Ondansetron exhibits potent antiviral activity against enterovirus 71 infection in vitro and in vivo

Figures

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  Fig. 1. Molecular structure of ondansetron and its antiviral effect against EV-A71 in vitro. (A) Chemical structures of ondansetron. (B) Dose-dependent antiviral efficacy of ondansetron against EV-A71 in rhabdomyosarcoma (RD) cells. Cells were infected with the EV-A71 H strain (100 × TCID₅₀) and treated with serial dilutions of the compound. At 72 h post-infection (hpi), cell viability was quantified using the CellTiter-Glo assay to determine the half-maximal effective concentration (EC₅₀) and half-maximal cytotoxic concentration (CC₅₀). Selectivity indices (SI = CC₅₀/EC₅₀) were calculated as >48.63 (ondansetron). (C-D) Suppression of viral replication and progeny virion production. RD cells were infected with EV-A71 (MOI = 0.001 PFU/mL) and treated with modulators. Viral RNA levels in cell lysates were quantified by qRT-PCR at 36hpi (C), while infectious viral particles in supernatants were assessed via plaque-forming unit (PFU) assays (D). Data represent mean ± SD of triplicate experiments. Statistical significance was analyzed by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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  Fig. 2. Effects of Ondansetron on EV-A71 VP1 Expression: Immunofluorescence and Immunoblot Analysis. (A) RD cells were inoculated with EV-A71 (MOI = 0.001 PFU/cell) and concomitantly treated with ondansetron. Following 48 h post-infection (h.p.i.), cells were fixed for immunofluorescence analysis. Intracellular VP1 proteins were immunolabeled with mouse anti-EV-A71 VP1 monoclonal antibody (12D7; green fluorescence), while nuclei were counterstained with Hoechst 33,342 (blue fluorescence). Representative confocal microscopy images are shown (scale bar: 200 μm). (B) EV-A71-infected RD cells (MOI = 0.001 PFU/cell) were pharmacologically treated with ondansetron. Cellular lysates were harvested at 36 h.p.i. and analyzed by SDS-PAGE followed by immunoblotting using anti-VP1 antibodies (12D7). α-Tubulin was employed as a loading control.

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  Fig. 3. Ondansetron Specifically Inhibits the Pre-entry Phase of EV-A71 Infection. (A) Temporal pharmacological intervention protocol. Schematic representation of compound administration regimens relative to viral infection timeline. (B) Intracellular viral RNA quantification. EV-A71-infected RD cells were subjected to experimental treatments following the temporal protocol in Panel A. Viral RNA was isolated at 12 h post-treatment and quantified by quantitative reverse transcription PCR (qRT-PCR). (C). In virus attachment assay, EV-A71 virus particles were pre-incubated with 0.1% DMSO vehicle control or Ondansetron (30 μM) and used to infect RD cells at M.O.I of 0.01 at 4 °C. After one hour, used DMEM medium to wash the cell and cultured the cell with new medium. The virus RNA was detected after 24 h. (D). In virus internalization assay, RD cells were infected with EV-A71 at M.O.I of 0.01 at 4 °C for one hour and washed the cell. Then the cell was treated with 0.1% DMSO vehicle control or Ondansetron (30 μM) at 37 °C. After one hour, washed the cell and cultured for another 24 h until to detect the virus RNA copies. Data represent mean ± standard deviation (SD) from three independent…

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  Fig. 4. In vivo antiviral efficacy of ondansetron against EV-A71 infection. (A) Survival and Bodyweight Assessment: Neonatal ICR mice (1-day-old, n = 3 experimental groups) were intraperitoneally (i.p.) inoculated with 10⁶ plaque-forming units (PFU) of EV-A71 (H strain). Subsequently, mice received daily i.p. administration of ondansetron or vehicle control at specified doses for six consecutive days. Survival rates (A), body weight dynamics (B) and clinical score were recorded daily until 21 days or 14 days in (C) post-infection (d.p.i.). Data in B was expressed as mean ± standard deviation (SD). Survival curve was analyzed by log-rank test. *p < 0.05, **p < 0.01, ****p < 0.0001. (C-E) Tissue-Specific Viral Load Analysis: Neonatal mice (n = 2 groups) were pretreated with ondansetron (1.5mg/kg) via i.p. injection at 12-hour intervals for three days, commencing 24 h prior to challenge with 10⁶ PFU of EV-A71 (H strain). Post-euthanasia, tissue samples (brain: D; hind limp muscle: E; lung: F; liver: G; spleen: H) were harvested for quantification of viral RNA copy numbers using quantitative reverse transcription PCR (qRT-PCR). Data represent mean ± SD. Statistical significance was…

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  Fig. 5. Cholesterol-related homeostasis is a critical determinant of EV-A71 pathogenesis. RD cells were inoculated into 12-well plates at 4 × 10⁵ cells/well and cultured overnight. Subsequently, cells were infected with 0.001 MOI of EV-A71 with the presence of 30 μM of ondansetron. At 24hpi, cells were harvested to extract total RNA and subjected to transcriptome analysis. (A). The common and unique differentially expressed genes were identified by a Venn diagram. (B). Minus-versus-add plot identified the complex upregulation and downregulation. (C). The number of differentially expressed genes (DEGs). Top enriched GO annotation analysis (D) and KEGG enrichment analysis (E) for differentially expressed genes that were upregulated by EV-A71 infection and restored by ondansetron treatment. (F). The heatmap of key DEGs related to EV-A71 infection and ondansetron intervention were visualized. The color shows the fold change of detected genes. (G). The relative expression of ABCA1, ABCG1 and HMGCS1genes were detected by RT-qPCR. (H). The RD cell was treated with EV-A71 or EV-A71 plus ondansetron (MOI: 0.001, ondansetron 30 μM) and the cell cholesterol was detected by Amplex Red Cholesterol…