Optimizing Human Intestinal Enteroids for Environmental Monitoring of Human Norovirus

Human noroviruses (HuNoV) are the leading cause of gastrointestinal illness and environmental monitoring is crucial to prevent HuNoV outbreaks. The recent development of a HuNoV cell culture assay in human intestinal enteroids (HIEs) has enabled detection of infectious HuNoV. However, this complex approach requires adaptation of HIEs to facilitate HuNoV replication from environmental matrixes. Integrating data from 200 experiments, we examined six variables: HIE age, HIE basement membrane compounds (BMC), HuNoV inoculum processing, HuNoV inoculum volume, treatment of data below limit of detection (LOD), and cutoff criteria for determining positive HuNoV growth. We infected HIEs with HuNoV GII.4 Sydney positive stool and determined 1.4 × 10³ genome equivalents per HIE well were required for HuNoV replication. HIE age had minimal effect on assay outcomes. LOD replacement and cutoff affected data interpretation, with lower values resulting in higher estimated HuNoV detection. Higher inoculum volumes lead to minimal decreases in HuNoV growth, with an optimal volume of 250uL facilitating capture of low concentrations of HuNoVs present in environmental isolates. Processing of HuNoV inoculum is valuable for disinfection studies and concentrating samples but is not necessary for all HIE applications. This work enhances the HuNoV HIE cell culture approach for environmental monitoring. Future HIE research should report cell age as days of growth and should clearly describe BMC choice, LOD handling, and positive cutoff.