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Human norovirus (HuNoV) is a major causative agent of foodborne illness and causes acute viral gastroenteritis. This study aimed to compare the virucidal efficacies of alcohol-based disinfectants against HuNoV and its surrogates for murine norovirus and feline calicivirus using a cell culture infectivity assay. Additionally, the study evaluated the validity of estimating virucidal efficacy on HuNoV from the results of virucidal efficacy on the surrogate virus. All disinfectants decreased the titer of each virus by >3 log10 and >4 log10 for an exposure duration of 30 s against murine norovirus and feline calicivirus, respectively. However, acidic alcohol-based disinfectants completely inactivated the HuNoV GII.17 strain for 30 or 60 s, whereas an alkaline alcohol-based disinfectant did not inactivate HuNoV GII.17 for 60 s. This finding indicates that the pH of alcohol disinfectants affects their virucidal effects against HuNoV, and acidity has a higher virucidal efficacy against HuNoV than alkalinity. Disinfectants showing virucidal efficacy against surrogates were not effective against HuNoV. Few studies have used cell culture infectivity assays to test the inactivating effects of hand sanitizers on HuNoV and its surrogates. Our study provides useful information for the development of disinfectants that are effective against HuNoV.

Foodborne viruses such as human norovirus (HuNoV) and hepatitis A virus (HAV) are the major causes of foodborne illnesses worldwide. These viruses have a low infectious dose and are persistent in the environment and food for weeks. Ready-to-eat (RTE) low moisture foods (LMFs) undergo minimal pathogen reduction processes. In recent years, multiple foodborne HAV outbreaks involving hundreds of individuals were associated with the consumption of dates, indicating that they could be important vehicles for foodborne infection. There is no standard method for the extraction and detection of foodborne viruses from dates, but herein we have compared the efficiency of three different protocols based on the ISO 15216 method in the extraction of murine norovirus (MNV) from whole Medjool dates and successfully employed the best performing method in the extraction of HAV, HuNoV GI, and GII and determined the limit of detection (LOD95) of 61, 148, and 184 genomic equivalent (gEq) per 25 g, respectively. Finally, we tested the adopted method on various varieties of dates including pitted ones and reported the detection of HuNoV GI and GII from four naturally contaminated date varieties. This ISO 15216 protocol could be employed for surveillance purposes and outbreak management related to dates.

Food contact surfaces can harbor and transmit pathogens leading to outbreaks. Decontamination strategies that are user- and environmentally-friendly without toxic by-product formation are needed. Novel UV-C light-emitting diode (LED) technologies are being explored to deliver the required dose to inactivate viruses in food-processing environments. The objective of this study was to compare the effects of 279 nm UV-C LED to 254 nm UV-C against hepatitis A virus (HAV) and feline calicivirus (FCV, a cultivable human norovirus surrogate) on stainless-steel, ceramic, and glass surfaces. Viruses were surface spread on sterile stainless-steel or ceramic coupons (100 μL on 2 × 2 cm2), or glass discs (50 μL on 1 × 1 cm2), air-dried, and UV-C-treated for up to 3.75 min (surface dose = 0–49.2 mJ/cm2 for HAV and 0–24.6 mJ/cm2 for FCV). Each triplicate treatment was assayed in duplicate, and data were statistically analyzed. The D10-values for HAV treated with UV-C at 254 nm on stainless-steel, ceramic, and glass were 9.48 ± 0.34, 14.53 ± 2.52, and 6.91 ± 1.93 mJ/cm2, while with UV-C LED at 279 nm were 19.53 ± 2.45, 26.05 ± 0.60, and 8.77 ± 2.08 mJ/cm2, respectively. The D10-values for FCV treated with UV-C at 254 nm on stainless-steel, ceramic, and glass were 3.65 ± 0.06, 6.25 ± 1.90, and 4.69 ± 0.03 mJ/cm2, while with UV-C LED at 279 nm were 7.097 ± 2.11, 8.31 ± 2.12, and 7.88 ± 0.86 mJ/cm2, respectively. Higher 279 nm UV-C doses were needed to inactivate HAV and FCV compared to 254 nm UV-C on the tested surfaces. Novel UV-C LED systems using appropriate doses show promise to inactivate foodborne viruses on food contact surfaces.

For nearly twenty years, gnotobiotic (Gn) pigs have been used as a model of human norovirus (HuNoV) infection and disease. Unique in their ability to develop diarrhea and shed virus post oral challenge, Gn pigs have since been used to evaluate the infectivity of several genogroup II HuNoV strains. Nearly all major pandemic GII.4 variants have been tested in Gn pigs, with varying rates of infectivity. Some induce an asymptomatic state despite being shed in large quantities in stool, and others induce high incidence of both diarrhea and virus shedding. Non-GII.4 strains, including GII.12 and GII.6, have also been evaluated in Gn pigs. Again, rates of diarrhea and virus shedding tend to vary between studies. Several factors may influence these findings, including age, dosage, biological host factors, or bacterial presence. The impact of these factors is nuanced and requires further evaluation to elucidate the exact mechanisms behind increases or decreases in infection rates. Regardless, the value of Gn pig models in HuNoV research cannot be understated, and the model will surely continue to contribute to the field in years to come.

Human norovirus (HuNoV) is a leading global cause of viral gastroenteritis, contributing to numerous outbreaks and illnesses annually. However, conventional cell culture systems cannot support the cultivation of infectious HuNoV, making its detection and study in food and water matrices particularly challenging. Recent advancements in HuNoV research, including the emergence of models such as human intestinal enteroids (HIEs) and zebrafish larvae/embryo, have significantly enhanced our understanding of HuNoV pathogenesis. This review provides an overview of current methods employed for HuNoV detection in food and water, along with their associated limitations. Furthermore, it explores the potential applications of the HIE and zebrafish larvae/embryo models in detecting infectious HuNoV within food and water matrices. Finally, this review also highlights the need for further optimization and exploration of these models and detection methods to improve our understanding of HuNoV and its presence in different matrices, ultimately contributing to improved intervention strategies and public health outcomes.

Human noroviruses are major causes of foodborne outbreaks linked to berries. The overall goal of this study was to investigate the persistence of a human norovirus surrogate, Tulane virus (TV), in berry smoothies and under simulated digestion through the gastrointestinal track. Two types of smoothies were prepared from blueberries and strawberries. Tulane virus was spiked into each smoothie and incubated either at 37 or 4 °C for 2, 60, and 120 min. Furthermore, the virus-spiked smoothies were subjected to sequential oral (2 min), gastric (10 and 60 min), and intestinal (15 and 120 min) digestion according to the standardized INFOGEST model. Quantification of infectious TV was carried out using the TCID50 assay. At 4 °C, in both berry smoothies, TV infectivity did not show significant changes throughout the 120 min period. At 37 °C, TV infectivity showed significant reduction (~0.5 log TCID50/mL) only in blueberry smoothies starting at 60 min. During the oral, gastric, and intestinal digestion phases, the mean log reduction in TV infectivity in blueberry did not exceed ~0.5 log, while infectious TV in strawberry smoothies under all phases was stable. Given the notable stability of infectious viruses in berry smoothies and the gastrointestinal tract, prevention of norovirus contamination of berries is paramount to reduce virus outbreaks linked to berries.

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.

Human noroviruses (HuNoVs) are a major cause of acute gastroenteritis, contributing significantly to annual foodborne illness cases. However, studying these viruses has been challenging due to limitations in tissue culture techniques for over four decades. Tulane virus (TV) has emerged as a crucial surrogate for HuNoVs due to its close resemblance in amino acid composition and the availability of a robust cell culture system. Initially isolated from rhesus macaques in 2008, TV represents a novel Calicivirus belonging to the Recovirus genus. Its significance lies in sharing the same host cell receptor, histo-blood group antigen (HBGA), as HuNoVs. In this study, we introduce, through cryo-electron microscopy (cryo-EM), the structure of a specific TV variant (the 9-6-17 TV) that has notably lost its ability to bind to its receptor, B-type HBGA—a finding confirmed using an enzyme-linked immunosorbent assay (ELISA). These results offer a profound insight into the genetic modifications occurring in TV that are necessary for adaptation to cell culture environments. This research significantly contributes to advancing our understanding of the genetic changes that are pivotal to successful adaptation, shedding light on fundamental aspects of Calicivirus evolution.

Norovirus (NoV) is the leading cause of viral foodborne gastroenteritis globally. Currently, the gold standard for detecting NoV in clinical, food, and environmental samples is via molecular-based methods, primarily RT-PCR. Nevertheless, there is a great need for confirmatory assays that can determine the infectivity of viral particles recovered from contaminated matrices. The use of the human intestinal enteroids system (HIEs) has allowed for the expansion of norovirus replication, although it still suffers from limitations of strain preferences and the requirement of high titer stocks for infection. In this study, we wanted to explore the feasibility of using the HIEs to support the replication of NoV that had been recovered from representative food matrices that have been associated with foodborne illness. We first confirmed that HIEs can support the replication of several strains of NoV as measured by RT-qPCR. We subsequently chose two of those strains that reproducibly replicated, GII.4 and GII.6, to evaluate in a TCID50 assay and for future experiments. Infectious NoV could be recovered and quantified in the HIEs from lettuce, frozen raspberries, or frozen strawberries seeded with high titers of either of these strains. While many experimental challenges still remain to be overcome, the results of this study represent an important step toward the detection of infectious norovirus from representative produce items.

To understand the evolution of GII.P6-GII.6 and GII.P7-GII.6 strains, the prevalent human norovirus genotypes, we analysed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than for the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains.

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.

Human norovirus and hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) are leading causes of foodborne disease worldwide. Among the various food products, different types of dairy products can be implicated in viral foodborne outbreaks and contamination can occur at different stages, such as preparation, contact with contaminated equipment or via other foods. The aim of this study was to characterise a proteinase K method adapted from the ISO 15216 method for the detection of HAV, HEV and norovirus in artificially contaminated dairy products, based on the recent international standard of ISO 16140-4. Results showed that the recovery yields obtained from pure RNA in dairy products ranged from 5.76% to 76.40% for HAV, from 35.09% to 100.00% for HEV, from 25.09% to 100.00% for norovirus GI and from 47.83% to 100.00% for norovirus GII. The process control MNV-1 was detected in all RNA extracts, with recovery yields between 36.83% and 100.00%. The limit of detection (LOD) of the method was between 184 and 642 genome copies/mL (or/g) for the LOD50 and 802 and 2800 genome copies/mL or/g for the LOD95 according to the virus analysed. This method proved to be suitable for detecting viruses in dairy products for routine diagnostic needs.

Human norovirus (HuNoV) is the leading foodborne pathogen causing nonbacterial gastroenteritis worldwide. The oyster is an important vehicle for HuNoV transmission, especially the GI.1 HuNoV. In our previous study, oyster heat shock protein 70 (oHSP 70) was identified as the first proteinaceous ligand of GII.4 HuNoV in Pacific oysters besides the commonly accepted carbohydrate ligands, a histo-blood group antigens (HBGAs)-like substance. However the mismatch of the distribution pattern between discovered ligands and GI.1 HuNoV suggests that other ligands may exist. In our study, proteinaceous ligands for the specific binding of GI.1 HuNoV were mined from oyster tissues using a bacterial cell surface display system. Fifty-five candidate ligands were identified and selected through mass spectrometry identification and bioinformatics analysis. Among them, the oyster tumor necrosis factor (oTNF) and oyster intraflagellar transport protein (oIFT) showed strong binding abilities with the P protein of GI.1 HuNoV. In addition, the highest mRNA level of these two proteins was found in the digestive glands, which is consistent with GI.1 HuNoV distribution. Overall the findings suggested that oTNF and oIFT may play important roles in the bioaccumulation of GI.1 HuNoV.

Viruses are a leading cause of foodborne disease worldwide. Hepatitis viruses (hepatitis A (HAV) and hepatitis E (HEV)) and human norovirus are recognized as the main viruses of public health concern in food hygiene. ISO 15216 approved procedures are not validated for detection of HAV and human norovirus in foodstuffs, such as fishes, leading to an inability to ensure the safety of these products. This study aimed to provide a rapid and sensitive method for detecting these targets in fish products. An existing method that includes proteinase K treatment was selected for further validation using artificially contaminated fish products, according to the recent international standard ISO 16140-4. Recovery efficiencies in pure RNA extracts of viruses ranged from 0.2% to 66.2% for HAV, 4.0% to 100.0% for HEV, 2.2% to 100.0% for norovirus GI, and 0.2% to 12.5% for norovirus GII. LOD50 values were between 144 and 8.4 × 104 genome copies/g for HAV and HEV, and 104 and 2.0 × 103 copies/g for norovirus GI and norovirus GII, respectively. LOD95 values were between 3.2 × 103 and 3.6 × 105 genome copies/g for HAV and HEV, and between 8.8 × 103 and 4.4 × 104 genome copies/g for norovirus GI and norovirus GII, respectively. The method developed here was successfully validated in various fish products and can be applied for routine diagnostic needs.

Human norovirus is the first cause of foodborne disease worldwide, leading to extensive outbreaks of acute gastroenteritis, and causing around 200,000 children to die annually in developing countries. No specific vaccines or antiviral agents are currently available, with therapeutic options limited to supportive care to prevent dehydration. The infection can become severe and lead to life-threatening complications in young children, the elderly and immunocompromised individuals, leading to a clear need for antiviral agents, to be used as treatments and as prophylactic measures in case of outbreaks. Due to the key role played by the viral RNA-dependent RNA polymerase (RdRp) in the virus life cycle, this enzyme is a promising target for antiviral drug discovery. In previous studies, following in silico investigations, we identified different small-molecule inhibitors of this enzyme. In this study, we rationally modified five identified scaffolds, to further explore structure–activity relationships, and to enhance binding to the RdRp. The newly designed compounds were synthesized according to multiple-step synthetic routes and evaluated for their inhibition of the enzyme in vitro. New inhibitors with low micromolar inhibitory activity of the RdRp were identified, which provide a promising basis for further hit-to-lead optimization.

Norovirus is the leading cause of acute viral gastroenteritis. Both human and murine noroviruses attach to commensal bacteria belonging to the mammalian gut flora, and binding levels are influenced by nutrients present in bacterial media. However, it is not known which nutrients are responsible for altering viral binding or why binding is altered. Gene expression of commensal bacteria can be changed by the external environment as well as by interaction with pathogens. For example, growth phase and incubation conditions impact expression levels of specific bacterial genes in Escherichia coli. We have previously shown that binding by both human and murine noroviruses to the commensal bacterium Enterobacter cloacae induces genome-wide changes in gene expression with a large number of differentially expressed genes associated with the surface structure of the bacterial cell. The current study evaluated norovirus binding under nutrient-limited conditions and assessed the expression of a select panel of these genes that are significantly altered by norovirus binding under these conditions. The goal of this work was to determine how norovirus attachment to Enterobacter cloacae affected the expression of these genes under varying nutrient and growth phase conditions. We found that the presence of glucose in minimal media reduced murine norovirus binding to E. cloacae and viral binding in the presence of glucose reduced gene expression for surface structures previously associated with norovirus attachment. Changes in viral binding and gene expression occurred in a growth phase-dependent manner. Collectively, these data demonstrate that both the growth phase and nutrient availability alter viral interactions with commensal bacteria and the subsequent changes in gene expression. Ultimately, this work advances our understanding of norovirus-bacterium interactions and provides a foundation for elucidating the conditions and surface structures that regulate norovirus attachment to bacteria.

Human norovirus (HuNoV) is one of the major pathogens of acute nonbacterial gastroenteritis. Due to the lack of a robust and reproducible in vitro culture system and an appropriate animal model, the mechanism underlying HuNoV-caused diarrhea remains unknown. In the current study, we found that HuNoV transfection induced the expression of aquaporin 1 (AQP1), which was further confirmed in the context of virus infection, whereas the enterovirus EV71 (enterovirus 71) did not have such an effect. We further revealed that VP1, the major capsid protein of HuNoV, was crucial in promoting AQP1 expression. Mechanistically, HuNoV induces AQP1 production through the NF-κB signaling pathway via inducing the expression, phosphorylation and nuclear translocation of p65. By using a model of human intestinal epithelial barrier (IEB), we demonstrated that HuNoV and VP1-mediated enhancement of small molecule permeability is associated with the AQP1 channel. Collectively, we revealed that HuNoV induced the production of AQP1 by activating the NF-κB signaling pathway. The findings in this study provide a basis for further understanding the significance of HuNoV-induced AQP1 expression and the potential mechanism underlying HuNoV-caused diarrhea.

Human noroviruses (HuNoVs) are a major cause of gastroenteritis and are associated with high morbidity because of their ability to survive in the environment and small inoculum size required for infection. Norovirus is transmitted through water, food, high touch-surfaces, and human-to-human contact. Ultraviolet Subtype C (UVC) light-emitting diodes (LEDs) can disrupt the norovirus transmission chain for water, food, and surfaces. Here, we illuminate considerations to be adhered to when picking norovirus surrogates for disinfection studies and shine light on effective use of UVC for norovirus infection control in water and air and validation for such systems and explore the blind spot of radiation safety considerations when using UVC disinfection strategies. This perspective also discusses the promise of UVC for norovirus mitigation to save and ease life.

Molecular characterization of human norovirus (HuNoV) genotypes enhances the understanding of viral features and illustrates distinctive evolutionary patterns. The aim of our study was to describe the prevalence of the genetic diversity and the epidemiology of the genotypes involved in HuNoV outbreaks in Catalonia (Spain) between 2017 and 2019. A total of 100 HuNoV outbreaks were notified with the predominance of GII (70%), followed by GI (27%) and mixed GI/GII (3%). Seasonality was observed for GII outbreaks only. The most prevalent genotypes identified were GII.4[P31] Sydney 2012, GII.4[P16] Sydney 2012 and GII.2[P16]. As compared to person-to-person (P/P) transmitted outbreaks, foodborne outbreaks showed significantly higher attack rates and lower duration. The average attack rate was higher in youth hostel/campgrounds compared to nursing homes. Only genotypes GI.4[P4], GII.2[P16], GII.4[P16], GII.4[P31] and GII.17[P17] were consistently detected every year, and only abundance of GII.2[P16] showed a negative trend over time. GII.4 Sydney 2012 outbreaks were significantly associated to nursing homes, while GII.2[P16] and GI.3[P3] were most frequently identified in youth hostel/campgrounds. The average attack rate was significantly higher when comparing GII.2[P16] vs. GI.4[P4], GII.2[P16] vs. GII.4[P31] Sydney 2012, and GII.6[P7] vs. GII.4[P31] Sydney 2012. No correlations were found between genotype and outbreak duration or age of affected individuals.

The RNA-dependent RNA polymerase (RdRp) receptor is an attractive target for treating human norovirus (HNV). A computer-aided approach like e-pharmacophore, molecular docking, and single point energy calculations were performed on the compounds retrieved from the Development Therapeutics Program (DTP) AIDS Antiviral Screen Database to identify the antiviral agent that could target the HNV RdRp receptor. Induced-fit docking (IFD) results showed that compounds ZINC1617939, ZINC1642549, ZINC6425208, ZINC5887658 and ZINC32068149 bind with the residues in the active site-B of HNV RdRp receptor via hydrogen bonds, salt bridge, and electrostatic interactions. During the molecular dynamic simulations, compounds ZINC6425208, ZINC5887658 and ZINC32068149 displayed an unbalanced backbone conformation with HNV RdRp protein, while ZINC1617939 and ZINC1642549 maintained stability with the protein backbone when interacting with the residues. Hence, the two new concluding compounds discovered by the computational approach can be used as a chemotype to design promising antiviral agents aimed at HNV RdRp.

Human noroviruses (HuNoVs) and the hepatitis A virus (HAV) are the main viral causes of foodborne illness worldwide. These viruses are frequently transmitted via fresh and frozen berries, such as strawberries and raspberries. ISO 15216:1 (2017), currently the preferred method for their detection, involves several steps and is time-consuming. Apolipoprotein H (ApoH) has been shown to have a strong affinity for several microorganisms, including HuNoVs. In this article, we report an ApoH-based method of capturing the HAV and HuNoVs adherent to berries and concentrating them for assay. The limit of detection of both viruses suspended in a buffer was low. On strawberries, the HAV was detected down to 104 genome copies/25 g in 100% of cases and down to 103 genome copies/25 g on raspberries in 50% of cases. This sensitivity was not significantly different from that of the ISO method 15216:1 (2017). HuNoV GII.4 was more difficult to detect using the ApoH method. The ApoH CaptoVIR kit does, nevertheless, appear to be usable in the near future as a single-test, multiple-detection method for viruses on fresh and frozen berries.

Human norovirus (HuNoV), which is the major causative agent of acute gastroenteritis, has broad antigenic diversity; thus, the development of a broad-spectrum vaccine is challenging. To establish the relationship between viral genetic diversity and antigenic diversity, capsid P proteins and antisera of seven GI and 16 GII HuNoV genotypes were analyzed. Enzyme-linked immunosorbent assays showed that HuNoV antisera strongly reacted with the homologous capsid P proteins (with titers > 5 × 104). However, 17 (73.9%) antisera had weak or no cross-reactivity with heterologous genotypes. Interestingly, the GII.5 antiserum cross-reacted with seven (30.4%) capsid P proteins (including pandemic genotypes GII.4 and GII.17), indicating its potential use for HuNoV vaccine development. Moreover, GI.2 and GI.6 antigens reacted widely with heterologous antisera (n ≥ 5). Sequence alignment and phylogenetic analyses of the P proteins revealed conserved regions, which may be responsible for the immune crossover reactivity observed. These findings may be helpful in identifying broad-spectrum epitopes with clinical value for the development of a future vaccine.

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.

Noroviruses (NoVs) are a leading cause of acute gastroenteritis worldwide. P particles are a potential vaccine candidate against NoV. Simvastatin is a cholesterol-reducing drug that is known to increase NoV infectivity. In this study, we examined simvastatin’s effects on P particle-induced protective efficacy and T-cell immunogenicity using the gnotobiotic pig model of human NoV infection and diarrhea. Pigs were intranasally inoculated with three doses (100 µg/dose) of GII.4/VA387-derived P particles together with monophosphoryl lipid A and chitosan adjuvants. Simvastatin-fed pigs received 8 mg/day orally for 11 days prior to challenge. A subset of pigs was orally challenged with 10 ID50 of a NoV GII.4/2006b variant at post-inoculation day (PID) 28 and monitored for 7 days post-challenge. Intestinal and systemic T cell responses were determined pre- and postchallenge. Simvastatin abolished the P particle’s protection and significantly increased diarrhea severity after NoV infection. Simvastatin decreased proliferation of virus-specific and non-specific CD8 T cells in duodenum and virus-specific CD4 and CD8 T cells in spleen and significantly reduced numbers of intestinal mononuclear cells in vaccinated pigs. Furthermore, simvastatin significantly decreased numbers of duodenal CD4+IFN-γ+, CD8+IFN-γ+ and regulatory T cells and total duodenal activated CD4+ and CD8+ T cells in vaccinated pigs pre-challenge at PID 28. Following challenge, simvastatin prevented the IFN-γ+ T cell response in spleen of vaccinated pigs. These results indicate that simvastatin abolished P particle vaccine-induced partial protection through, at least in part, impairing T cell immunity. The findings have specific implications for the development of preventive and therapeutic strategies against NoV gastroenteritis, especially for the elderly population who takes statin-type drugs.

Human noroviruses (huNoVs) cause epidemic acute gastroenteritis with significant mortality and morbidity worldwide. However, there are no commercial vaccines or antivirals against these important pathogens so far. In this study, we found that bovine colostrum (bCM) inhibited huNoV VLPs and their capsid-protruding (P) domains binding to histo-blood group antigens (HBGAs) that are huNoV receptor or attachment factors for infection, suggesting that bCM may function as a natural antiviral against huNoVs. We then characterized the bCM for the functional inhibition components by sequentially separating bCM into multiple fractions through various chromatography approaches, followed by determining their inhibitory abilities against huNoV receptor-binding P protein interacting with HBGAs. The protein components of bCM functional fractions were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Our data suggested that some milk proteins, likely in the form of glycoproteins, contribute to the observed blocking effects of bCM. Our findings lay an important foundation to further develop bCM into a potential natural antiviral against huNoVs.

Human norovirus (HuNoV) is the leading cause of acute gastroenteritis (AGE) worldwide, which is highly stable and contagious, with a few virus particles being sufficient to establish infection. Although the World Health Organization in 2016 stated that it should be an absolute priority to develop a HuNoV vaccine, unfortunately, there is currently no licensed HuNoV vaccine available. The major barrier to the development of an effective HuNoV vaccine is the lack of a robust and reproducible in vitro cultivation system. To develop a HuNoV vaccine, HuNoV immunogen alone or in combination with other viral immunogens have been designed to assess whether they can simultaneously induce protective immune responses against different viruses. Additionally, monovalent and multivalent vaccines from different HuNoV genotypes, including GI and GII HuNoV virus-like particles (VLPs), have been assessed in order to induce broad protection. Although there are several HuNoV vaccine candidates based on VLPs that are being tested in clinical trials, the challenges to develop effective HuNoV vaccines remain largely unresolved. In this review, we summarize the advances of the HuNoV cultivation system and HuNoV vaccine research and discuss current challenges and future perspectives in HuNoV vaccine development.

Every year, millions of children are infected with viruses that target the gastrointestinal tract, causing acute gastroenteritis and diarrheal illness. Indeed, approximately 700 million episodes of diarrhea occur in children under five annually, with RNA viruses norovirus, rotavirus, and astrovirus serving as major causative pathogens. Numerous methodological advancements in recent years, including the establishment of novel cultivation systems using enteroids as well as the development of murine and other animal models of infection, have helped provide insight into many features of viral pathogenesis. However, many aspects of enteric viral infections remain elusive, demanding further study. Here, we describe the different in vitro and in vivo tools available to explore different pathophysiological attributes of human enteric RNA viruses, highlighting their advantages and limitations depending upon the question being explored. In addition, we discuss key areas and opportunities that would benefit from further methodological progress.

Human noroviruses (HuNoVs) are one of the leading causes of foodborne illnesses globally. The viral genome is the most essential information for viral source tracing and viral transmission pattern monitoring. However, whole genome sequencing of HuNoVs is still challenging due to the sequence heterogeneity among different genotypes and low titer in samples. To address this need, in this study, the Transposase assisted RNA/DNA hybrid Co-tagmentation (TRACE-seq) method was established for next generation sequencing library preparation of HuNoVs. Our data demonstrated that almost the whole HuNoVs genome (>7 kb) could be obtained from all of the 11 clinical samples tested. Twelve genotypes including GI.3, GI.4, GI.5, GI.8, GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, and GII.21 were involved. Compared with the traditional method for viral metagenomics library preparation, optimized TRACE-seq greatly reduced the interference from the host’s and bacterial RNAs. In addition, viral genome sequences can be assembled by using less raw data with sufficient depth along the whole genome. Therefore, for the high versatility and reliability, this method is promising for whole viral genome attainment. It is particularly applicable for the viruses with a low titer that are mixed with a complicated host background and are unable to be cultured in vitro, like the HuNoVs utilized in this study.

Norovirus genogroup II (NoV GII) induces acute gastrointestinal food-borne illness in humans. Because gnotobiotic pigs can be infected with human norovirus (HuNoV) GII, they are frequently used to analyze the associated pathogenic mechanisms and immune responses, which remain poorly understood. Recently, mRNA sequencing analysis (RNA-Seq) has been used to identify cellular responses to viruses. In this study, we investigated the host immune response and possible mechanisms involved in virus evasion in the ileum of gnotobiotic pigs infected with HuNoV by RNA-Seq. HuNoV was detected in the feces, blood, and tissues of the jejunum, ileum, colon, mesenteric lymph node, and spleen of pigs infected with HuNoV. In analysis of mRNA sequencing, expression of anti-viral protein genes such as OAS1, MX1, and MX2 were largely decreased, whereas type I IFN was increased in pigs infected with HuNoV. In addition, expression of TNF and associated anti-inflammatory cytokine genes such as IL10 was increased in HuNoV-infected pigs. Expression of genes related to natural killer (NK) cell cytotoxicity and CD8+ T cell exhaustion was increased, whereas that of MHC class I genes was decreased. Expression profiles of selected genes were further confirmed by qRT-PCR and Western blot. These results suggest that infection with HuNoV induces NK cell-mediated cytotoxicity but suppresses type I IFN- and CD8+ T cell-mediated antiviral responses.

Norovirus is the leading cause of acute gastroenteritis worldwide. The pathogenesis of norovirus and the induced immune response remain poorly understood due to the lack of a robust virus culture system. The monolayers of two secretor-positive Chinese human intestinal enteroid (HIE) lines were challenged with two norovirus pandemic GII.4 Sydney strains. Norovirus RNA replication in supernatants and cell lysates were quantified by RT-qPCR. RNA expression levels of immune-related genes were profiled using PCR arrays. The secreted protein levels of shortlisted upregulated genes were measured in supernatants using analyte-specific enzyme-linked immunosorbent assay (ELISA). Productive norovirus replications were achieved in three (75%) out of four inoculations. The two most upregulated immune-related genes were CXCL10 (93-folds) and IFI44L (580-folds). Gene expressions of CXCL10 and IFI44L were positively correlated with the level of norovirus RNA replication (CXCL10: Spearman’s r = 0.779, p < 0.05; IFI44L: r = 0.881, p < 0.01). The higher level of secreted CXCL10 and IFI44L proteins confirmed their elevated gene expression. The two genes have been reported to be upregulated in norovirus volunteer challenges and natural human infections by other viruses. Our data suggested that HIE could mimic the innate immune response elicited in natural norovirus infection and, therefore, could serve as an experimental model for future virus-host interaction and antiviral studies.

This study investigates the effects of dielectric barrier discharge (DBD) plasma treatment (1.1 kV, 43 kHz, N2 1.5 L/min, 10~60 min) on human norovirus (HuNoV) GII.4 infectivity in fresh oysters. HuNoV viability in oysters was assessed by using propidium monoazide (PMA) as a nucleic acid intercalating dye before performing a real-time reverse transcription–quantitative polymerase chain reaction (RT-qPCR). Additionally, the impact of the DBD plasma treatment on pH and Hunter colors was assessed. When DBD plasma was treated for 60 min, the HuNoV genomic titer reduction without PMA pretreatment was negligible (1 log copy number/µL in just 30 min. D1 and D2-value of HuNoV infectivity were calculated as 36.5 and 73.0 min of the DBD plasma treatment, respectively, using the first-order kinetics model (R2 = 0.98). The pH and Hunter colors were not significantly different (p > 0.05) between the untreated and DBD-plasma-treated oysters. The results suggest that PMA/RT-qPCR could help distinguish HuNoV infectivity without negatively affecting oyster quality following >30 min treatment with DBD plasma. Moreover, the inactivation kinetics of nonthermal DBD plasma against HuNoV in fresh oysters might provide basic information for oyster processing and distribution.

Norovirus infections are a leading cause of acute gastroenteritis outbreaks worldwide and across all age groups, with two main genogroups (GI and GII) infecting humans. The aim of our study was to investigate the occurrence of norovirus in saliva samples from individuals involved in outbreaks of acute gastroenteritis in closed and semiclosed institutions, and its relationship with the virus strain, virus shedding in stool, the occurrence of symptoms, age, and the secretor status of the individual. Epidemiological and clinical information was gathered from norovirus outbreaks occurring in Catalonia, Spain during 2017–2018, and stool and saliva samples were collected from affected and exposed resident individuals and workers. A total of 347 saliva specimens from 25 outbreaks were analyzed. Further, 84% of individuals also provided a paired stool sample. For GII infections, norovirus was detected in 17.9% of saliva samples from symptomatic cases and 5.2% of asymptomatic individuals. Positivity in saliva occurred in both secretors and nonsecretors. None of the individuals infected by norovirus GI was positive for the virus in saliva. Saliva positivity did not correlate with any of the studied symptoms but did correlate with age ≥ 65 years old. Individuals who were positive in saliva showed higher levels of virus shedding in stool. Mean viral load in positive saliva was 3.16 ± 1.08 log10 genome copies/mL, and the predominance of encapsidated genomes was confirmed by propidium monoazide (PMA)xx-viability RTqPCR assay. The detection of norovirus in saliva raises the possibility of oral-to-oral norovirus transmission during the symptomatic phase and, although to a lesser extent, even in cases of asymptomatic infections.

Human norovirus (HuNoV) is a principal cause of acute gastroenteritis worldwide, particularly in developing countries. Its global prevalence is underscored by more serious morbidity and some mortality in the young (

As the leading cause of acute gastroenteritis worldwide, human noroviruses (HuNoVs) have caused around 685 million cases of infection and nearly $60 billion in losses every year. Despite their highly contagious nature, an effective vaccine for HuNoVs has yet to become commercially available. Therefore, rapid detection and subtyping of noroviruses is crucial for preventing viral spread. Over the past half century, there has been monumental progress in the development of techniques for the detection and analysis of noroviruses. However, currently no rapid, portable assays are available to detect and subtype infectious HuNoVs. The purpose of this review is to survey and present different analytical techniques for the detection and characterization of noroviruses.

Lettuce is often implicated in human norovirus (HuNoV) foodborne outbreaks. We identified H-like histo-blood group antigens (HBGAs) on lettuce leaves as specific binding moieties for virus-like particles (VLPs) of HuNoV GII.4/HS194/2009 strain. The objective of this study was to determine whether HuNoV-lettuce binding is mediated through the virus HBGA binding sites (HBS). Toward this objective, VLPs of historical HuNoV GII.4 strains (1987, 1997, 2002, 2004 and 2006) with known natural mutations in their HBS, two newly generated VLP mutants of GII.4/HS194/2009 (D374A and G443A) and a VLP mutant (W375A) of GI.1/Norwalk/1968 along with its wild type VLPs, which displays distinct HBS, were investigated for their binding to lettuce. ELISA revealed that historical GII.4 strains binding to lettuce was dependent on their HBGAs profiles. The VLP mutants D374A and G443A lost binding to HBGAs and displayed no to minimal binding to lettuce, respectively. The VLPs of GI.1/Norwalk/1968 strain bound to lettuce through an H-like HBGA and the binding was inhibited by fucosidase digestion. Mutant W375A which was previously shown not to bind to HBGAs, displayed significantly reduced binding to lettuce. We conclude that the binding of HuNoV GII.4 and GI.1 strains to lettuce is mediated through the virus HBS.

Human norovirus (HuNoV) infection is a major cause of gastroenteritis all over the world. Despite this, these non-enveloped RNA viruses are poorly characterized due to the lack of robust and widely available HuNoV culture systems. The two published systems (B cell line and stem cell-derived enteroids) support replication of HuNoVs but the levels of replication are not sufficient for the generation of highly purified virus stocks or the development of culture-based quantification assays. Therefore, improvement of HuNoV in vitro replication is still needed. Murine norovirus and other caliciviruses have provided insights into norovirus replication that paved the way for the development of the current HuNoV culture systems and may also aid in the improvement of these systems. This review will highlight ways in which previous research guided and impacted the development of HuNoV culture systems and discuss ways in which more recent discoveries might be utilized to improve the quality of the HuNoV in vitro replication.

Human norovirus (HuNoV) is responsible for more than 95% of outbreaks of acute nonbacterial gastroenteritis worldwide. Despite major efforts, there are no vaccines or effective therapeutic interventions against this virus. Chicken immunoglobulin Y (IgY)-based passive immunization has been shown to be an effective strategy to prevent and treat many enteric viral diseases. Here, we developed a highly efficient bioreactor to generate high titers of HuNoV-specific IgY in chicken yolks using a recombinant vesicular stomatitis virus expressing HuNoV capsid protein (rVSV-VP1) as an antigen. We first demonstrated that HuNoV VP1 protein was highly expressed in chicken cells infected by rVSV-VP1. Subsequently, we found that White Leghorn hens immunized intramuscularly with rVSV-VP1 triggered a high level of HuNoV-specific yolk IgY antibodies. The purified yolk IgY was efficiently recognized by HuNoV virus-like particles (VLPs). Importantly, HuNoV-specific IgY efficiently blocked the binding of HuNoV VLPs to all three types (A, B, and O) of histo-blood group antigens (HBGAs), the attachment factors for HuNoV. In addition, the receptor blocking activity of IgY remained stable at temperature below 70 °C and at pH ranging from 4 to 9. Thus, immunization of hens with VSV-VP1 could be a cost-effective and practical strategy for large-scale production of anti-HuNoV IgY antibodies for potential use as prophylactic and therapeutic treatment against HuNoV infection.

Human noroviruses (HuNoVs) are responsible for more than 95% of the non-bacterial acute gastroenteritis epidemics in the world. The CDC estimates that every year 21 million individuals suffer from HuNoV-induced gastroenteritis in the United States. Currently, there is no FDA-approved vaccine for HuNoVs. Development of an effective vaccine has been hampered by the lack of an efficient cell culture system for HuNoVs and a suitable small animal model for pathogenesis study. In this study, we developed lactic acid bacteria (LAB) as a vector to deliver HuNoV antigen. A LAB strain (Lactococcus lactis) carrying VP1 gene of a HuNoV GII.4 virus (LAB-VP1) was constructed. It was found that HuNoV VP1 protein was highly expressed by LAB vector and was secreted into media supernatants. To test whether LAB-based HuNoV vaccine candidate is immunogenic, 4-day-old gnotobiotic piglets were orally inoculated with various doses of LAB-VP1. It was found that LABs were persistent in the small intestine of piglets and shed in pig feces for at least 25 days post inoculation. LAB DNA and VP1 were detected in mesenteric lymph nodes and spleen tissue in LAB-VP1 inoculated groups. HuNoV-specific IgG and IgA were detectable in serum and feces respectively at day 13 post-inoculation, and further increased at later time points. After being challenged with HuNoV GII.4 strain, a large amount of HuNoV antigens were observed in the duodenum, jejunum, and ileum sections of the intestine in the LAB control group. In contrast, significantly less, or no, HuNoV antigens were detected in the LAB-VP1 immunized groups. Collectively, these results demonstrate that a LAB-based HuNoV vaccine induces protective immunity in gnotobiotic piglets.

Human norovirus is the major cause of non-bacterial epidemic gastroenteritis. Human norovirus binds to environmental solids via specific and non-specific interactions, and several specific receptors for human norovirus have been reported. Among them, histo-blood group antigens (HBGA) are the most studied specific receptor. Studies have identified the presence of HBGA-like substances in the extracellular polymeric substances (EPS) and lipopolysaccharides (LPS) of human enteric bacteria present in aquatic environments, gastrointestinal cells, gills, and palps of shellfish, and cell walls, leaves, and veins of lettuce. These HBGA-like substances also interact with human norovirus in a genotype-dependent manner. Specific interactions between human norovirus and environmental matrices can affect norovirus removal, infectivity, inactivation, persistence, and circulation. This review summarizes the current knowledge and future directions related to the specific interactions between human norovirus and HBGA-like substances in environmental matrices and their possible effects on the fate and circulation of human norovirus.

The Caliciviridae family of viruses contains clinically important human and animal pathogens, as well as vesivirus 2117, a known contaminant of biopharmaceutical manufacturing processes employing Chinese hamster cells. An extensive literature exists for inactivation of various animal caliciviruses, especially feline calicivirus and murine norovirus. The caliciviruses are susceptible to wet heat inactivation at temperatures in excess of 60 °C with contact times of 30 min or greater, to UV-C inactivation at fluence ≥30 mJ/cm2, to high pressure processing >200 MPa for >5 min at 4 °C, and to certain photodynamic inactivation approaches. The enteric caliciviruses (e.g.; noroviruses) display resistance to inactivation by low pH, while the non-enteric species (e.g.; feline calicivirus) are much more susceptible. The caliciviruses are inactivated by a variety of chemicals, including alcohols, oxidizing agents, aldehydes, and β-propiolactone. As with inactivation of viruses in general, inactivation of caliciviruses by the various approaches may be matrix-, temperature-, and/or contact time-dependent. The susceptibilities of the caliciviruses to the various physical and chemical inactivation approaches are generally similar to those displayed by other small, non-enveloped viruses, with the exception that the parvoviruses and circoviruses may require higher temperatures for inactivation, while these families appear to be more susceptible to UV-C inactivation than are the caliciviruses.

Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. HuNoV infections lead to substantial societal and economic burdens. There are currently no licensed vaccines or therapeutics for the prevention or treatment of HuNoVs. A lack of well-characterized in vitro and in vivo infection models has limited the development of HuNoV countermeasures. Experimental infection of human volunteers and the use of related viruses such as murine NoV have provided helpful insights into HuNoV biology and vaccine and therapeutic development. There remains a need for robust animal models and reverse genetic systems to further HuNoV research. This review summarizes available HuNoV animal models and reverse genetic systems, while providing insight into their usefulness for vaccine and therapeutic development.

Human norovirus (HuNoV) is a foremost cause of domestically acquired foodborne acute gastroenteritis and outbreaks. Despite industrial efforts to control HuNoV contamination of foods, its prevalence in foodstuffs at retail is significant. HuNoV infections are often associated with the consumption of contaminated produce, including ready-to-eat (RTE) salads. Decontamination of produce by washing with disinfectants is a consumer habit which could significantly contribute to mitigate the risk of infection. The aim of our study was to measure the effectiveness of chemical sanitizers in inactivating genogroup I and II HuNoV strains on mixed salads using a propidium monoazide (PMAxx)-viability RTqPCR assay. Addition of sodium hypochlorite, peracetic acid, or chlorine dioxide significantly enhanced viral removal as compared with water alone. Peracetic acid provided the highest effectiveness, with log 10 reductions on virus levels of 3.66 ± 0.40 and 3.33 ± 0.19 for genogroup I and II, respectively. Chlorine dioxide showed lower disinfection efficiency. Our results provide information useful to the food industry and final consumers for improving the microbiological safety of fresh products in relation to foodborne viruses., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.