In a parallel experiment, the route of entry of MNV in the ileum was monitored. Fluorescence microscopic analysis of Q-dot-conjugated viruses detected MNV particles in compartment II. No prominent sign of virion entry was detected through the villi at the ileum without PPs (Fig. 3d). These results demonstrate that MNV primarily enters into the ileum bearing PPs through intestinal villi, possibly via M cells or transepithelial dendritic cells. To confirm the entry of MNV through this part of the ileum, viral genome was monitored upon delivering Q-dot-conjugated MNV directly to the small intestine of mouse ex vivo. High levels of viral genome was detected in the ileum with PPs [in both compartment I representing PPs and compartment II] 8 h after virus delivery (Supplementary Fig. 3a), in agreement with our results showing MNV particles within villi. Consequently, enhanced IFN-β mRNA levels were detected in both ileal compartments while greater induction was observed in ileal PPs (iPPs) early during virus infection (Supplementary Fig. 3b,c), suggesting that MNV entering through villi reached to the PPs to infect ileal sentinel cells. These results are consistent with the previous findings indicating entry of norovirus through the M cells within the follicle-associated epithelium overlying PPs or in villi to infect macrophages and dendritic cells37,39,40.