INTRA- AND INTER-HOST EVOLUTION OF HUMAN NOROVIRUS IN HEALTHY ADULTS.

Background: Human noroviruses are a leading cause of acute and sporadic gastroenteritis worldwide. The evolution of human noroviruses in immunocompromised persons has been evaluated in many studies. Much less is known about the evolutionary dynamics of human norovirus in healthy adults. Methods: We used sequential samples collected from a controlled human infection study with GI.1/Norwalk/US/68 virus to evaluate intra- and inter-host evolution of a human norovirus in healthy adults. Up to 12 samples from day 1 to day 56 post-challenge were sequenced using a norovirus-specific capture probe method. Results: Complete genomes were assembled, even in samples that were below the limit of detection of standard RT-qPCR assays, up to 28 days post-challenge. Analysis of 123 complete genomes showed changes in the GI.1 genome in all persons, but there were no conserved changes across all persons. Single nucleotide variants resulting in non-synonymous amino acid changes were observed in all proteins, with the capsid VP1 and nonstructural protein NS3 having the largest numbers of changes. Conclusions: These data highlight the potential of a new capture-based sequencing approach to assemble human norovirus genomes with high sensitivity and demonstrate limited conserved immune pressure-driven evolution of GI.1 virus in healthy adults.

RNA-dependent RNA polymerase of predominant human norovirus forms liquid-liquid phase condensates as viral replication factories.

Many viral proteins form biomolecular condensates via liquid-liquid phase separation (LLPS) to support viral replication and evade host antiviral responses, and thus, they are potential targets for designing antivirals. In the case of non-enveloped positive-sense RNA viruses, forming such condensates for viral replication is unclear and less understood. Human noroviruses (HuNoV) are positive-sense RNA viruses that cause epidemic and sporadic gastroenteritis worldwide. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of pandemic GII.4 HuNoV forms distinct condensates that exhibit all the signature properties of LLPS with sustained polymerase activity and the capability of recruiting components essential for viral replication. We show that such condensates are formed in HuNoV-infected human intestinal enteroid cultures and are the sites for genome replication. Our studies demonstrate the formation of phase separated condensates as replication factories in a positive-sense RNA virus, which plausibly is an effective mechanism to dynamically isolate RdRp replicating the genomic RNA from interfering with the ribosomal translation of the same RNA.

CONFORMATIONAL FLEXIBILITY IS A CRITICAL FACTOR IN DESIGNING BROAD-SPECTRUM HUMAN NOROVIRUS PROTEASE INHIBITORS.

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is, therefore, an excellent target for developing small molecule inhibitors. While rupintrivir, a potent small-molecule inhibitor of several picornavirus proteases, effectively inhibits GI.1 protease, it is an order of magnitude less effective against GII protease. Other GI.1 protease inhibitors also tend to be less effective against GII proteases. To understand the structural basis for the potency difference, we determined the crystal structures of proteases of GI.1, pandemic GII.4 (Houston and Sydney), and GII.3 in complex with rupintrivir. These structures show that the open substrate pocket in GI protease binds rupintrivir without requiring significant conformational changes, whereas, in GII proteases, the closed pocket flexibly extends, reorienting arginine-112 in the BII-CII loop to accommodate rupintrivir. Structures of R112A protease mutants with rupintrivir, coupled with enzymatic and inhibition studies, suggest R112 is involved in displacing both substrate and ligands from the active site, implying a role in the release of cleaved products during polyprotein processing. Thus, the primary determinant for differential inhibitor potency between the GI and GII proteases is the increased flexibility in the BII-CII loop of the GII proteases caused by H-G mutation in this loop. Therefore, the inherent flexibility of the BII-CII loop in GII proteases is a critical factor to consider when developing broad-spectrum inhibitors for HuNoV proteases.

Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples-a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.

Infant and adult human intestinal enteroids are morphologically and functionally distinct.

Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population. IMPORTANCE: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction.

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease.

Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays.

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.

Insights into Human Norovirus Cultivation in Human Intestinal Enteroids.

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 to 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 into intestinal organoids that were transplanted and matured in mice before making enteroids (H9tHIEs), genetically-engineered (J4 FUT2 knock-in [ KI ], J2 STAT1 knock-out [ 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 GI and GII 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: HuNoVs cause global diarrheal illness and chronic infections in immunocompromised patients. This manuscript 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 chronic 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-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.

Bile acid-sensitive human norovirus strains are susceptible to sphingosine-1-phosphate receptor 2 inhibition.

Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause endemic and pandemic acute viral gastroenteritis. Previously, we reported that many HuNoV strains require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. BA was not essential for the replication of a pandemic-causing GII.4 HuNoV strain. We found the hydrophobic BA glycochenodeoxycholic acid (GCDCA) promotes the replication of the BA-dependent strain GII.3 in jejunal enteroids. Furthermore, we found that inhibition of the G-protein-coupled BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), by JTE-013, reduced GII.3 infection dose-dependently and inhibited GII.3 cellular uptake in enteroids. Herein, we sought to determine whether S1PR2 is required for other BA-dependent HuNoV strains, the BA-independent GII.4, and whether S1PR2 is required for BA-dependent HuNoV infection in HIEs from other small intestinal segments. We found a second S1PR2 inhibitor, GLPG2938, reduces GII.3 infection dose-dependently, and an S1PR2 agonist (CYM-5520) enhances GII.3 replication in the absence of GCDCA. GII.3 replication also is abrogated in the presence of JTE-013 and CYM-5520. JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not GII.4 Sydney (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. Finally, GII.3 infection of duodenal, jejunal, and ileal lines derived from the same individual is reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoVs exploit BA effects on S1PR2 to infect the entire small intestine.IMPORTANCEHuman noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA-independent strain, all require S1PR2 for infection. In addition, BA-dependent infection requires S1PR2 in multiple segments of the small intestine. Together, these results indicate that S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.

GI.1 Norovirus Neutralizing Antibody Levels Are Correlated with GI.1 Histo-blood Group Antigen-Blocking Antibody Levels.

BACKGROUND: The in vitro cultivation of human noroviruses allows a comparison of antibody levels measured in neutralization and histoblood group antigen (HBGA)-blocking assays. METHODS: Serum samples collected during the evaluation of an investigational norovirus vaccine (HIL-214 [formerly TAK-214]) were assayed for neutralizing antibody levels against the vaccine's prototype Norwalk virus/GI.1 (P1) virus strain. Results were compared to those previously determined using HBGA-blocking assays. RESULTS: Neutralizing antibody seroresponses were observed in 83% of 24 vaccinated adults, and antibody levels were highly correlated (r=0.81, P<0.001) with those measured by HBGA-blocking. CONCLUSIONS: GI.1-specific HBGA-blocking antibodies are a surrogate for neutralization of GI.1 norovirus.

2'-Fucosyllactose Inhibits Human Norovirus Replication in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Currently, there are no targeted antivirals for the treatment of HuNoV infection. Histo-blood group antigens (HBGAs) on the intestinal epithelium are cellular attachment factors for HuNoVs; molecules that block the binding of HuNoVs to HBGAs thus have the potential to be developed as antivirals. Human milk oligosaccharides (HMOs) are glycans in human milk with structures analogous to HBGAs. HMOs have been shown to act as decoy receptors to prevent the attachment of multiple enteric pathogens to host cells. Previous X-ray crystallography studies have demonstrated the binding of HMO 2'-fucosyllactose (2'FL) in the same pocket as HBGAs for some HuNoV strains. We evaluated the effect of 2'FL on the replication of a globally dominant GII.4 Sydney [P16] HuNoV strain using human intestinal enteroids (HIEs) from adults and children. A significant reduction in GII.4 Sydney [P16] replication was seen in duodenal and jejunal HIEs from multiple adult donors, all segments of the small intestine from an adult organ donor and in two pediatric duodenal HIEs. However, 2'FL did not inhibit HuNoV replication in two infant jejunal HIEs that had significantly lower expression of α1-2-fucosylated glycans. 2'FL can be synthesized in large scale, and safety and tolerance have been assessed previously. Our data suggest that 2'FL has the potential to be developed as a therapeutic for HuNoV gastroenteritis.

N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Bile acid-sensitive human norovirus strains are susceptible to sphingosine-1-phosphate receptor 2 inhibition.

Human noroviruses (HuNoVs) are a diverse group of RNA viruses that cause both endemic and pandemic acute viral gastroenteritis. Previously we reported that many strains of HuNoV require bile or bile acid (BA) to infect human jejunal intestinal enteroid cultures. Of note, BA was not essential for replication of a pandemic-causing GII.4 HuNoV strain. Using the BA-requiring strain GII.3, we found that the hydrophobic BA GCDCA induces multiple cellular responses that promote replication in jejunal enteroids. Further, we found that chemical inhibition of the G-protein coupled receptor, sphingosine-1- phosphate receptor 2 (S1PR2), by JTE-013 reduced both GII.3 infection in a dose- dependent manner and cellular uptake in enteroids. Herein, we sought to determine if S1PR2 is required by other BA-dependent HuNoV strains and BA-independent GII.4, and if S1PR2 is required for BA-dependent HuNoV infection in other segments of the small intestine. We found JTE-013 inhibition of S1PR2 in jejunal HIEs reduces GI.1, GII.3, and GII.17 (BA-dependent) but not the GII.4 Sydney variant (BA-independent) infection, providing additional evidence of strain-specific differences in HuNoV infection. GII.3 infection of duodenal, jejunal and ileal lines derived from the same individual was also reduced with S1PR2 inhibition, indicating a common mechanism of BA-dependent infection among multiple segments of the small intestine. Our results support a model where BA-dependent HuNoV exploit the activation of S1PR2 by BA to infect the entire small intestine. Importance: Human noroviruses (HuNoVs) are important viral human pathogens that cause both outbreaks and sporadic gastroenteritis. These viruses are diverse, and many strains are capable of infecting humans. Our previous studies have identified strain-specific requirements for hydrophobic bile acids (BAs) to infect intestinal epithelial cells. Moreover, we identified a BA receptor, sphingosine-1-phosphate receptor 2 (S1PR2), required for infection by a BA-dependent strain. To better understand how various HuNoV strains enter and infect the small intestine and the role of S1PR2 in HuNoV infection, we evaluated infection by additional HuNoV strains using an expanded repertoire of intestinal enteroid cell lines. We found that multiple BA-dependent strains, but not a BA- independent strain, all required S1PR2 for infection. Additionally, BA-dependent infection required S1PR2 in multiple segments of the small intestine. Together these results indicate S1PR2 has value as a potential therapeutic target for BA-dependent HuNoV infection.

Infant and Adult Human Intestinal Enteroids are Morphologically and Functionally Distinct.

Background & Aims: Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. Methods: We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. Results: RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. Conclusions: HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population.

Rotavirus-mediated DGAT1 degradation: A pathophysiological mechanism of viral-induced malabsorptive diarrhea.

Gastroenteritis is among the leading causes of mortality globally in infants and young children, with rotavirus (RV) causing ~258 million episodes of diarrhea and ~128,000 deaths annually in infants and children. RV-induced mechanisms that result in diarrhea are not completely understood, but malabsorption is a contributing factor. RV alters cellular lipid metabolism by inducing lipid droplet (LD) formation as a platform for replication factories named viroplasms. A link between LD formation and gastroenteritis has not been identified. We found that diacylglycerol O-acyltransferase 1 (DGAT1), the terminal step in triacylglycerol synthesis required for LD biogenesis, is degraded in RV-infected cells by a proteasome-mediated mechanism. RV-infected DGAT1-silenced cells show earlier and increased numbers of LD-associated viroplasms per cell that translate into a fourfold-to-fivefold increase in viral yield (P < 0.05). Interestingly, DGAT1 deficiency in children is associated with diarrhea due to altered trafficking of key ion transporters to the apical brush border of enterocytes. Confocal microscopy and immunoblot analyses of RV-infected cells and DGAT1-/- human intestinal enteroids (HIEs) show a decrease in expression of nutrient transporters, ion transporters, tight junctional proteins, and cytoskeletal proteins. Increased phospho-eIF2α (eukaryotic initiation factor 2 alpha) in DGAT1-/- HIEs, and RV-infected cells, indicates a mechanism for malabsorptive diarrhea, namely inhibition of translation of cellular proteins critical for nutrient digestion and intestinal absorption. Our study elucidates a pathophysiological mechanism of RV-induced DGAT1 deficiency by protein degradation that mediates malabsorptive diarrhea, as well as a role for lipid metabolism, in the pathogenesis of gastroenteritis.

Human intestinal organoids from Cronkhite-Canada syndrome patients reveal link between serotonin and proliferation.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation.

Standardization of an antiviral pipeline for human norovirus in human intestinal enteroids demonstrates nitazoxanide has no to weak antiviral activity.

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.

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity.

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.

A Bivalent Human Norovirus Vaccine Induces Homotypic and Heterotypic Neutralizing Antibodies.

A GII.2 outbreak in an efficacy study of a bivalent virus-like particle (VLP) norovirus vaccine, TAK-214, in healthy US adults provided an opportunity to examine GII.4 homotypic vs. GII.2 heterotypic responses to vaccination and infection. Three serological assays (VLP-binding, histoblood group antigen-blocking, and neutralizing) were performed for each genotype. Results were highly correlated within a genotype but not between genotypes. Although the vaccine provided protection from GII.2-associated disease, little GII.2-specific neutralization occurred after vaccination. Choice of antibody assay can affect assessments of human norovirus vaccine immunogenicity.