Primary human intestinal organoids model enteric infection of monkeypox virus and enable scalable drug discovery.

Monkeypox virus (MPXV) infection-associated intestinal manifestations, including diarrhea and proctitis, have been frequently reported during mpox outbreaks. Here, we present clinical evidence that MPXV can directly infect the human intestine and induce lesions. Intriguingly, primary organoids cultured from human ileum and rectum support productive infections by MPXV strains from clade IIb, Ia, and Ib, which are responsible for the 2022-2023 global outbreak and concurrent outbreaks in Africa. Given that primary intestinal organoids can be rapidly expanded at large scale, we were able to screen a broad-spectrum antiviral drug library. We identified 12 leading candidates of safe-in-human agents, including clinically used drugs such as clofarabine. We extensively validated the anti-MPXV activity of clofarabine in human intestinal and skin organoids, consistently demonstrating potent antiviral activity against clade Ia, Ib, and IIb strains. These findings are important for better understanding the clinical manifestations of mpox. Primary intestinal organoid-based infection models and the established antiviral drug discovery pipeline bear major implications for responding to the current mpox global health emergency and sustaining epidemic poxvirus preparedness.