Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.
mSphere. 2021 Jan 27;6(1):e01136-20. doi: 10.1128/mSphere.01136-20.
Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide. We previously demonstrated human intestinal stem cell-derived enteroids (HIEs) support cultivation of several HuNoV strains. However, HIEs did not support virus replication from every HuNoV-positive stool sample, which led us to test and optimize new medium conditions, identify characteristics of stool samples that allow replication, and evaluate consistency of replication over time. Optimization of our HIE-HuNoV culture system has shown the following: (i) a new HIE culture medium made with conditioned medium from a single cell line and commercial media promotes robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture medium made with conditioned media from 3 separate cell lines; (ii) GI.1, 11 GII genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.12, GII.13, GII.14, and GII.17), and six GII.4 variants can be cultivated in HIEs; (iii) successful replication is more likely with virus in stools with higher virus titers; (iv) GII.4_Sydney_2012 virus replication was reproducible over 3 years; and (v) HuNoV infection is restricted to the small intestine, based on replication of two viral strains in duodenal and ileal HIEs, but not colonoids, from two susceptible donors. These results improve the HIE culture system for HuNoV replication. Use of HIEs by several laboratories worldwide to study the molecular mechanisms that regulate HuNoV replication confirms the usefulness of this culture system, and our optimized methods for virus replication will advance the development of effective therapies and methods for virus control. Human noroviruses (HuNoVs) are highly contagious and cause acute and sporadic diarrheal illness in all age groups. In addition, chronic infections occur in immunocompromised cancer and transplant patients. These viruses are antigenically and genetically diverse, and there are strain-specific differences in binding to cellular attachment factors. In addition, new discoveries are being made on strain-specific differences in virus entry and replication and the epithelial cell response to infection in human intestinal enteroids. Human intestinal enteroids are a biologically relevant model to study HuNoVs; however, not all strains can be cultivated at this time. A complete understanding of HuNoV biology thus requires cultivation conditions that will allow the replication of multiple strains. We report optimization of HuNoV cultivation in human intestinal enteroid cultures to increase the numbers of cultivatable strains and the magnitude of replication, which is critical for testing antivirals, neutralizing antibodies, and methods of virus inactivation.
人类诺如病毒（HuNoVs）是导致全球暴发和散发急性胃肠炎的主要病原体。我们之前证明，人肠干细胞衍生的类器官（HIEs）可支持多种 HuNoV 株的培养。然而，HIEs 并不能支持来自每个 HuNoV 阳性粪便样本的病毒复制，这促使我们测试和优化新的培养基条件，确定允许复制的粪便样本特征，并评估随时间推移的复制一致性。我们的 HIE-HuNoV 培养系统的优化表明：（i）使用来自单个细胞系的条件培养基和商业培养基制成的新 HIE 培养基可促进 HuNoV 株的强烈复制，这些 HuNoV 株在我们使用来自 3 个独立细胞系的条件培养基制成的原始培养基中生长时复制能力较差；（ii）GI.1、11 GII 基因型（GII.1、GII.2、GII.3、GII.4、GII.6、GII.7、GII.8、GII.12、GII.13、GII.14 和 GII.17）和六种 GII.4 变体可在 HIE 中培养；（iii）病毒载量较高的粪便中，成功复制的可能性更大；（iv）GII.4_Sydney_2012 病毒的复制可在 3 年内重现；（v）基于来自两个易感供体的十二指肠和回肠 HIE 中两种病毒株的复制，HuNoV 感染仅限于小肠，而不是结肠类器官。这些结果改进了 HuNoV 复制的 HIE 培养系统。全世界的几个实验室使用 HIE 来研究调节 HuNoV 复制的分子机制，这证实了该培养系统的有用性，我们优化的病毒复制方法将推进有效治疗方法和病毒控制方法的发展。人类诺如病毒（HuNoVs）具有高度传染性，可导致所有年龄段人群发生急性和散发的腹泻病。此外，免疫功能低下的癌症和移植患者会发生慢性感染。这些病毒具有抗原和遗传多样性，并且在与细胞附着因子结合方面存在株特异性差异。此外，新的发现是关于病毒进入和复制以及人类肠内类器官中上皮细胞对感染的反应的株特异性差异。人类肠内类器官是研究 HuNoVs 的一种生物学相关模型；然而，目前并非所有菌株都可培养。因此，要全面了解 HuNoV 生物学，就需要开发出可复制多种菌株的培养条件。我们报告了 HuNoV 在人肠内类器官培养中的培养优化，以增加可培养菌株的数量和复制程度，这对于测试抗病毒药物、中和抗体和病毒灭活方法至关重要。
