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.

人类诺如病毒 (HuNoV) 是胃肠道疾病的主要原因，环境监测对于预防 HuNoV 爆发至关重要。最近在人肠类肠 (HIEs) 中开发的 HuNoV 细胞培养试验已经能够检测传染性 HuNoV。然而，这种复杂的方法需要适应 HIE，以促进从环境基质中复制 HuNoV。整合来自 200 个实验的数据，我们检查了六个变量：HIE 年龄、HIE 基底膜化合物 (BMC)、HuNoV 接种处理、HuNoV 接种量、低于检测限 (LOD) 的数据处理以及确定 HuNoV 阳性生长的截止标准。我们用 HuNoV GII.4 悉尼阳性粪便感染 HIE，并确定 1.4 × 10 ³HuNoV 复制需要每个 HIE 孔的基因组当量。HIE 年龄对检测结果的影响很小。LOD 替换和截止影响数据解释，较低的值导致较高的估计 HuNoV 检测。较高的接种量导致 HuNoV 生长的最小减少，最佳体积为 250uL，有助于捕获环境分离物中存在的低浓度 HuNoV。处理 HuNoV 接种物对于消毒研究和浓缩样品很有价值，但并非所有 HIE 应用都必需。这项工作增强了用于环境监测的 HuNoV HIE 细胞培养方法。未来的 HIE 研究应将细胞年龄报告为生长天数，并应清楚地描述 BMC 选择、LOD 处理和阳性截止值。