HSBDF-Derived Bioactive Components Broadly Inhibit Enteroviruses by Targeting 3C Protease and Attenuating Inflammatory Responses

Figures

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  Figure 1. HSBDF effectively inhibited CV-A9, CV-B3, and EV-A71. (a) Dose-dependent antiviral activity of HSBDF against CV-A9 (left, RD cells), CV-B3 (middle, Vero E6 cells), and EV-A71 (right, RD cells) (left y-axis), with parallel cytotoxicity profiles (right y-axis). Antiviral activity was analyzed by quantifying viral nucleic acid load in cell lysates; cytotoxicity was assessed via the CellTiter-Blue cell viability assay. (b) Viral particle titers of CV-A9 (left, RD cells), CV-B3 (middle, Vero E6 cells), and EV-A71 (right, RD cells) in supernatants post-0.8 mg/mL HSBDF-treatment, quantified by plaque assay. (c) Dose-dependent antiviral activity of HSBDF against CV-A9 (upper) and EV-A71 (lower) in Huh-7 cells (left y-axis), with corresponding cytotoxicity (right y-axis). (d) Schematic of the time-of-addition assay design. Full-Time: cells were incubated with the drug-virus mixture throughout the infection period. Entry: cells were incubated with the drug-virus mixture for 2 h and washed 2 h after viral infection. Post-Entry: HSBDF was added 2 h after viral infection. Cells were collected for RT-qPCR detection at 12 h post-infection. (e) Intracellular viral RNA levels of CV-A9 (left),…

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  Figure 2. Transcriptome analysis of HSBDF against CV-A9 infection in RD cells. (a) Volcano plot of DEGs between CV-A9-infected RD cells and RD cells. (b) Volcano plot of DEGs between CV-A9-infected RD cells and CV-A9-infected RD cells with HSBDF treatment. The concentration of HSBDF was 1 mg/mL. (c) Heatmap of DEGs between the four groups. (d) Network of enriched terms colored by cluster ID, where nodes that share the same cluster ID are typically close to each other. (e) GO analysis (above) and KEGG pathway enrichment (below) of DEGs in CV-A9-infected cells after HSBDF treatment. (f) Venn diagram reflecting overlapping genes. Green indicates DEGs between CV-A9-infected RD cells and RD cells. Blue indicates DEGs between CV-A9-infected RD cells with HSBDF treatment and CV-A9-infected RD cells. Red indicates DEGs between RD cells with HSBDF treatment and RD cells.

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  Figure 3. Evaluation of effective antiviral components of HSBDF at CV-A9 level. (a) Schematic diagram of screening for effective antiviral components. A total of 119 commercially available components obtained by mass spectrometry analysis of HSBDF were used for anti-CV-A9 activity evaluation. (b) Cytopathic effect (CPE) morphology of RD cells infected with CV-A9 (MOI = 0.001) and treated with compounds (10 μM) for 36 h. Positive control: CV-A9 infection without treatment; negative control: uninfected and untreated cells. (c,e) Dose–response curves of velutin, pyrogallol, isorhamnetin, and (−)-epicatechin gallate against CV-A9 in RD cells (c) and Huh 7 cells (e) (left y-axis: inhibition; right y-axis: cytotoxicity). (d) Viral titers in supernatants after 50 μM compound treatment were quantified by plaque assay. (f) Time-of-addition assays of CV-A9 (MOI = 0.001) for four compounds (25 μM) on RD cells. Cells were collected for RT-qPCR detection at 12 h post-infection. ns, no significant difference; **** p < 0.0001 (g) The plaque assay of supernatants from the time-of-addition assays of Figure (f); a 10−3 gradient is selected in the figure for comparison. Representative curves of at least two…

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  Figure 4. Inhibitory activities of bioactive compounds against other important enteroviruses. Dose–response curves of velutin (a), pyrogallol (b), isorhamnetin (c), and (−)-epicatechin gallate (d) against CV-B3 (MOI = 0.001), EV-A71 (MOI = 0.1), PV1 (MOI = 0.003), Echo-11 (MOI = 0.005). Left y-axis: inhibition; right y-axis: cytotoxicity. Representative curves of at least two independent experiments presented as mean ± SD of three technical replicates. (e) Summary of anti-enterovirus activity of velutin, pyrogallol, isorhamnetin, and (−)-epicatechin gallate, × indicated no antiviral effect at concentrations below 100 μM.

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  Figure 5. Velutin, isorhamnetin, and (−)-epicatechin gallate inhibited enterovirus 3Cpro enzyme activities and alleviated high expression of inflammatory factors. (a–c) Effect of different concentrations of velutin (a), (−)-epicatechin gallate (b), and isorhamnetin (c) on CV-A9 3Cpro cleavage activity in vitro, with rutin as the positive control. (d–f) Molecular docking of CV-A9 3Cpro (green surface) with velutin (d), (−)-epicatechin gallate (e), and isorhamnetin (f). Yellow dashed lines denote hydrogen bonds and attached residues; interacting residues and binding energies (ΔG) are tabulated. (g) Inhibitory effects of velutin, isorhamnetin, and (−)-epicatechin gallate on in vitro cleavage activity of CV-B3 and EV-A71 3Cpro (rutin as positive control). (h–j) mRNA expression levels of IL-6 (top) and TNF-α (bottom) in CV-A9-infected cells (MOI = 0.001) treated with 50 μM velutin (h), isorhamnetin (i), and (−)-epicatechin gallate (j) for 24 h. Data mean ± SD of three technical replicates. ns, no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

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  Figure 6. HSBDF and velutin effectively inhibited EV-A71 in vivo in the BALB/c mouse model. (a) Schematic diagram of the animal experiment. Seven-day-old BALB/c WT mice were intraperitoneally injected with 50 μL EV-A71 per mouse and separately treated intraperitoneally with PBS or compounds. At 5 d.p.i., the viral loads in various tissues of mice were quantified by qRT-PCR. (b) Viral load in spine, colon, heart, or intercostal muscle for each group. ns, no significant difference; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (c) The intestines/heart of mice were stained with hematoxylin and eosin (H&E). The orange arrow indicates epithelial cell detachment; the green arrow indicates intestinal gland necrosis/disappearance, replaced by connective tissue hyperplasia; the blue arrow indicates scattered infiltration of lymphocytes and granulocytes; the yellow arrow indicates the intestinal gland dilatation; and the red arrow indicates vacuolar degeneration of cardiomyocytes.