Development of rhesus macaque enteroids and enteroid-derived cell lines for enteric calicivirus infections

Recoviruses (ReCV) serve as valuable surrogates for fastidious human noroviruses (HuNoV). Although HuNoVs can be propagated in small intestinal enteroids, this system has several limitations, including low replication efficiency and an inability to sustain continuous propagation for generating infectious virus stocks beyond certain strains, such as GII.3. In contrast, ReCVs can be efficiently propagated in conventional cell lines (e.g., LLC-MK2), which has significantly advanced understanding of their biological characteristics. As a result, ReCVs are widely used as HuNoV surrogates. However, a physiologically relevant ReCV propagation system that closely mimics HuNoV enteroid cultures has not yet been described. Here, we report the establishment of intestinal stem cell-derived rhesus macaque enteroid lines representing both the small intestine and colon, along with two enteroid-derived cell lines. We further characterize these systems and evaluate their susceptibility to infection with three diverse ReCV isolates. Both small intestine- and colon-derived enteroids, as well as their corresponding cell lines, supported robust ReCV infection. The enteroids contained all major intestinal cell types. The duodenum-derived cell line comprised enteroendocrine cells (chromogranin A) and absorptive enterocytes (villin), whereas the colon-derived cell line included enteroendocrine cells and goblet cells (MUC2). Importantly, both cell lines formed tight barriers (transepithelial electrical resistance [TEER] > 3,000 Ω·cm²), making them far superior to LLC-MK2 cells (TEER ~ 100 Ω·cm²) for studies of viral entry and pathogenesis. The rhesus enteroids and intestinal cell lines developed in this study will enable comparative studies of HuNoV and ReCV replication in a physiologically relevant system and may have direct applications for culturing HuNoVs.IMPORTANCEHuman intestinal stem cell-derived enteroids have enabled the propagation of human noroviruses; however, this system has several important limitations, including high cost, low replication efficiency, and (except for a recent report for GII.3 human norovirus) the inability to sustain continuous virus propagation for the generation of infectious virus stocks. As a result, research still relies on human norovirus-containing clinical samples as the primary virus source. Here, we developed host-specific rhesus macaque enteroid cultures and two enteroid-derived cell lines that support robust recovirus replication. Studying recovirus replication in rhesus enteroids may help overcome key limitations of the human norovirus enteroid cultures. Moreover, because both natural and experimental human norovirus infections have been described in rhesus macaques, the rhesus enteroids and enteroid-derived cell lines described in this study may be directly applicable to human norovirus cultivation.