Establishment of a Novel Caco-2-Based Cell Culture System for Human Sapovirus Propagation

Human sapovirus (HuSaV), first identified in the 1970s, is a significant cause of acute gastroenteritis, particularly in young children. Despite its clinical significance, research on HuSaV has been limited due to the absence of a reliable cell culture system. In 2020, a breakthrough study reported that HuSaV GI.1 and GII.3 strains could be cultured and serially propagated using HuTu80 cells in the presence of bile acids. However, in 2024, a subsequent study reported that effective replication in HuTu80 cells requires specialized cells that have undergone over 100 passages. In this study, we sought to identify an alternative cell culture system for HuSaV. HuSaV GI.1 can replicate and be serially propagated using Caco-2 cells under bile acid supplementation. Importantly, the Caco-2 cells were freshly sourced from the American Type Culture Collection, ensuring reproducibility for laboratories worldwide. Furthermore, Caco-2MC cells were established via single-cell cloning from in-house Caco-2/Cas9 cells with 91.5% HuSaV-susceptible. HuSaV strains GI.1, GI.2, GI.3, GII.1, GII.3, and GV.1 were successfully propagated using Caco-2MC cells, with RNA copy numbers increasing up to 4.4 log₁₀-fold within 5 days post-infection. This efficient HuSaV cell culture system represents a significant advancement in HuSaV research.