Next Generation Sequencing analysis of OC43 and EV1 viral infection on macrophage-augmented intestinal organoids following treatment with nirmatrelvir and acetate

The pathogenesis of enteric viral infections arises from dual mechanisms: intrinsic viral replication dynamics and dysregulated host immune-inflammatory cascades. To bridge this knowledge gap, we developed macrophage-augmented intestinal organoids (MaugOs), a physiologically relevant model engineered by co-culturing primary human intestinal organoids with autologous macrophages. Using this platform, we interrogated five enteric viruses with distinct intestinal tropisms: Echovirus 1, Echovirus 6, Rotavirus, seasonal coronavirus OC43, and *SARS-CoV-2*. Key findings revealed that all tested viruses exhibited robust replication in MaugOs and activated canonical antiviral pathways (e.g., IFN-β/ISG15). Notably, Rotavirus, OC43, and *SARS-CoV-2* uniquely elicited inflammatory responses (IL-6/IL-8 upregulation), contrasting with the non-inflammatory phenotype of echoviruses. Mechanistically, acetate—a dominant gut microbial metabolite—dose-dependently attenuated virus-induced inflammation (NF-κB/STAT3 inhibition), while exhibiting cell-compartment-specific effects on viral replication: suppressing OC43 replication in macrophages but enhancing it in organoids. Furthermore, we established a combinatorial therapeutic strategy in MaugOs by pairing the antiviral agent nirmatrelvir with either anti-inflammatory drugs or acetate, achieving dual suppression of viral load (↓90%) and cytokine storm (IL-6/IL-8 ↓70%). These results position MaugOs as a transformative platform for 1) dissecting spatial virus-immune-epithelial interactions and 2) accelerating drug discovery targeting both viral pathogenesis and immunopathology.