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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.

Human norovirus (NoV) is a leading cause of fresh produce associated outbreaks. Previous research indicates that the roots of growing leafy greens and berries internalize human NoV. However the effect of plant type and inoculum level on internalization rates has not been directly compared. In this study we compared the internalization and dissemination rates of human NoV and its surrogate, Tulane virus (TV) in green onion, radishes, and Romaine lettuce. We also evaluated the effect inoculum level and plant growth matrix on the rate of viral internalization. In the hydroponic growth system, we detected internalization and dissemination of human NoV RNA in green onions. In hydroponically growing green onions inoculated with high titer TV, we found higher rates of internalization and dissemination compared to green onions inoculated with low titer TV. In soil growth systems, no infectious TV was detected in either green onion or radishes. However, in Romaine lettuce plants grown in soil approximately 4 log10 PFU/g was recovered from all tissues on day 14 p.i. Overall, we found that the type of plant, growth matrix, and the inoculum level influences the internalization and dissemination of human NoV and TV.

ABSTRACT 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.

Human norovirus (HNoV) accounts for one-fifth of all acute viral gastroenteritis worldwide and an economic burden of ~60 billion globally. The lack of treatment options against HNoV is in part due to the lack of cultivation systems. Recently, a model of infection in biopsy-derived human intestinal enteroids (HIE) has been described: 3D-HIE are first dispersed in 2D-monolayers and differentiated prior to infection, resulting in a labor-intensive, time-consuming procedure. Here, we present an alternative protocol for HNoV infection of 3D-HIE. We found that 3D-HIE differentiated as efficiently as 2D-monolayers. In addition, immunofluorescence-based quantification of UEA-1, a lectin that stains the villus brush border, revealed that ~80% of differentiated 3D-HIE spontaneously undergo polarity inversion, allowing for viral infection without the need for microinjection. Infection with HNoV GII.4-positive stool samples attained a fold-increase over inoculum of ~2 Log10 at 2 days postinfection or up to 3.5 Log10 when ruxolitinib, a JAK1/2-inhibitor, was added. Treatment of GII.4-infected 3D-HIE with the polymerase inhibitor 2'-C-Methylcytidine (2CMC) and other antivirals showed a reduction in viral infection, suggesting that 3D-HIE are an excellent platform to test anti-infectives. The transcriptional host response to HNoV was then investigated by RNA sequencing in infected versus uninfected 3D-HIE in the presence of ruxolitinib to focus on virus-associated signatures while limiting interferon-stimulated gene signatures. The analysis revealed upregulated hormone and neurotransmitter signal transduction pathways and downregulated glycolysis and hypoxiaresponse pathways upon HNoV infection. Overall, 3D-HIE have proven to be a highly robust model to study HNoV infection, screen antivirals, and to investigate the host response to HNoV infection. [ABSTRACT FROM AUTHOR]

Background: Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens. Methods: RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored. Results: The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/μL within approximately 30–40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement. Conclusions: Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings. [ABSTRACT FROM AUTHOR]

Commonly used surface sanitizers often lack activity against human noroviruses (hNoVs). The impact of inactivation versus removal when these products are applied via wiping is poorly characterized. The purpose of this work was to assess the anti-hNoV efficacy of various surface sanitizer chemistries, as applied to a laminate material commonly used for restaurant tabletops, using standard surface assays (ASTM E1053-11) and a newly developed wiping protocol. Four commercially available products with different active ingredient(s) (i.e., ethanol [EtOH], acid 1 anionic surfactant [AAS], quaternary ammonium compound [QAC], and sodium hypochlorite [NaOCl]) and a water control were evaluated against hNoV GII.4 Sydney, hNoV GI.6, and the cultivable surrogate Tulane virus (TuV). Virus concentration was evaluated using RNase-reverse transcriptase (RT)-quantitative PCR (qPCR) (hNoV) and infectivity assay (TuV). Only the EtOH-based product significantly reduced virus concentration (.3.5 log10 reduction [LR]) by surface assay, with all other products producing #0.5 LR. The inclusion of a wiping step enhanced the efficacy of all products, producing complete virus elimination for the EtOH-based product and 1.6 to 3.8 LR for the other chemistries. For hNoVs, no detectable residual virus could be recovered from paper towels used to wipe the EtOH-based product, while high concentrations of virus could be recovered from the used paper towel and the wiped coupon (1.5 to 2.5 log10 lower genome equivalent copies [GEC] compared to control) for the QAC- and AAS-based products and for water. These results illustrate the variability in anti-hNoV activity of representative surface sanitizers and highlights the value of wiping, the efficacy of which appears to be driven by a combination of virus inactivation and removal. [ABSTRACT FROM AUTHOR]

In this study, the effect of sodium hypochlorite (10-200 ppm of Cl 2 ) on the inactivation of human norovirus (HuNV) GII.4 and hepatitis A virus (HAV) in groundwater was investigated using propidium monoazide (PMA)/reverse transcription quantitative real-time PCR (RT-qPCR). Initially, 4.00 log 10 genome copies/μL of HuNV GII.4 or 5.50 log 10 genome copies/μL of HAV were artificially inoculated in groundwater. The titers of HuNV GII.4 and HAV decreased significantly ( P < 0.05) with increasing Cl 2 concentrations. Groundwater was treated with 10, 30, 50, 100, 150, and 200 ppm of Cl 2 , and the viable HuNV GII.4 was significantly ( P < 0.05) reduced to 3.28 (0.21-log reduction), 3.18 (0.31-log reduction), 3.01 (0.48 log reduction), 2.75 (0.74 log reduction), 2.54 (0.95 log reduction), and 2.34 (1.15 log reduction) log 10 genome copies/μL, respectively. The viable HAV was also significantly ( P < 0.05) reduced to 4.99 (0.23 log reduction), 4.76 (0.46 log reduction), 4.55 (0.67 log reduction), 4.21 (1.01-log reduction), 3.89 (1.33 log reduction), and 3.64 (1.58 log reduction) log 10 genome copies/μL, respectively. The decimal reduction times ( D values) (1-log 10 genome reduction) of HuNV GII.4 and HAV infectivity in groundwater were predicted as 116.7 and 98.9 ppm of Cl 2 , respectively, using the first-order kinetics model (HuNV GII.4: y = -0.0054 x + 3.3585, correlation coefficient ( R 2 ) = 0.97; HAV: y = -0.0091 x + 5.0470, R 2 = 0.97). The result specifically suggests that 150- to 200-ppm Cl 2 can potentially be used for the inactivation of >1-log 10 genome copy/μL HuNV GII.4 and HAV in groundwater.IMPORTANCEGroundwater represents a vital component of the global water supply, serving as a crucial source of potable water for humans. It serves as a source of potable water for up to 50% of the global population and accounts for 43% of all water used for irrigation. It thus follows that the sustainable management of groundwater represents a pivotal solution. However, the regrowth of pathogens in water that is not treated with chlorine or where proper residual chlorine is not maintained represents a risk to public health., Competing Interests: The authors declare no conflict of interest

Despite regulations set up to monitor the microbiological quality of shellfish in producing areas, shellfish-borne gastroenteritis outbreaks still occur. Indeed, oyster depuration practices that are efficient to eliminate bacteria, fail to eliminate human norovirus from oyster flesh. In order to evaluate the impact of seawater temperature on the elimination of norovirus particles from oysters, large batches of oysters were contaminated using raw sewage containing norovirus and subjected to depuration at 8°C or 18°C. Over the experiment, quantitative RT-qPCR showed a one-log decrease of norovirus (both genogroups combined) genome copies per gram of digestive tissue after 41 days for oysters depurated at 8°C and 24 days at 18°C. The decrease of norovirus (both genogroups combined) in two batches of field-contaminated oysters depurated for two weeks at 18°C was in the same range (21 and 23 days respectively). All experiments showed a difference in genomic decay between the two norovirus genogroups, with norovirus genogroup I being more persistent in March/April compared to April/May., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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.

ABSTRACT: Objective: To compare the intestinal microbiota profiles in infants following rotavirus (RV) and human norovirus (HNoV) infection.Methods: Faecal specimens from 18 infants {mean age 11.8 months [standard deviation (SD) 3.0] months} with acute gastroenteritis caused by RV (G9P8) and 24 infants [mean age 8.8 (SD 6.4) months] with acute gastroenteritis caused by HNoV (GII) infection were collected prospectively. The faecal microbiome was assessed by 16S rRNA amplicon pyrosequencing. Alpha diversity, beta diversity, deferentially abundant taxa and microbial functions were assessed by bioinformatic analysis.Results: The Chao1 index for the HNoV group was significantly higher compared with the control group (P=0.0003), and was lower for the RV group compared with the HNoV group (P=0.0078). No significant difference in beta diversity was observed between the RV and HNoV groups. The RV group showed greater abundance of Actinobacteria at phylum level and Bifidobacterium spp., Streptococcus spp., Enterococcus spp. and Lactobacillus spp. at genus level. The HNoV group showed richness in Fusobacteria and Cyanobacteria at phylum level, and Enterococcus spp. and Streptococcus spp. at genus level. Bacillus was the characteristic genus in infected infants. In comparison with the control group, the viral group (P≤0.01), the RV group (P=0.002) and the HNoV group (P≤0.01) showed significant differences in potentially pathogenic bacteria.Conclusions: Changes in microbiotic structure were observed in infants following RV and HNoV infection. The Chao 1 index of alpha diversity increased significantly in the HNoV group. Bacillus was the characteristic genus in infected infants. An increase in pathogenic bacteria, particularly Streptococcus spp. and Enterococcus spp., was detected in infected infants.

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.

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. [ABSTRACT FROM AUTHOR]

Human norovirus (HuNoV) is the common cause of acute gastroenteritis worldwide. Norovirus-like particles (VLPs) have been applied to analyze virus–host cell interactions. This study established a highly quantitative detection system for HuNoV VLP entry into live cells based on the NanoLuc luciferase complementation. First, we generated a HiBiT-tagged VLP and examined VLP-HiBiT entry into Vero cells expressing LgBiT. The entry was saturable and competed by a non-labeled VLP, indicating the VLP-specific entry into the host cells. Upon comparison of the VLP-HiBiT entry among some cell lines, Caco-2 cells were found to be the most susceptible. The VLP-HiBiT entry was unaffected by treatment at an acidic pH value above 3.0 and was significantly reduced by heat treatment at 75°C for more than 2 min. These results reflected those of previous studies, indicating the stability of VLPs and binding to histo-blood group antigens at various pH ranges and temperatures and cellular tropism. • This study established quantitative detection system for the entry of norovirus. • HiBiT-tagged VLP (VLP-HiBiT) entered into LgBiT-expressing live cells. • VLP-HiBiT entry was unaffected by treatment at pH value above 3.0. • VLP-HiBiT entry was significantly reduced by heat treatment at 75°C for 2 min. • Caco-2 cells were the most susceptible among some cell lines examined. [ABSTRACT FROM AUTHOR]

Human enteric viruses, as adenovirus (HAdV), norovirus (HuNoV) and rotavirus (RVA) are significant causes of gastroenteritis associated with consumption of contaminated water worldwide. Various methods have been described for their detection and monitoring in water. The aim of this study was to compare the performance of four conditions for concentrating HAdV, HuNoV and RVA from water matrices, in order to develop a single protocol that could simultaneously concentrate all target viruses from tap water. The tested conditions were based on the adsorption-elution using electronegative filters, in which we evaluated cation-coated filtration by MgCl 2 with or without acid rinse by H 2 SO 4 and two elution buffers, namely NaOH and tris-glycine-beef extract. Genomic material was extracted and amplified by real-time PCR and real-time RT-PCR using commercial kits. Based on the statistical analysis of amplification results (cycles of quantification), the condition involving cation-coated filtration by MgCl 2 using electronegative filters with acid rinse by H 2 SO 4 combined with NaOH elution allowed efficient recovery of both HAdV, HuNoV and RVA from tap water compared to the other conditions. These findings confirm the effectiveness of the approach used to monitor three major enteric viruses in tap water. • The performance of four concentration conditions based on adsorption and elution through negatively charged filters were tested in detecting Adenovirus (HAdV), Norovirus (HuNoV) and Rotavirus (RVA) in tap water • These conditions were: (1) Filtration with preconditioning using MgCl2 and elution with NaOH, (2) Filtration with preconditioning using MgCl2 and elution with Tris-Glycine-Beef extract (TGBE), (3) Filtration without preconditioning and elution with NaOH, and (4) Filtration without preconditioning and elution with TGBE. • The ranking of concentration conditions varied depending on the specific virus being tested. • Based on statistical analysis of amplification results (Cycles of quantification), the condition involving cation-coated filtration by MgCl2 using electronegative filters, coupled with acid rinse using H2SO4 and NaOH elution, enabled efficient recovery of HAdV, HuNoV, and RVA from tap water, outperforming other conditions. This method consistently produces reproducible and consistent Cq values, rendering it a promising choice for routine virus research in water samples within virology laboratories. [ABSTRACT FROM AUTHOR]

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.

Aim: To evaluate the anti‐noroviral efficacy of PURELL® surface sanitizer and disinfectant spray (PSS, an alcohol‐based formulation) using human norovirus GII.4 Sydney [hNoV, by RT‐qPCR and human intestinal enteroid (HIE) infectivity assay] and its cultivable surrogate, Tulane virus (TuV, infectivity assay), compared to sodium hypochlorite (NaOCl) solutions. Methods and Results: PSS efficacy was evaluated in suspension and on surfaces [stainless steel (SS)] using ASTM methods. Results were expressed as log10 reduction (LR) of genome equivalent copy number (GEC, for hNoV, assayed by RT‐qPCR) and plaque forming units (PFU, for TuV, per infectivity assay). In suspension, PSS achieved a 2.9 ± 0.04 LR hNoV GEC irrespective of contact time (30 or 60 s) and soil load (2.5% or 5%). Under all treatment conditions, infectious TuV could not be recovered following exposure to PSS, corresponding to the assay limit of detection (3.1–5.2 log10 PFU). Infectious hNoV could not be detected in the HIE model after exposure to PSS. On SS and 2.5% soil, PSS produced a 3.1 ± 0.1 LR hNoV GEC, comparable to 500 ppm NaOCl for 60 s. With 5.0% soil, PSS produced a 2.5 ± 0.2 LR hNoV GEC, which was similar to 1000–5000 ppm NaOCl for 60 s. Conclusions: PSS showed high anti‐hNoV efficacy by RT‐qPCR and in in vitro (TuV) and ex vivo (HIE) infectivity assays and performed similar to 1000–5000 ppm NaOCl for a 60‐s contact time on SS with added soil. Significance and Impact of Study: hNoV remains a significant cause of morbidity globally, partly due to its resistance to numerous surface disinfectants. RT‐qPCR results from this study indicate PSS efficacy against hNoV is comparable to NaOCl efficacy. Infectivity assays leveraging TuV and the HIE model for hNoV support and confirm loss of virus infectivity. Collectively, these results indicate the product's ability to inactivate hNoV quickly, which could be beneficial in settings having elevated risk for hNoV transmission. [ABSTRACT FROM AUTHOR]

Slightly acidic electrolyzed water (SAEW), an effective non-thermal virucidal treatment, is used widely to prevent infectious viral cross-contamination. Surface disinfection technologies using ultraviolet C-light-emitting diode (UVC-LED) irradiation have recently attracted considerable attention. The SAEW sprayer technique is an efficient approach to preventing the spread of infectious viral pathogens in the public healthcare sector. Therefore, we investigated a small-scale system comprising sprayed SAEW disinfection combined with UVC-LED irradiation to inactivate the human norovirus (HuNoV) in the environment. A stainless-steel surface was inoculated with a HuNoV genogroup II genotype 4 (GII.4) to achieve maximum reduction values of 3.21 log10 genomic copies. For optimal disinfection conditions, the response surface methodology based on the Box–Behnken design revealed that the specific treatment conditions for inactivation of HuNoV GII.4 were an SAEW droplet volume of 180 μL, 30 ppm available chlorine concentration of SAEW, and a UVC-LED exposure dose of 2 mJ/cm2. The results indicate that the combined disinfection treatment could efficiently prevent the spread of HuNoVs in environment. Furthermore, the quadratic polynomial equations of the 3-D response surface can be employed to predict the effects of combined disinfection treatment on HuNoV contamination on environmental surfaces. Therefore, sprayed SAEW disinfection combined with UVC-LED irradiation proposed in this study may offer insights for designing optimal control strategies and techniques to prevent the transmission of infectious diseases, particularly HuNoV. [ABSTRACT FROM AUTHOR]

Fresh foods like fruits, vegetables and shellfish are potential sources for viral infections such as human norovirus (NoV). Chemical treatment like chlorination is a well-known process for food pathogens and virus elimination. However, with the increase of the consumer demand for less toxic treatment, the use of natural antimicrobials like essential oils from spice or plants, fruit extracts, and cold pasteurization treatments (fermentation, irradiation, ozonation and high pressure) could be considered. The aim of this review is to discuss these technologies and their efficacy to eliminate NoV on the surface of fresh food. [ABSTRACT FROM AUTHOR]

Human noroviruses (hNoV) are the leading cause of acute non-bacterial gastroenteritis worldwide and contaminated hands play a significant role in the spread of disease. Some hand sanitizers claim to interrupt hNoV transmission, but their antiviral efficacy on human hands is poorly characterized. The purpose of this work was to characterize the efficacy of representative commercial hand sanitizers against hNoV using an in vivo fingerpad method (ASTM E1838-17). Eight products [seven ethanol-based and one benzalkonium chloride (BAK)-based], and a benchmark 60% ethanol solution, were each evaluated on 10 human volunteers using the epidemic GII.4 hNoV strain. Virus titers before and after treatment were evaluated by RT-qPCR preceded by RNase treatment; product efficacy was characterized by log10 reduction (LR) in hNoV genome equivalent copies after treatment. The benchmark treatment produced a 1.7 ± 0.5 LR, compared with Product A (containing 85% ethanol) which produced a 3.3 ± 0.3 LR and was the most efficacious (p < 0.05). Product B (containing 70% ethanol), while less efficacious than Product A (p < 0.05), performed better than the benchmark with a LR of 2.4 ± 0.4. Five of the other ethanol-based products (labeled ethanol concentration ranges of 62–80%) showed similar efficacy to the 60% ethanol benchmark with LR ranging from 1.3 to 2.0 (p > 0.05). Product H (0.1% BAK) was less effective than the benchmark with a LR of 0.3 ± 0.2 (p < 0.05). None of the products screened were able to completely eliminate hNoV (maximum assay resolution 5.0 LR). Product performance was variable and appears driven by overall formulation. There remains a need for more hand sanitizer formulations having greater activity against hNoV, a virus that is comparatively recalcitrant relative to other pathogens of concern in community, healthcare, and food preparation environments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all age groups worldwide. HuNoVs can be detected in vitro using molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced personnel, and extended time to obtain results. Besides, the diversity of viral genotypes is high. Therefore, methods that are rapid, broad-range and effective in the detection of HuNoVs are desiderated for screening the feces or vomit of infected people during outbreaks. Results In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2 × 106 genomic copies per gram of stool sample (gc/g) and 4.4 × 105 gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against RT-qPCR. The relative sensitivity, specificity and agreement of ICA was 84.2% (95% CI: 83.6–84.8%), 100.0% (95% CI: 98.5–100.0%) and 87.7% (95% CI: 85.6–89.8%), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA detected a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs. Conclusions This study demonstrates that ICA targeting the S domain of VP1 is a promising candidate for effectively identifying the different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.

Slightly acidic electrolyzed water (SAEW), an effective non-thermal virucidal treatment, is used widely to prevent infectious viral cross-contamination. Surface disinfection technologies using ultraviolet C-light-emitting diode (UVC-LED) irradiation have recently attracted considerable attention. The SAEW sprayer technique is an efficient approach to preventing the spread of infectious viral pathogens in the public healthcare sector. Therefore, we investigated a small-scale system comprising sprayed SAEW disinfection combined with UVC-LED irradiation to inactivate the human norovirus (HuNoV) in the environment. A stainless-steel surface was inoculated with a HuNoV genogroup II genotype 4 (GII.4) to achieve maximum reduction values of 3.21 log10 genomic copies. For optimal disinfection conditions, the response surface methodology based on the Box–Behnken design revealed that the specific treatment conditions for inactivation of HuNoV GII.4 were an SAEW droplet volume of 180 μL, 30 ppm available chlorine concentration of SAEW, and a UVC-LED exposure dose of 2 mJ/cm2. The results indicate that the combined disinfection treatment could efficiently prevent the spread of HuNoVs in environment. Furthermore, the quadratic polynomial equations of the 3-D response surface can be employed to predict the effects of combined disinfection treatment on HuNoV contamination on environmental surfaces. Therefore, sprayed SAEW disinfection combined with UVC-LED irradiation proposed in this study may offer insights for designing optimal control strategies and techniques to prevent the transmission of infectious diseases, particularly HuNoV.

Human noroviruses (hNoV) are the leading cause of acute non-bacterial gastroenteritis worldwide and contaminated hands play a significant role in the spread of disease. Some hand sanitizers claim to interrupt hNoV transmission, but their antiviral efficacy on human hands is poorly characterized. The purpose of this work was to characterize the efficacy of representative commercial hand sanitizers against hNoV using an in vivo fingerpad method (ASTM E1838-17). Eight products [seven ethanol-based and one benzalkonium chloride (BAK)-based], and a benchmark 60% ethanol solution, were each evaluated on 10 human volunteers using the epidemic GII.4 hNoV strain. Virus titers before and after treatment were evaluated by RT-qPCR preceded by RNase treatment; product efficacy was characterized by log10 reduction (LR) in hNoV genome equivalent copies after treatment. The benchmark treatment produced a 1.7 ± 0.5 LR, compared with Product A (containing 85% ethanol) which produced a 3.3 ± 0.3 LR and was the most efficacious (p < 0.05). Product B (containing 70% ethanol), while less efficacious than Product A (p < 0.05), performed better than the benchmark with a LR of 2.4 ± 0.4. Five of the other ethanol-based products (labeled ethanol concentration ranges of 62–80%) showed similar efficacy to the 60% ethanol benchmark with LR ranging from 1.3 to 2.0 (p > 0.05). Product H (0.1% BAK) was less effective than the benchmark with a LR of 0.3 ± 0.2 (p < 0.05). None of the products screened were able to completely eliminate hNoV (maximum assay resolution 5.0 LR). Product performance was variable and appears driven by overall formulation. There remains a need for more hand sanitizer formulations having greater activity against hNoV, a virus that is comparatively recalcitrant relative to other pathogens of concern in community, healthcare, and food preparation environments.

Human noroviruses (HuNoVs) are the major non-bacterial pathogens causing acute gastroenteritis in people of all ages worldwide. No stable culture system in vitro is available for routing the detection of multiple strains of HuNoVs. A simple and rapid method for detection of HuNoVs is of great significance for preventing and controlling this pathogen. In this work, an electrochemical biosensor for sensitive and fast detection of HuNoVs was constructed based on a screen-printed carbon electrode (SPCE). Gold nanoparticles and protein-A were applied on the SPCE surface for enhancement of the electrical signals and the linkage of antibodies with a fixed orientation, respectively. A monoclonal antibody (MAb) against the S domain protein of the viral capsid (VP1) was further immobilized on the SPCE to bind HuNoVs specifically. The binding of VP1 to the coated MAbs resulted in the reduction of conductivity (current) measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The reduction in the current was correlated to the concentration of VP1/HuNoVs. The detection limitation of Genogroup I.1 (GI.1) VP1 and Genogroup II.4 (GII.4) VP1 was 0.37 ng/ml (≈1.93×107 HuNoVs/mL) and 0.22 ng/ml (≈1.15×107 HuNoVs/mL), respectively. The detection limitation of both GI and GII HuNoVs in clinical fecal samples was 104 genomic copies/mL. The results could be obtained in 1 h. We demonstrated that this disposable electrochemical biosensor was a good candidate for rapid detection of different genogroup and genotype HuNoVs.

Human norovirus (HuNoV) is a leading cause of infectious gastroenteritis. Since HuNoV shows resistance to alcohol, chlorine-based sanitizers are applied to decontaminate the virus on environmental surfaces. Chlorous acid water (CA) has been recently approved as a novel chlorine-based disinfectant categorized as a Type 2 OTC medicine in Japan. In this study, we aimed to evaluate the capability of CA to inactivate HuNoV. HuNoV (genogroups GII.2 and GII.4) was exposed to the test disinfectants including CA and sodium hypochlorite (NaClO), and the residual RNA copy was measured by reverse transcription quantitative PCR (RT-qPCR) after pretreatment with RNase. In addition, the log 10 reduction of HuNoV RNA copy number by CA and NaClO was compared in the presence of bovine serum albumin (BSA), sheep red blood cells (SRBC), polypeptone, meat extract or amino acids to evaluate the stability of these disinfectants under organic-matter-rich conditions. In the absence of organic substances, CA with 200 ppm free available chlorine provided >3.0 log 10 reduction in the HuNoV RNA copy number within 5 min. Even under high organic matter load (0.3% each of BSA and SRBC or 0.5% polypeptone), 200 ppm CA achieved >3.0 log 10 reduction in HuNoV RNA copy number while less than 1.0 log 10 reduction was observed with 1,000 ppm sodium hypochlorite (NaClO) in the presence of 0.5% polypeptone. CA reacted with only cysteine, histidine and glutathione while NaClO reacted with all of the amino acids tested. CA is an effective disinfectant to inactivate HuNoV under organic-matter-rich conditions. [ABSTRACT FROM AUTHOR]

Intestinal commensal bacteria contribute to maintaining gut homeostasis. Disruptions to the commensal flora are linked to the development and persistence of disease. The importance of these organisms is further demonstrated by the widespread ability of enteric viruses to exploit commensal bacteria to enhance viral infection. These viruses interact directly with commensal bacteria, and while the impact of this interaction on viral infection is well described for several viruses, the impact on the commensal bacteria has yet to be explored. In this article, we demonstrate, for the first time, that enteric viruses alter the gene expression and phenotype of individual commensal bacteria. Human and murine norovirus interaction with bacteria resulted in genome‐wide differential gene expression and marked changes in the surface architecture of the bacterial cells. Furthermore, the interaction of the virus with bacteria led to increased production of smaller outer membrane vesicles (OMVs). Enhanced production of smaller vesicles was also observed when noroviruses were incubated with other commensal bacteria, indicating a potentially broad impact of norovirus interaction. The vesicle production observed in the in vivo model followed a similar trend where an increased quantity of smaller bacterial vesicles was observed in stool collected from virus‐infected mice compared to mock‐infected mice. Furthermore, changes in vesicle size were linked to changes in protein content and abundance, indicating that viral binding induced a shift in the mechanism of the OMV biogenesis. Collectively, these data demonstrate that enteric viruses induce specific changes in bacterial gene expression, leading to changes in bacterial extracellular vesicle production that can potentially impact host responses to infection. [ABSTRACT FROM AUTHOR]

GII.2[P16] and GII.4 Sydney [P16] are currently the two predominant norovirus genotypes. This study sought to clarify their evolutionary patterns by analyzing the major capsid VP1 and RNA-dependent RNA polymerase (RdRp) genes. Sequence diversities were analyzed at both nucleotide and amino acid levels. Selective pressures were evaluated with the Hyphy package in different models. Phylogenetic trees were constructed by the maximum likelihood method from full VP1 sequences, and evolutionary rates were estimated by the Bayesian Markov Chain Monte Carlo approach. The results showed that (1) several groups of tightly linked mutations between the RdRp and VP1 genes were detected in the GII.2[P16] and GII.4[P16] noroviruses, and most of these mutations were synonymous, which may lead to a better viral fitness to the host; (2) although the pattern of having new GII.4 variants every 2–4 years has been broken, both the pre- and the post-2015 Sydney VP1 had comparable evolutionary rates to previously epidemic GII.4 variants, and half of the major antigenic sites on GII.4 Sydney had residue substitutions and several caused obvious changes in the carbohydrate-binding surface that may potentially alter the property of the virus; and (3) GII.4 Sydney variants during 2018–21 showed geographical specificity in East Asia, South Asia, and North America; the antigenic sites of GII.2 are strictly conserved, but the GII.2 VP1 chronologically evolved into nine different sublineages over time, with sublineage IX being the most prevalent one since 2018. This study suggested that both VP1 and RdRp of the GII.2[P16] and GII.4 Sydney [P16] noroviruses exhibited different evolutionary directions. GII.4[P16] is likely to generate potential novel epidemic variants by accumulating mutations in the P2 domain, similar to previously epidemic GII.4 variants, while GII.2[P16] has conserved predicted antigenicity and may evolve by changing the properties of nonstructural proteins, such as polymerase replicational fidelity and efficiency. This study expands the understanding of the evolutionary dynamics of GII.2[P16] and GII.4[P16] noroviruses and may predict the emergence of new variants. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the virucidal effect and potential use as a disinfectant of undiluted and diluted slightly acidic electrolyzed water (SAEW) on human noroviruses (HuNoVs) using the in vitro suspension test and in food test. The oxidization reduction potential of SAEW gradually decreased with the increase in distilled water volume. Moreover, as the volume of distilled water and the dilution ratio increased, the available chlorine concentration of the samples significantly decreased from 31.2 ± 0.63 (SAEW) to 1.3 ± 0.21 (1:10 dilution of SAEW solution) (p < 0.05). Undiluted SAEW presented the lowest pH (5.56 ± 0.02) and as SAEW was diluted in distilled water, the pH of the sample increased. Considering the standard reduction values of pathogenic virus (> 4.00 log reduction), the reduction value of HuNoVs in cabbage samples was 4.65 (GI.6) and 4.28 (GII.4) log. These results suggest the potential application of SAEW for inactivating HuNoVs in the food industry. [ABSTRACT FROM AUTHOR]

This study investigated an outbreak in a kindergarten in Wuyi County of acute gastroenteritis concerning a large number of students and teachers. We performed a case-control study, and collected information on the layout of the school, symptoms, onset time of all cases and vomiting sites. A total of 62 individuals fit the definition of probable cases; among these, there were 19 cases of laboratory-confirmed norovirus infection. Nausea and vomiting were the most common symptoms in the outbreak. Seven student norovirus patients vomited in the school. The odds ratio (OR) of norovirus illness was 15.75 times higher among teachers who handled or interacted with student vomitus without respiratory protection than compared to those without this type of exposure (OR 15.75, 95% CI 1.75–141.40). Nine samples were successfully genotyped; eight samples were norovirus GII.2[P16], one sample was norovirus GII.4 Sydney[P16]. This study revealed that improper handling of vomitus is a risk factor of norovirus infection. Therefore, more attention should be given to train school staff in knowledge of disinfection. [ABSTRACT FROM AUTHOR]

Human norovirus (HuNoV) is the leading cause of foodborne illness outbreaks and the second most common cause of waterborne infections in the United States. The goal of this research was to investigate the antiviral activity of chitosan microparticles (CMs) against HuNoV GII.4 Sydney and its cultivable surrogate Tulane virus (TuV) in suspensions mimicking fecally contaminated water. CMs were prepared by cross-linking chitosan molecules with sodium sulfate, and the antiviral activity of CMs was assessed with an infectivity assay on TuV and by quantitative reverse transcription PCR on TuV and HuNoV. A 3% CM suspension in phosphate-buffered saline (pH 7.2) bound to TuV particles but had a negligible impact on virus infectivity (P > 0.05). A 10-min contact time resulted in a 1.5-log reduction in genomic copies per mL of TuV and HuNoV in fecal suspensions (P < 0.05). Despite the negligible impact on viral infectivity, CMs can moderately bind to infectious virus particles and help purify environmental water by removing these particles. In this study, TuV was a suitable surrogate for HuNoV with similar log reductions in fecal suspension. These findings highlight the potential application of CM as a novel treatment to minimize the spread of waterborne viral pathogens. CMs had antiviral activity against TuV and HuNoV in fecal suspensions. CMs impact on TuV infectivity was negligible. CMs bound to virus particles regardless of the fecal load. CMs have the potential to remove HuNoV from water. [ABSTRACT FROM AUTHOR]

Human norovirus (NOV) is a common and serious virus that accounts for sporadic cases and outbreaks of gastroenteritis. This study aimed to develop rapid, reliable and portable detection systems by coupling reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for NOV genotype GII.4. Here, three primers for RNA-dependent RNA polymerase gene of NOV were designed and screened. Then, RT-RPA products were detected using CRISPR-Cas12a system by combing with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 40 min, with the detection limit of up to 9.65 × 102copies/mL and no cross-reactivity with metapneumovirus, bocavirus, seoul virus, and respiratory syncytial virus. Furthermore, the detection coincidence rates of RT-RPA-Cas12a-based fluorescence and LF with qRT-PCR were 98.3%. Therefore, the present study suggests that both RT-RPA-Cas12a-based fluorescence and LF are promising sensitive, specific and alternative method for diagnosis of NOV genotype GII.4 without ancillary equipment. • RT-RPA-Cas12a-fluorescence and lateral flow assay developed are rapid and reliable method for norovirus diagnosis. • RT-RPA-Cas12a-fluorescence and lateral flow assay can be achieved within 40 min. • RT-RPA-Cas12a-fluorescence and lateral flow assay display high specificity and sensitivity of 9.65 × 102 copies/mL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Human noroviruses (HuNoV) are the leading cause of gastrointestinal illness and environmental monitoring is crucial to prevent HuNoV outbreaks. The recent development of a HuNoV cell culture assay in human intestinal enteroids (HIEs) has enabled detection of infectious HuNoV. However, this complex approach requires adaptation of HIEs to facilitate HuNoV replication from environmental matrixes. Integrating data from 200 experiments, we examined six variables: HIE age, HIE basement membrane compounds (BMC), HuNoV inoculum processing, HuNoV inoculum volume, treatment of data below limit of detection (LOD), and cutoff criteria for determining positive HuNoV growth. We infected HIEs with HuNoV GII.4 Sydney positive stool and determined 1.4 × 103 genome equivalents per HIE well were required for HuNoV replication. HIE age had minimal effect on assay outcomes. LOD replacement and cutoff affected data interpretation, with lower values resulting in higher estimated HuNoV detection. Higher inoculum volumes lead to minimal decreases in HuNoV growth, with an optimal volume of 250uL facilitating capture of low concentrations of HuNoVs present in environmental isolates. Processing of HuNoV inoculum is valuable for disinfection studies and concentrating samples but is not necessary for all HIE applications. This work enhances the HuNoV HIE cell culture approach for environmental monitoring. Future HIE research should report cell age as days of growth and should clearly describe BMC choice, LOD handling, and positive cutoff. [ABSTRACT FROM AUTHOR]

Human norovirus (HuNoV) is one of the leading causes of acute gastroenteritis globally. HuNoV outbreaks have been recently reported during air travels. Contaminated surfaces are known as a critical transmission route at various settings. The aim of this study was to provide key information about the survival and the decontamination of HuNoV on three commonly touched airplane cabin surfaces. In this study, we monitored the survival of HuNoV on seat leather, plastic tray table, and seatbelt for 30 days, with and without additional organic load (simulated gastric fluid). The efficacy of two EPA registered anti-norovirus disinfectants were also evaluated. Results showed that HuNoV was detected at high titers (>4 log 10 genomic copy number) for up to 30 days when additional organic load was present. Both tested disinfectants were found highly ineffective against HuNoV when the surface was soiled. The study showed that when the organic load was present, HuNoV was highly stable and resistant against disinfectants. Findings from this study indicated that appropriate procedures should be developed by airline companies with the help of public health authorities to decrease passengers’ exposure risk to HuNoV.

In July 2018, recombinant norovirus GII.Pe-GII.4 Sydney was detected in dogs who had diarrhea in a kennel and in children living on the same premises in Thailand. Whole-genome sequencing and phylogenetic analysis of 4 noroviruses from Thailand showed that the canine norovirus was closely related to human norovirus GII.Pe-GII.4 Sydney, suggesting human-to-canine transmission.

Abstract Intestinal commensal bacteria contribute to maintaining gut homeostasis. Disruptions to the commensal flora are linked to the development and persistence of disease. The importance of these organisms is further demonstrated by the widespread ability of enteric viruses to exploit commensal bacteria to enhance viral infection. These viruses interact directly with commensal bacteria, and while the impact of this interaction on viral infection is well described for several viruses, the impact on the commensal bacteria has yet to be explored. In this article, we demonstrate, for the first time, that enteric viruses alter the gene expression and phenotype of individual commensal bacteria. Human and murine norovirus interaction with bacteria resulted in genome‐wide differential gene expression and marked changes in the surface architecture of the bacterial cells. Furthermore, the interaction of the virus with bacteria led to increased production of smaller outer membrane vesicles (OMVs). Enhanced production of smaller vesicles was also observed when noroviruses were incubated with other commensal bacteria, indicating a potentially broad impact of norovirus interaction. The vesicle production observed in the in vivo model followed a similar trend where an increased quantity of smaller bacterial vesicles was observed in stool collected from virus‐infected mice compared to mock‐infected mice. Furthermore, changes in vesicle size were linked to changes in protein content and abundance, indicating that viral binding induced a shift in the mechanism of the OMV biogenesis. Collectively, these data demonstrate that enteric viruses induce specific changes in bacterial gene expression, leading to changes in bacterial extracellular vesicle production that can potentially impact host responses to infection.

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 (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.

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.

Carpet cleaning guidelines currently do not include the use of an antimicrobial, except after a bodily fluid event. To address this gap, we compared the efficacy of three antimicrobials-two hydrogen peroxide-based (H 2 O 2 ) products (A and B) and one chlorine-based product (C)-and a steam treatment against two norovirus surrogates, specifically feline calicivirus (FCV) and Tulane virus (TuV). These tests were performed on nylon carpets with either water-permeable or waterproof backing types. The effect of repeated antimicrobial use on carpet properties was also evaluated. For a carpet with water-permeable backing, products A, B, and C achieved a 0.8, 3.1, and 0.9 log 10 PFU/coupon reduction of FCV and 0.3, 2.5, and 0.4 log 10 TCID 50 /coupon reduction of TuV, respectively, following a 30 min contact time. For carpet with waterproof backing, only product B achieved a 5.0 log 10 PFU/coupon reduction of FCV and >3.0 log 10 TCID 50 /coupon reduction of TuV, whereas products A and C achieved a 2.4 and 1.6 log 10 PFU/coupon reduction of FCV and a 1.2 and 1.2 log 10 TCID 50 /coupon reduction of TuV, respectively. Steam treatment achieved a ≥ 5.2 log 10 PFU/coupon reduction of FCV and a > 3.2 log 10 TCID 50 /coupon reduction of TuV in 15 seconds on the carpet with both backing types. The repeated use of products A and B decreased the tensile strength of the carpet backing, while use of product B resulted in cracks on carpet fibers. Overall, steam treatment for 15 seconds was efficacious on both carpet types, but only product B achieved efficacy after a 30-minute exposure on the carpet with waterproof backing.IMPORTANCECarpets are common in long-term care facilities, despite its potential as a vehicle for transmission of agents associated with healthcare-associated infections, including human norovirus (NoV). Presently, our understanding of carpet disinfection is limited; hence, there are no commercial antimicrobials against norovirus available for use on carpets. Our findings showed that steam treatment, which minimally affected the properties of carpet fibers and backing, was more efficacious against human norovirus surrogates on carpets compared to the three chemical antimicrobials tested. Additionally, the two surrogates were more sensitive to chemical antimicrobials on the carpet with waterproof backing compared to carpets with water-permeable backing. These findings can inform development of antimicrobials for use on carpets contaminated with human norovirus., Competing Interests: The authors declare no conflict of interest.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

• Rotavirus and human norovirus had different effects on intestinal microbiota in infants with acute gastroenteritis. • Bacillus was detected as the characteristic genus in infected infants. • Disturbance of microbiota may increase the risk of potentially pathogenic bacteria. Objective : To compare the intestinal microbiota profiles in infants following rotavirus (RV) and human norovirus (HNoV) infection. Methods: Faecal specimens from 18 infants {mean age 11.8 months [standard deviation (SD) 3.0] months} with acute gastroenteritis caused by RV (G9P8) and 24 infants [mean age 8.8 (SD 6.4) months] with acute gastroenteritis caused by HNoV (GII) infection were collected prospectively. The faecal microbiome was assessed by 16S rRNA amplicon pyrosequencing. Alpha diversity, beta diversity, deferentially abundant taxa and microbial functions were assessed by bioinformatic analysis. Results: The Chao1 index for the HNoV group was significantly higher compared with the control group (P =0.0003), and was lower for the RV group compared with the HNoV group (P =0.0078). No significant difference in beta diversity was observed between the RV and HNoV groups. The RV group showed greater abundance of Actinobacteria at phylum level and Bifidobacterium spp., Streptococcus spp., Enterococcus spp. and Lactobacillus spp. at genus level. The HNoV group showed richness in Fusobacteria and Cyanobacteria at phylum level, and Enterococcus spp. and Streptococcus spp. at genus level. Bacillus was the characteristic genus in infected infants. In comparison with the control group, the viral group (P ≤0.01), the RV group (P =0.002) and the HNoV group (P ≤0.01) showed significant differences in potentially pathogenic bacteria. Conclusions: Changes in microbiotic structure were observed in infants following RV and HNoV infection. The Chao 1 index of alpha diversity increased significantly in the HNoV group. Bacillus was the characteristic genus in infected infants. An increase in pathogenic bacteria, particularly Streptococcus spp. and Enterococcus spp. , was detected in infected infants. [ABSTRACT FROM AUTHOR]

Human noroviruses (HuNoVs) are important foodborne pathogens causing acute gastroenteritis. Oysters are an important vehicle for the transmission of HuNoVs. Histo-blood group antigen (HBGA)-like substances are considered the primary ligands for bioaccumulation of HuNoVs in oyster tissues. In this study, proteinaceous ligands for specific binding of HuNoVs were mined from oyster tissues using a bacterial cell surface display system. The macromolecular target was captured and identified in proteomic analysis. The distribution of viral particles, oyster heat shock protein 70 (oHSP 70), and type A HBGA (positive control) in oyster tissue was investigated by multiplex immunofluorescence assays after artificial contamination with HuNoVs (GII.4). Our results demonstrated that oHSP 70 is a candidate vital ligand for specific binding of HuNoVs in oyster tissues. In addition, P proteins (GI.1 and GII.4) and viral particles (GI.1 and GII.4) were captured by recombinant oHSP 70 in an enzyme-linked immunosorbent assay with a sample signal/negative signal of 7.8, 6.3, 17.0, and 8.8, respectively. The findings suggested that oHSP 70 plays an important role in the binding of these foodborne viruses. [ABSTRACT FROM AUTHOR]

Contamination of fomites by human norovirus (HuNoV) can initiate and prolong outbreaks. Fomite swabbing is necessary to predict HuNoV exposure and target interventions. Historically, swab recovered HuNoV has been measured by molecular methods that detect viral RNA but not infectious HuNoV. The recent development of HuNoV cultivation in human intestinal enteroids (HIEs) enables detection of infectious HuNoV. It is unknown if the swabbing process and swab matrix will allow for cultivation of fomite recovered HuNoV. We used HIEs to culture swab-recovered HuNoV GII.4 Sydney from experimentally infected surfaces—a hospital bed tray (N = 32), door handle (N = 10), and sanitizer dispenser (N = 11). Each surface was swabbed with macrofoam swabs premoistened in PBS plus 0.02% Tween80. Swab eluate was tested for infectious HuNoV by cultivation in HIE monolayers. Infectious HuNoV can be recovered from surfaces inoculated with at least 105 HuNoV genome equivalents/3 cm2. In total, 57% (N = 53) of recovered swabs contained infectious HuNoV detected by HIEs. No difference in percent positive swabs was observed between the three surfaces at p = 0.2. We demonstrate that fomite swabbing can be combined with the HIE method to cultivate high titer infectious HuNoV from the environment, filling a significant gap in HuNoV detection. Currently, high titers of HuNoV are required to measure growth in HIEs and the HIE system precludes absolute quantification of infectious viruses. However, the HIE system can provide a binary indication of infectious HuNoV which enhances existing detection methods. Identification of infectious HuNoVs from swabs can increase monitoring accuracy, enhance risk estimates, and help prevent outbreaks.

Human norovirus (HuNoV) is the leading cause of epidemic acute gastroenteritis worldwide. Type I interferons (IFN)-α/β are highly potent cytokines that are initially identified for their essential roles in antiviral defense. It was reported that HuNoV infection did not induce IFN-β expression but was controlled in the presence of IFN-β in human intestinal enteroids and a gnotobiotic pig model, suggesting that HuNoV has likely developed evasion countermeasures. In this study, we found that a cDNA clone of GII.4 HuNoV, the predominantly circulating genotype worldwide, inhibits the production of IFN-β and identified the viral NTPase as a key component responsible for such inhibition. HuNoV NTPase not only inhibits the activity of IFN-β promoter but also the mRNA and protein production of IFN-β. Additional studies indicate that NTPase inhibits the phosphorylation and nuclear translocation of interferon-regulatory factor-3 (IRF-3), leading to the suppression of IFN-β promoter activation. Mechanistically, NTPase interacts with IkB kinase ε (IKKε), an important factor for IRF-3 phosphorylation, and such interaction blocks the association of IKKε with unanchored K48-linked polyubiquitin chains, resulting in the inhibition of IKKε phosphorylation. Further studies demonstrated that the 1-179 aa domain of NTPase which interacts with IKKε is critical for the suppression of IFN-β production. Our findings highlight the role of HuNoV NTPase in the inhibition of IFN-β production, providing insights into a novel mechanism underlying how HuNoV evades the host innate immunity.

Human norovirus (HuNoV) is the leading cause of epidemic acute gastroenteritis worldwide. Type I interferons (IFN)-α/β are highly potent cytokines that are initially identified for their essential roles in antiviral defense. It was reported that HuNoV infection did not induce IFN-β expression but was controlled in the presence of IFN-β in human intestinal enteroids and a gnotobiotic pig model, suggesting that HuNoV has likely developed evasion countermeasures. In this study, we found that a cDNA clone of GII.4 HuNoV, the predominantly circulating genotype worldwide, inhibits the production of IFN-β and identified the viral NTPase as a key component responsible for such inhibition. HuNoV NTPase not only inhibits the activity of IFN-β promoter but also the mRNA and protein production of IFN-β. Additional studies indicate that NTPase inhibits the phosphorylation and nuclear translocation of interferon-regulatory factor-3 (IRF-3), leading to the suppression of IFN-β promoter activation. Mechanistically, NTPase interacts with IkB kinase ε (IKKε), an important factor for IRF-3 phosphorylation, and such interaction blocks the association of IKKε with unanchored K48-linked polyubiquitin chains, resulting in the inhibition of IKKε phosphorylation. Further studies demonstrated that the 1-179 aa domain of NTPase which interacts with IKKε is critical for the suppression of IFN-β production. Our findings highlight the role of HuNoV NTPase in the inhibition of IFN-β production, providing insights into a novel mechanism underlying how HuNoV evades the host innate immunity. [ABSTRACT FROM AUTHOR]