Department of Molecular Virology and Microbiology, Baylor College of Medicine (BCM), Houston, Texas, USA.
Department of Pediatric Surgery, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, USA.
mSphere. 2024 Nov 21;9(11):e0044824. doi: 10.1128/msphere.00448-24. Epub 2024 Oct 15.
Human noroviruses (HuNoVs) are a significant cause of epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system hindered the study of HuNoV replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We optimized culture media conditions and generated genetically modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present new insights into this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs from human intestinal organoids produced from directed differentiation of human embryonic stem cells that were then transplanted and matured in mice before making enteroids (H9tHIEs), genetically engineered (J4 knock-in [] J2 knockout []) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4 HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of genogroup I and II HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research.Human noroviruses (HuNoVs) cause global diarrheal illness and chronic infections in immunocompromised patients. This paper reports approaches for cultivating HuNoVs in secretor positive human intestinal enteroids (HIEs). HuNoV infectivity was compared in new HIE models, including ones from (i) different intestinal segments of single donors, (ii) human embryonic stem cell-derived organoids transplanted into mice, (iii) genetically modified lines, and (iv) a patient with common variable immunodeficiency disease. HIEs from small intestine, but not colon, support HuNoV replication with donor, segment, and strain-specific variations. Unexpectedly, HIEs from one donor are resistant to GII.3 infection. The genetically modified J4 knock-in () HIEs enable cultivation of a broad range of GI and GII genotypes. New insights into strain-specific differences in HuNoV replication in HIEs support this platform for advancing understanding of HuNoV biology and developing potential therapeutics.
人类诺如病毒（HuNoVs）是全球流行性和散发性急性胃肠炎的重要病因。由于缺乏可复制的培养体系，HuNoV 的复制和发病机制研究几乎停滞了半个世纪。我们成功地在人类肠类器官（HIEs）中培养了多种 HuNoV 株，从而克服了这一障碍，这极大地推动了 HuNoV 的研究。我们优化了培养条件，并生成了基因修饰的 HIE 培养物，以增强 HuNoV 在 HIE 中的复制。在此基础上，我们现在对该培养系统提出了新的见解，包括测试不同的培养基、独特的 HIE 系和其他病毒株。我们评估并比较了 HuNoV 在新的 HIE 模型中的感染性，这些模型包括来自个体成人供体不同肠段的 HIEs、来自人类胚胎干细胞定向分化产生的、然后移植到小鼠中成熟的肠类器官（H9tHIEs）、基因工程（J4 敲入 [] J2 敲除 []）HIEs，以及来自普通变异性免疫缺陷（CVID）患者和婴儿的 HIEs。我们的研究结果表明，来自成人的小肠 HIEs（而非结肠类器官）、H9tHIEs、来自 CVID 患者的 HIEs 和来自婴儿的 HIEs 支持 HuNoV 复制，但病毒感染存在节段和株特异性差异。J4 HIEs 对 HuNoV 感染的敏感性最高，允许培养更广泛的 I 型和 II 型 HuNoV 株，这是以前未曾报道过的。总之，这些结果有助于深入了解 HuNoVs，并突出了 HIE 培养在 HuNoV 研究中的变革潜力。人类诺如病毒（HuNoVs）可导致全球腹泻病和免疫功能低下患者的慢性感染。本文报道了在分泌型阳性的人类肠类器官（HIEs）中培养 HuNoVs 的方法。我们比较了 HuNoV 在新的 HIE 模型中的感染性，这些模型包括（i）来自单个供体的不同肠段、（ii）移植到小鼠中的人类胚胎干细胞衍生的类器官、（iii）基因修饰的系和（iv）普通变异性免疫缺陷病患者的 HIEs。来自小肠而非结肠的 HIEs 支持 HuNoV 复制，但存在供体、节段和株特异性差异。出人意料的是，一个供体的 HIEs 对 GII.3 感染具有抗性。基因修饰的 J4 敲入（）HIEs 可培养广泛的 GI 和 GII 基因型。HIEs 中 HuNoV 复制的株特异性差异的新见解支持了该平台，有助于深入了解 HuNoV 生物学并开发潜在的治疗方法。
