (c) Schematic workflow for functional component inactivation in CBNK-EVs (e.g., by heat, protease, or nuclease treatment). (d) Vero-E6 cells were treated with inactivated CBNK-EVs as per the scheme in (c), followed by infection with ZIKV (MOI = 1) for 1 h. ZIKV E and ZIKV NS5 protein levels were analyzed by Western blot after 24 h. Densitometric analysis of the protein bands is shown (right, n = 3). (e) Represent images of the co-localization of CBNK-EVs, ZIKV E and host cells by confocal microscope, scale bar: 5 μm. (f) Fluorescence intensity of CBNK-EV and β-actin co-localization signals (white arrows) in the EVs group. (g) Fluorescence co-localization signals (yellow arrows) of CBNK-EVs and ZIKV in the co-culture group. (h) TEM images of CBNK-EVs, ZIKV particles, and their interactions. Scale bar: 100 nm. (i) Membrane disruption assay. ZIKV particles were co-incubated with CBNK-EVs, followed by treatment with micrococcal nuclease to digest unprotected RNA. Intact viral RNA, indicative of membrane integrity, was then measured by RT-qPCR targeting the ZIKV E. Mean ± SD (n = 3). ∗∗P < 0.01, ∗∗∗P < 0.001 (one-way ANOVA).

Article Snippet: Vero E6, BHK21, 293T and HTR-8/Svneo cells were purchased from ATCC.

Techniques: Functional Assay, Infection, Virus, Incubation, Western Blot, Microscopy, Fluorescence, Co-Culture Assay, Membrane, Disruption, Quantitative RT-PCR

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Perforin mediates the direct virion-disrupting activity of CBNK-EVs. (a) Quantification of perforin and granzyme B in CBNK-EVs by ELISA. (b) Vero-E6 cells were infected with ZIKV (MOI = 1) after pre-incubation of viral particles with 40 ng/L of recombinant perforin or granzyme B for 2 h. ZIKV E protein levels were assessed by Western blot after 24 h, densitometric analysis of the protein bands is shown (right, n = 3). (c) CBNK-EVs were pre-incubated with ZIKV in the presence or absence of 10 mM EGTA, followed by micrococcal nuclease digestion. Protected ZIKV E RNA was quantified by RT-qPCR to assess virion integrity. (d) Western blot analysis of perforin expression in CBNK cells after transfection with the indicated siRNAs. (e) Quantification of perforin levels from (d). (f) Perforin levels in CBNK-EVs collected from control or perforin-knockdown cells, measured by ELISA and normalized to particle count (per 10 11 particles, n = 6). (g) Vero-E6 cells were infected with ZIKV (MOI = 1) that had been pre-incubated with control or perforin-knockdown CBNK-EVs. ZIKV E protein levels were evaluated by Western blot. (h) Analysis of ZIKV E protein levels from (g). (i) Characterization of ITGB2-EVs and control EVs from 293T cells by NTA and TEM. Scale bar, 100 nm. (j) Zeta potential measurements of ITGB2-EVs and control EVs. (k) Western blot analysis of EV markers and ITGB2 expression in ITGB2-EVs and control EVs. (l) Antiviral activity of CBNK-EVs and ITGB2-EVs evaluated by cell-based ZIKV E protein ELISA. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗P < 0.001 (one-way ANOVA).

Article Snippet: Vero E6, BHK21, 293T and HTR-8/Svneo cells were purchased from ATCC.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Recombinant, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Control, Knockdown, Zeta Potential Analyzer

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: ITGB2 facilitates CBNK-EVs binding to Zika virions and enhances cellular susceptibility to ZIKV. (a) Multicolor immunofluorescence staining of 293T cells transfected with ITGB2, showing co-localization (yellow) between ITGB2 (green) and ZIKV E protein (red). Scale bar: 20 μm. (b) Quantitative analysis of ZIKV E-positive 293T cells from (a). (c) Fluorescence intensity profile along white arrows in (a), indicating sites of ITGB2 and ZIKV E co-localization. (d) 293T cells were transfected with increasing amounts (0.5, 1, 2 μg) of ITGB2 plasmid and infected with ZIKV (MOI = 1) for 1 h. ZIKV RNA levels were measured by qPCR to assess infection susceptibility (mean ± SD, n = 3). (e) Molecular docking model predicting the interaction interface between ITGB2 and ZIKV E protein. (f) Co-IP assay in 293T cells, followed by immunoblotting with anti-ZIKV E protein antibody. (g) CBNK-EVs were pre-incubated with ITGB2 mAb (0, 5, 20 μg/mL) before being applied to Vero-E6 or BHK-21 cells. Cells were then infected with ZIKV (MOI = 1) for 1 h, and antiviral activity was assessed by measuring ZIKV RNA levels at 24 h post-infection. Data are presented as mean ± SD (n = 3 for d and g; n = 6 for b). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (one-way ANOVA).

Article Snippet: Vero E6, BHK21, 293T and HTR-8/Svneo cells were purchased from ATCC.

Techniques: Binding Assay, Multicolor Immunofluorescence Staining, Transfection, Fluorescence, Plasmid Preparation, Infection, Co-Immunoprecipitation Assay, Western Blot, Incubation, Activity Assay