Noroviruses, in the genus Norovirus, are a significant cause of viral gastroenteritis in humans and animals. For almost 50 years, the lack of a cultivation system for human noroviruses (HuNoVs) was a major barrier to understanding virus biology and the development of effective antiviral strategies. This review presents a historical perspective of the development of a cultivation system for HuNoVs in human intestinal epithelial cell cultures. Successful cultivation was based on the discovery of genetically-encoded host factors required for infection, knowledge of the site of infection in humans, and advances in the cultivation of human intestinal epithelial cells achieved by developmental and stem cell biologists. The human stem cell-derived enteroid cultivation system recapitulates the multicellular, physiologically active human intestinal epithelium, and allows studies of virus-specific replication requirements, evaluation of human host-pathogen interactions, and supports the pre-clinical assessment of methods to prevent and treat HuNoV infections.

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.

Globally, most cases of gastroenteritis are caused by pandemic GII.4 human norovirus (HuNoV) strains with no approved therapies or vaccines available. The cellular pathways that these strains exploit for cell entry and internalization are unknown. Here, using nontransformed human jejunal enteroids (HIEs) that recapitulate the physiology of the gastrointestinal tract, we show that infectious GII.4 virions and virus-like particles are endocytosed using a unique combination of endosomal acidification-dependent clathrin-independent carriers (CLIC), acid sphingomyelinase (ASM)-mediated lysosomal exocytosis, and membrane wound repair pathways. We found that besides the known interaction of the viral capsid Protruding (P) domain with host glycans, the Shell (S) domain interacts with both galectin-3 (gal-3) and apoptosis-linked gene 2-interacting protein X (ALIX), to orchestrate GII.4 cell entry. Recognition of the viral and cellular determinants regulating HuNoV entry provides insight into the infection process of a non-enveloped virus highlighting unique pathways and targets for developing effective therapeutics.

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 FUT2 knock-in [ KI ] , J2 STAT1 knockout [ KO ]) 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 FUT2-KI 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. IMPORTANCE: 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 FUT2 knock-in ( KI ) 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.

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a “raised” conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis. In immunocompetent hosts, symptoms usually resolve within 3 days; however, in immunocompromised persons, HuNoV infection can become persistent, debilitating, and sometimes life-threatening. There are no licensed therapeutics for HuNoV due to a near half-century delay in its cultivation. Treatment for chronic HuNoV infection in immunosuppressed patients anecdotally includes nitazoxanide, a broad-spectrum antimicrobial licensed for treatment of parasite-induced gastroenteritis. Despite its off-label use for chronic HuNoV infection, nitazoxanide has not been clearly demonstrated to be an effective treatment. In this study, we standardized a pipeline for antiviral testing using multiple human small intestinal enteroid lines representing different intestinal segments and evaluated whether nitazoxanide inhibits replication of five HuNoV strains in vitro . Nitazoxanide did not exhibit high selective antiviral activity against any HuNoV strain tested, indicating it is not an effective antiviral for HuNoV infection. Human intestinal enteroids are further demonstrated as a model to serve as a preclinical platform to test antivirals against HuNoVs to treat gastrointestinal disease. Abstr