Human liver-derived organoids recapitulate Oropouche virus infection and manifestation, enabling antiviral drug discovery

Oropouche virus (OROV) is a neglected, re-emerging arbovirus that typically causes self-limiting febrile illness but can also result in severe complications. Currently, there are no approved vaccines or treatments for OROV infection. We integrated clinical data from OROV-infected patients with human liver-derived organoid models to investigate the virus's impact on the liver and evaluate repurposed antivirals. Patient blood tests show elevated liver enzymes, suggesting OROV-associated hepatic dysfunction. OROV isolates productively infect liver organoids and induce severe cellular damage. Transcriptomic profiling reveals strong virus-host interactions, including activation of interferon-stimulated genes and cell death pathways. Pharmacological inhibition of the interferon pathway enhances OROV replication, whereas treatment with therapeutic interferon-a suppresses the infection. Molnupiravir, a clinically approved antiviral targeting viral RNA-dependent RNA polymerase, markedly inhibits OROV replication and mitigates virus-induced cytopathology. Combining molnupiravir with interferon-a resulted in synergistic antiviral activity, indicating the complementarity of virus-targeted and host-directed strategies. Overall design: Organoids cultured from adult human liver tissue were infected with both OROV isolates, OROV-1967 (strain RIVM/OROV/TRVL9760) and OROV-2024 (strain IRCCS-SCDC_1/2024_OROV-2024). Infected organoids were harvested at 1- 48-, and 96-hours post-infection. A treatment group was established using organoids infected with the OROV-2024 isolate and treated with 1 µM NHC treatment from 1 hour to 96 hours post-inoculation. As negative controls, uninfected organoids were cultured under same conditions for 96 hours. Total RNA was extracted from all samples using the MachereyNagel NucleoSpin RNA II Kit (Bioke, Netherlands) and quantified with the Bioanalyzer RNA 6000 Picochip. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads for cDNA synthesis and library construction by Novogene. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. RNA sequencing was conducted using a paired-end 150 bp (PE 150) sequencing strategy via Illumina platforms.