Human Norovirus Replication Research Articles

Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Norovirus-mediated translation repression promotes macrophage cell death.

Norovirus infection is characterised by a rapid onset of disease and the development of debilitating symptoms including projectile vomiting and diffuse diarrhoea. Vaccines and antivirals are sorely lacking and developments in these areas are hampered by the lack of an adequate cell culture system to investigate human norovirus replication and pathogenesis. Herein, we describe how the model norovirus, Mouse norovirus (MNV), produces a viral protein, NS3, with the functional capacity to attenuate host protein translation which invokes the activation of cell death via apoptosis. We show that this function of NS3 is conserved between human and mouse viruses and map the protein domain attributable to this function. Our study highlights a critical viral protein that mediates crucial activities during replication, potentially identifying NS3 as a worthy target for antiviral drug development.

Development of a broad-spectrum therapeutic Fc-nanobody for human noroviruses.

Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.

Human norovirus cultivation systems and their use in antiviral research.

Human norovirus (HuNoV) is a major cause of acute gastroenteritis and foodborne diseases, affecting all age groups. Despite its clinical needs, no approved antiviral therapies are available. Since the discovery of HuNoV in 1972, studies on anti-norovirals, mechanism of HuNoV infection, viral inactivation, etc., have been hampered by the lack of a robust laboratory-based cultivation system for HuNoV. A recent breakthrough in the development of HuNoV cultivation systems has opened opportunities for researchers to investigate HuNoV biology in the context of de novo HuNoV infections. A tissue stem cell-derived human intestinal organoid/enteroid (HIO) culture system is one of those that supports HuNoV replication reproducibly and, to our knowledge, is most widely distributed to laboratories worldwide to study HuNoV and develop therapeutic strategies. This review summarizes recently developed HuNoV cultivation systems, including HIO, and their use in antiviral studies.

Current trends and new approaches for human norovirus replication in cell culture: a literature review.

Human norovirus (HuNoV) is one of the world's leading causes of acute gastroenteritis. At present, effective reproduction of the virus in cell cultures remains a challenge for virologists, as there is a lack of a permissive cell line that allows the entire viral life cycle to be reproduced. This is a barrier to the study of the HuNoV life cycle, its tropism, and virus-host interactions. It is also a major hurdle for the development of viral detection platforms, and ultimately for the development of therapeutics. The lack of an inexpensive, technically simple, and easily implemented cultivation method also negatively affects our ability to evaluate the efficacy of a variety of control measures (disinfectants, food processes) for human norovirus. In the process of monitoring this pathogen, it is necessary to detect infectious viral particles in water, food, and other environmental samples. Therefore, improvement of in vitro replication of HuNoV is still needed. In this review, we discuss current trends and new approaches to HuNoV replication in cell culture. We highlight ways in which previous research on HuNoV and other noroviruses has guided and influenced the development of new HuNoV culture systems and discuss the improvement of in vitro replication of HuNoV.

Molnupiravir inhibits human norovirus and rotavirus replication in 3D human intestinal enteroids

Human norovirus (HuNoV) and human rotavirus (HRV) are the leading causes of gastrointestinal diarrhea. There are no approved antivirals and rotavirus vaccines are insufficient to cease HRV associated mortality. Furthermore, treatment of chronically infected immunocompromised patients is limited to off-label compassionate use of repurposed antivirals with limited efficacy, highlighting the urgent need of potent and specific antivirals for HuNoV and HRV. Recently, a major breakthrough in the in vitro cultivation of HuNoV and HRV derived from the use of human intestinal enteroids (HIEs). The replication of multiple circulating HuNoV and HRV genotypes can finally be studied and both in the same non-transformed and physiologically relevant model. Activity of previously described anti-norovirus or anti-rotavirus drugs, such as 2′-C-methylcytidine (2CMC), 7-deaza-2′-C-methyladenosine (7DMA), nitazoxanide, favipiravir and dasabuvir, was assessed against clinically relevant human genotypes using 3D-HIEs. 2CMC showed the best activity against HuNoV GII.4, while 7DMA was the most potent antiviral against HRV. We identified the anti-norovirus and -rotavirus activity of molnupiravir and its active metabolite, N4-hydroxycytidine (NHC), a broad-spectrum antiviral used to treat coronavirus disease 2019 (COVID-19). Molnupiravir and NHC inhibit HuNoV GII.4, HRV G1P[8], G2P[4] and G4P[6] in 3D-HIEs with high selectivity and show a potency comparable to 2CMC against HuNoV. Moreover, molnupiravir and NHC block HRV viroplasm formation, but do not alter its size or subcellular localization. Taken together, molnupiravir inhibits both HuNoV and HRV replication, suggesting that the drug could be a candidate for the treatment of patients chronically infected with either one of these diarrhea causing viruses.

Replication of Human Norovirus in Human Intestinal Enteroids Is Affected by Fecal Sample Processing.

Human intestinal enteroids (HIEs) culture is an emerging model for assessing the infectivity of human noroviruses (HuNoVs). The model is based on detecting an increase in HuNoV RNA post-infection of HIEs. HuNoV fecal samples used for HIE infection are traditionally processed by serial filtration. Recently, processing HuNoV fecal samples by serial centrifugation was shown to retain vesicles containing HuNoV. The objective of this study was to investigate whether serially centrifuged fecal samples, RNA extraction kit (QIAamp versus MagMaX) and HIE age (newer versus older) affect HuNoV RNA fold increase in HIE. HuNoV GII.1, GII.4 and GII.6 fecal samples were prepared by serial centrifugation and filtration and the viral RNA in HIE was quantified at 1 and 72 h post-infection (hpi) following RNA extraction and RT-qPCR. The serially filtered GII.1, GII.4 and GII.6 showed successful replication in HIE, resulting in mean log increases of 2.2, 2 and 1.2, respectively, at 72 vs. 1 hpi. In contrast, only serially centrifuged GII.1 showed consistently successful replication. However, using newer HIE passages and the MagMAX kit resulted in mean log fold increases for serially centrifuged GII.1, GII.4 and GII.6 (1.6, 2.3 and 1.8 log, respectively) that were similar to serially filtered samples. Therefore, HuNoV fecal sample processing and HIE age can affect virus replication in the HIE model.

Trends on Human Norovirus Virus-like Particles (HuNoV-VLPs) and Strategies for the Construction of Infectious Viral Clones toward In Vitro Replication.

Most acute gastroenteritis (AGE) outbreaks and sporadic cases in developing countries are attributable to infection by human norovirus (HuNoV), the enteric virus mainly transmitted via fecal-contaminated water. However, it has been challenging to study HuNoV due to the lack of suitable systems to cultivate and replicate the virus, hindering the development of treatments and vaccines. Researchers have been using virus-like particles (VLPs) and infectious viral clones to overcome this challenge as alternatives to fresh virus isolates in various in vitro and ex vivo models. VLPs are multiprotein structures that mimic the wild-type virus but cannot replicate in host cells due to the lack of genetic materials for replication, limiting downstream analysis of the virus life cycle and pathogenesis. The development of in vitro cloning systems has shown promise for HuNoV replication studies. This review discusses the approaches for constructing HuNoV-VLPs and infectious viral clones, the techniques involved, and the challenges faced. It also highlights the relationship between viral genes and their protein products and provides a perspective on technical considerations for producing efficient HuNoV-VLPs and infectious viral clones, which could substitute for native human noroviruses in future studies.

Chemokine CCL6 Plays Key Role in the Inhibitory Effect of Vitamin A on Norovirus Infection.

Norovirus (NoV) is the most common viral cause of acute gastroenteritis worldwide. Vitamin A has demonstrated the potential to protect against gastrointestinal infections. However, the effects of vitamin A on human norovirus (HuNoV) infections remain poorly understood. This study aimed to investigate how vitamin A administration affects NoV replication. We demonstrated that treatment with retinol or retinoic acid (RA) inhibited NoV replication in vitro based on their effects on HuNoV replicon-bearing cells and murine norovirus-1 (MNV-1) replication in murine cells. MNV replication in vitro showed significant transcriptomic changes, which were partially reversed by retinol treatment. RNAi knockdown of CCL6, a chemokine gene that was downregulated by MNV infection but upregulated by retinol administration, resulted in increased MNV replication in vitro. This suggested a role of CCL6 in the host response to MNV infections. Similar gene expression patterns were observed in the murine intestine after oral administration of RA and/or MNV-1.CW1. CCL6 directly decreased HuNoV replication in HG23 cells, and might indirectly regulate the immune response against NoV infection. Finally, relative replication levels of MNV-1.CW1 and MNV-1.CR6 were significantly increased in CCL6 knockout RAW 264.7 cells. This study is the first to comprehensively profile transcriptomes in response to NoV infection and vitamin A treatment in vitro, and thus may provide new insights into dietary prophylaxis and NoV infections.

HuNoV Non-Structural Protein P22 Induces Maturation of IL-1β and IL-18 and N-GSDMD-Dependent Pyroptosis through Activating NLRP3 Inflammasome.

Norovirus infection is the leading cause of foodborne gastroenteritis worldwide, causing more than 200,000 deaths each year. As a result of a lack of reproducible and robust in vitro culture systems and suitable animal models for human norovirus (HuNoV) infection, the pathogenesis of HuNoV is still poorly understood. In recent years, human intestinal enteroids (HIEs) have been successfully constructed and demonstrated to be able to support the replication of HuNoV. The NLRP3 inflammasome plays a key role in host innate immune responses by activating caspase1 to facilitate IL-1β and IL-18 secretion and N-GSDMD-driven apoptosis, while NLRP3 inflammasome overactivation plays an important role in the development of various inflammatory diseases. Here, we found that HuNoV activated enteric stem cell-derived human intestinal enteroids (HIEs) NLRP3 inflammasome, which was confirmed by transfection of Caco2 cells with full-length cDNA clones of HuNoV. Further, we found that HuNoV non-structural protein P22 activated the NLRP3 inflammasome and then matured IL-1β and IL-18 and processed the cleavage of gasdermin-D (GSDMD) to N-GSDMD, leading to pyroptosis. Besides, berberine (BBR) could ameliorate the pyroptosis caused by HuNoV and P22 by inhibiting NLRP3 inflammasome activation. Together, these results reveal new insights into the mechanisms of inflammation and cell death caused by HuNoV and provide potential treatments.

Use of Zebrafish Embryos To Reproduce Human Norovirus and To Evaluate Human Norovirus Infectivity Decay after UV Treatment.

This study reports an essential improvement of the method for replication of human norovirus (HNoV) with the use of zebrafish (Danio rerio) embryos. With three HNoV genotypes and P-types GII.2[P16], GII.4[P16], and GII.17[P31], we demonstrated that this tool had higher efficiency and robustness than the zebrafish larvae as reported previously. When zebrafish larvae were injected with virus (1.6 ± 0.3 log genome copies/10 larvae), a significant increase of virus genome copies was detected at 2 days postinfection (dpi; 4.4 ± 0.8 log genome copies/10 larvae, P < 0.05) and the viral loads started to decrease gradually from 3 dpi. In comparison, when the viruses were injected into the zebrafish embryos, significant virus replication was noticed from 1 dpi and lasted to 6 dpi (P < 0.05). The virus levels detected at 3 dpi had the highest mean value and the smallest variation (7.7 ± 0.2 log genome copies/10 larvae). The high levels of virus replication enabled continuous passaging for all three strains up to four passages. The zebrafish embryo-generated HNoVs showed clear patterns of binding to human histo-blood group antigens (HBGAs) in human saliva by a simple saliva-binding reverse transcription-quantitative PCR (RT-qPCR). Last, in a disinfection study, it was shown that a dose of 6 mJ/cm2 UV254 was able induce a >2-log reduction in HNoV infectivity for all three HNoV strains tested, suggesting that HNoVs were more UV susceptible than multiple enteric viruses and commonly used HNoV surrogates as tested before. IMPORTANCE HNoVs are a leading cause of gastroenteritis outbreaks worldwide. The zebrafish embryo tool as developed in this study serves as an efficient way to generate viruses with high titers and clean background and a straightforward platform to evaluate HNoV inactivation efficacies. It is expected that this tool will not only benefit epidemiological research on HNoV but also be used to generate HNoV inactivation parameters which are highly needed by the water treatment and food industries.

The Role of Histo-Blood Group Antigens and Microbiota in Human Norovirus Replication in Zebrafish Larvae.

Human norovirus (HuNoV) is the major agent for viral gastroenteritis, causing >700 million infections yearly. Fucose-containing carbohydrates named histo-blood group antigens (HBGAs) are known (co)receptors for HuNoV. Moreover, bacteria of the gut microbiota expressing HBGA-like structures have shown an enhancing effect on HuNoV replication in an in vitro model. Here, we studied the role of HBGAs and the host microbiota during HuNoV infection in zebrafish larvae. Using whole-mount immunohistochemistry, we visualized the fucose expression in the zebrafish gut for the HBGA Lewis X [LeX, α(1,3)-fucose] and core fucose [α(1,6)-fucose]. Costaining of HuNoV-infected larvae proved colocalization of LeX and to a lower extent core fucose with the viral capsid protein VP1, indicating the presence of fucose residues on infected cells. Upon blocking of fucose expression by a fluorinated fucose analogue, HuNoV replication was strongly reduced. Furthermore, by comparing HuNoV replication in conventional and germfree zebrafish larvae, we found that the natural zebrafish microbiome does not have an effect on HuNoV replication, contrary to earlier reports about the human gut microbiome. Interestingly, monoassociation with the HBGA-expressing Enterobacter cloacae resulted in a minor decrease in HuNoV replication, which was not triggered by a stronger innate immune response. Overall, we show here that fucose has an essential role for HuNoV infection in zebrafish larvae, as in the human host, but their natural gut microbiome does not affect viral replication. IMPORTANCE Despite causing over 700 million infections yearly, many gaps remain in the knowledge of human norovirus (HuNoV) biology due to an historical lack of efficient cultivation systems. Fucose-containing carbohydrate structures, named histo-blood group antigens, are known to be important (co)receptors for viral entry in humans, while the natural gut microbiota is suggested to enhance viral replication. This study shows a conserved mechanism of entry for HuNoV in the novel zebrafish infection model, highlighting the pivotal opportunity this model represents to study entry mechanisms and identify the cellular receptor of HuNoV. Our results shed light on the interaction of HuNoV with the zebrafish microbiota, contributing to the understanding of the interplay between gut microbiota and enteric viruses. The ease of generating germfree animals that can be colonized with human gut bacteria is an additional advantage of using zebrafish larvae in virology. This small animal model constitutes an innovative alternative to high-severity animal models.

Replication of Human Norovirus in Mice after Antibiotic-Mediated Intestinal Bacteria Depletion.

Human noroviruses (HuNoVs) are the main cause of acute gastroenteritis causing more than 50,000 deaths per year. Recent evidence shows that the gut microbiota plays a key role in enteric virus infectivity. In this context, we tested whether microbiota depletion or microbiota replacement with that of human individuals susceptible to HuNoVs infection could favor viral replication in mice. Four groups of mice (n = 5) were used, including a control group and three groups that were treated with antibiotics to eliminate the autochthonous intestinal microbiota. Two of the antibiotic-treated groups received fecal microbiota transplantation from a pool of feces from infants (age 1–3 months) or an auto-transplantation with mouse feces that obtained prior antibiotic treatment. The inoculation of the different mouse groups with a HuNoVs strain (GII.4 Sydney [P16] genotype) showed that the virus replicated more efficiently in animals only treated with antibiotics but not subject to microbiota transplantation. Viral replication in animals receiving fecal microbiota from newborn infants was intermediate, whereas virus excretion in feces from auto-transplanted mice was as low as in the control mice. The analysis of the fecal microbiota by 16S rDNA NGS showed deep variations in the composition in the different mice groups. Furthermore, differences were observed in the gene expression of relevant immunological mediators, such as IL4, CXCL15, IL13, TNFα and TLR2, at the small intestine. Our results suggest that microbiota depletion eliminates bacteria that restrict HuNoVs infectivity and that the mechanism(s) could involve immune mediators.

Fucoidan from Fucus vesiculosus can inhibit human norovirus replication by enhancing the host innate immune response

• IFN signaling-related genes and ISGs upregulated in hNoV infected zebrafish larvae. • Fucoidan inhibited hNoV replication in zebrafish larvae. • Fucoidan enhanced the innate immune response of zebrafish larvae. Fucoidan extracted from Fucus vesiculosus was able to inhibit the replication of human noroviruses (hNoV) GII.4[P16] in zebrafish ( Danio rerio ) larvae. In the hNoV infected zebrafish larvae, a transcriptomic analysis showed significant upregulation (p < 0.01) of the interferons (IFNs) signaling related genes as well as a series of IFN-stimulated genes (ISGs) encoding antiviral effectors compared to the mock-infected controls. Accordingly, injection of fucoidan together with hNoV resulted in significant upregulation of 834 genes (p < 0.01) and the innate immune system was calculated as the top cluster with 94 gene counts involved and a log 10 (p) value of −25.41. Tulane virus (TV), a very commonly used surrogate for hNoV, served as a complementary tool showing that the fucoidan was not able to block the virions irreversibly. Enhancement of the host innate immune response could be one of the important mechanisms behind the anti-hNoV effect of fucoidan.

Human Norovirus–Induced Gene Expression Biomarkers in Zebrafish

The challenges associated with development of an animal model system to replicate human norovirus (HuNoV) has hampered the study of the pathogenesis and therapeutic interventions for this virus. In this study, we replicated HuNoV GII.4 and evaluated virus gene expression in infected zebrafish. Three doses of inoculation resulted in successful virus replication. Genes for transmembrane transporters (tfa, cftr, slc26a3, and slc26a6), a heat shock chaperone (hspa8), and immune response cytokines (ifng1 and il1b) were highly expressed in HuNoV-infected zebrafish; however, expression levels of genes were reduced in zebrafish infected with thermally inactivated HuNoV. These results confirm HuNoV replication in juvenile zebrafish and will facilitate the investigation of biomarker gene expression during HuNoV infection.

Generation of CRISPR-Cas9-mediated genetic knockout human intestinal tissue-derived enteroid lines by lentivirus transduction and single-cell cloning.

Human intestinal tissue-derived enteroids (HIEs; also called organoids) are a powerful ex vivo model for gastrointestinal research. Genetic modification of these nontransformed cultures allows new insights into gene function and biological processes involved in intestinal diseases as well as gastrointestinal and donor segment-specific function. Here we provide a detailed technical pipeline and protocol for using the CRISPR-Cas9 genome editing system to knock out a gene of interest specifically in HIEs by lentiviral transduction and single-cell cloning. This protocol differs from a previously published alternative using electroporation of human colonoids to deliver piggyback transposons or CRISPR-Cas9 constructs, as this protocol uses a modified, fused LentiCRISPRv2-small-guiding RNA to express Cas9 and small-guiding RNA in a lentivirus. The protocol also includes the steps of gene delivery and subsequent single-cell cloning of the knockout cells as well as verification of clones and sequence identification of the mutation sites to establish knockout clones. An overview flowchart, step-by-step guidelines and troubleshooting suggestions are provided to aid the researcher in obtaining the genetic knockout HIE line within 2-3 months. In this protocol, we further describe how to use HIEs as an ex vivo model to assess host restriction factors for viral replication (using human norovirus replication as an example) by knocking out host attachment factors or innate immunity genes. Other applications are discussed to broaden the utility of this system, for example, to generate knockin or conditional knockout HIE lines to investigate the function of essential genes in many biological processes including other types of organoids.

Advances in understanding of the innate immune response to human norovirus infection using organoid models.

Norovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and the most frequent cause of foodborne illness in the United States. There is no specific treatment for norovirus infections and therapeutic interventions are based on alleviating symptoms and limiting viral transmission. The immune response to norovirus is not completely understood and mechanistic studies have been hindered by lack of a robust cell culture system. In recent years, the human intestinal enteroid/human intestinal organoid system (HIE/HIO) has enabled successful human norovirus replication. Cells derived from HIE have also successfully been subjected to genetic manipulation using viral vectors as well as CRISPR/Cas9 technology, thereby allowing studies to identify antiviral signaling pathways important in controlling norovirus infection. RNA sequencing using HIE cells has been used to investigate the transcriptional landscape during norovirus infection and to identify antiviral genes important in infection. Other cell culture platforms such as the microfluidics-based gut-on-chip technology in combination with the HIE/HIO system also have the potential to address fundamental questions on innate immunity to human norovirus. In this review, we highlight the recent advances in understanding the innate immune response to human norovirus infections in the HIE system, including the application of advanced molecular technologies that have become available in recent years such as the CRISPR/Cas9 and RNA sequencing, as well as the potential application of single cell transcriptomics, viral proteomics, and gut-on-a-chip technology to further elucidate innate immunity to norovirus.

Structure-Activity Relationship Studies on Novel Antiviral Agents for Norovirus Infections.

Human norovirus is the leading cause of acute gastroenteritis worldwide, affecting every year 685 million people. Norovirus outbreaks are associated with very significant economic losses, with an estimated societal cost of 60 billion USD per year. Despite this, no therapeutic options or vaccines are currently available to treat or prevent this infection. An antiviral therapy that can be used as treatment and as a prophylactic measure in the case of outbreaks is urgently needed. We previously described the computer-aided design and synthesis of novel small-molecule agents able to inhibit the replication of human norovirus in cell-based systems. These compounds are non-nucleoside inhibitors of the viral polymerase and are characterized by a terminal para-substituted phenyl group connected to a central phenyl ring by an amide-thioamide linker, and a terminal thiophene ring. Here we describe new modifications of these scaffolds focused on exploring the role of the substituent at the para position of the terminal phenyl ring and on removing the thioamide portion of the amide-thioamide linker, to further explore structure-activity relationships (SARs) and improve antiviral properties. According to three to four-step synthetic routes, we prepared thirty novel compounds, which were then evaluated against the replication of both murine (MNV) and human (HuNoV) norovirus in cells. Derivatives in which the terminal phenyl group has been replaced by an unsubstituted benzoxazole or indole, and the thioamide component of the amide-thioamide linker has been removed, showed promising results in inhibiting HuNoV replication at low micromolar concentrations. Particularly, compound 28 was found to have an EC50 against HuNoV of 0.9 µM. Although the most active novel derivatives were also associated with an increased cytotoxicity in the human cell line, these compounds represent a very promising starting point for the development of new analogues with reduced cytotoxicity and improved selectivity indexes. In addition, the experimental biological data have been used to create an initial 3D quantitative structure-activity relationship model, which could be used to guide the future design of novel potential anti-norovirus agents.

Efficacy of alcohol-based hand sanitizers against human norovirus using RNase-RT-qPCR with validation by human intestinal enteroid replication.

Successful human norovirus (HuNoV) cultivation in stem cell‐derived human intestinal enteroids (HIE) was recently reported. The purpose of this study was to evaluate the anti‐HuNoV efficacy of two alcohol‐based commercial hand sanitizers and 60% ethanol by suspension assay using RNase‐RT‐qPCR, with subsequent validation of efficacy by HuNoV cultivation using the HIE model. In suspension, when evaluated by RNase‐RT‐qPCR, 60% ethanol resulted in less than one log10 reduction in HuNoV genome equivalent copies (GEC) regardless of contact time (30 or 60s) or soil load. The two commercial products outperformed 60% ethanol regardless of contact time or soil load, providing 2·2–3·2 log10 HuNoV GEC reductions by suspension assay. Product B could not be validated in the HIE model due to cytotoxicity. Following a 60s exposure, viral replication in the HIE model increased 1·9 ± 0·2 log10 HuNoV GEC for the neutralization (positive) control and increased 0·9 ± 0·2 log10 HuNoV GEC in challenged HIE after treatment with 60% ethanol. No HuNoV replication in HIE was observed after a 60 s exposure to Product A.

Alcohol abrogates human norovirus infectivity in a pH-dependent manner

Alcohol-based disinfectants are widely used for the sanitization of microorganisms, especially those that cause infectious diseases, including viruses. However, since the germicidal mechanism of alcohol is lipolysis, alcohol-based disinfectants appear to have a minimal effect on non-enveloped viruses, such as noroviruses. Because there is no cultivation method for human norovirus (HuNoV) in vitro, murine norovirus and feline calicivirus have been used as surrogates for HuNoV to analyze the efficacy of disinfectant regents. Therefore, whether these disinfectants and their conditions are effective against HuNoVs remain unknown. In this study, we report that ethanol or isopropanol alone can sufficiently suppress GII.4 genotype HuNoV replication in human iPSC-derived intestinal epithelial cells. Additionally, pH adjustments and salting-out may contribute toward the virucidal effect of alcohol against other HuNoV genotypes and cancel the impediment of organic substance contamination, respectively. Therefore, similar to sodium hypochlorite, alcohol-based disinfectants containing electrolytes can be used for HuNoV inactivation.