Human intestinal organoid-derived epithelium reveals donor-dependent antiviral responses to therapeutics against enteric picornaviruses

Human picornavirus infections remain without approved antiviral therapies. Antiviral testing performed in immortalised cell lines often fails to predict antiviral efficacy in vivo and subsequently clinical outcomes. Here, we assessed how antiviral responses to clinically approved compounds differ between immortalised intestinal cell lines and a physiologically relevant human intestinal organoid-derived epithelium (HIE) model. Posaconazole, itraconazole, ribavirin, remdesivir, and pocapavir, compounds with reported anti-picornaviral activity in cell lines, were evaluated against enterovirus A71 (EV-A71) and human parechovirus 1 (HPeV-1) in Caco-2 and HT-29 cell lines, and human foetal HIE monolayers from multiple donors. Remdesivir consistently inhibited EV-A71 replication in Caco-2 and HT-29 cell lines, and HPeV-1 replication in Caco-2 cells, whereas the other compounds showed limited or variable effects. In contrast, in HIE, remdesivir and ribavirin reduced EV-A71 replication in only one donor, while itraconazole and posaconazole exhibited donor-specific cytotoxicity. No compound consistently inhibited HPeV-1 replication across HIE donors despite detectable antiviral effect in cell lines. Comparative analysis of drug-metabolising enzyme expression did not fully account for donor-specific response variability. Collectively, these findings reveal substantial heterogeneity in antiviral responses that is not captured by standard cell lines and underscore the value of incorporating genetically and physiologically diverse human organoid-based models into preclinical antiviral screening pipelines to improve translational fidelity.