Primary human intestinal organoids recapitulate 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. The clade IIb MPXV strain has caused the 2022-2023 global outbreak, whereas the Ia and Ib strains are causing the concurrent outbreaks in Africa. Here, we found clinical evidence that MPXV can directly infect human intestine to induce lesions. Intriguingly, primary organoids cultured from human ileum and rectum support productive infections of MPXV clade IIb, Ia and Ib strains. Upon differentiation, we found that enterocytes and goblet cells but not enteroendocrine cells are capable of supporting viral replication. Given that primary intestinal organoids can be rapidly expanded in 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, and consistently demonstrated the potent antiviral activity against clade Ia, Ib and IIb strains. These findings are important for better understanding the clinical manifestations of mpox. Primary intestinal organoids-based infection models and the established antiviral drug discovery pipeline bear major implications in responding to the current mpox global health emergency, and sustaining epidemic poxvirus preparedness. Overall design: RNA-seq profiling of healthy control of human intestinal organoids, Monkeypox virus infected intestinal organoids harvested at 1, 24, 48 and 96 hours respectively, and intestinal organoids infected with Monkeypox virus treated with 1uM clofarabine for 96 hours.