Virucidal activity of chlorine dioxide in combination with acetic acid or citric acid and a surfactant, in presence of interfering substances, against polio-, adeno- and murine norovirus in suspension-, carrier- and four-field tests.

Introduction: The aim of the study was to investigate whether the virucidal effectiveness of chlorine dioxid against adenovirus and murine norovirus can be improved by combining it with carboxylic acids and surfactants. Method: The virucidal efficacy against polio-, adeno- and murine norovirus has been tested in presence of interfering substances in the quantitative suspension test according to EN 14476, the carrier test without mechanical action according to EN 16777, and in the four-field test according to EN 16615.Three chlorine-dioxide-based surface disinfectants were tested: a two-component cleaning disinfectant concentrate for large surfaces, a ready-to-use (RTU) foam, and an RTU gel. Results: Cleaning and disinfecting preparations based on chlorine dioxide, applied at various concentrations, in combination with acetic acid or citric acid and surfactants, are virucidally active against polio-, adeno-, and norovirus after an exposure time of 5 minutes in presence of interfering substances.

The first complete genome sequence and genetic evolution analysis of bovine norovirus in Xinjiang, China.

Introduction: Viruses are among the main pathogens causing diarrhoea in calves. The current study found that bovine norovirus (BNoV) is one of the principal viruses causing diarrhoea in calves in Xinjiang, China. Material and Methods: A total of 974 calf faecal samples from six regions in Xinjiang were tested for BNoV using reverse-transcriptase PCR. The genomic characteristics of BNoV and the genetic evolution of the VP1 gene, protein three-dimensional structure characteristics and amino acid variation were analysed using bioinformatics methods. Results: Epidemiological survey results showed that the infection rate of BNoV was 19.82%, and all samples tested positive in five regions. The results of the genetic evolution analysis showed that BNoV strains from Tacheng of northern Xinjiang and Kashgar of southern Xinjiang both belonged to the GIII.2 genotype of BNoV but were not on the same cluster of evolutionary branches. Additionally, the amino acid variation of the VP1 protein was not observed to significantly affect its spatial structure. Conclusion: This study is the first to report the genetic characteristics of the BNoV complete genome sequence in Xinjiang and provides a scientific basis for BNoV vaccine development and pathogenesis research.

Norovirus outbreaks in a nursery school in Buenos Aires, Argentina.

Norovirus (NoV) is the leading cause of outbreaks of acute gastroenteritis worldwide. These are non-enveloped viruses that are classified into 10 genogroups, of which genogroup I (GI), II (GII), IV (GIV), VIII (GVIII), and IX (GIX) are the ones that infect humans. Two outbreaks (A and B) of acute gastroenteritis that occurred in a nursery school are described. The first outbreak (A) occurred in November 2018, and the second (B) in February 2020. The detection of viral and bacterial pathogens was performed to study both outbreaks. Additionally, an epidemiological investigation of the outbreaks was conducted. In the analyzed fecal and vomit samples from both children and adults in the nursery school, NoV GII.4 [P16] Sydney 2012 and NoV GI.3 [P13] were detected in outbreaks A and B, respectively. Since the study of acute gastroenteritis outbreaks is underestimated in Argentina, it is necessary to design prevention, study, and control protocols, as well as to improve the outbreak notification system in our country.

Association between detection rate of norovirus GII and climatic factors in the Northwest Amazon region.

Worldwide, approximately one fifth of all cases of diarrhea are associated with norovirus, mainly in children, with a defined seasonality in temperate climates, but seasonal dynamics are less known in tropical climates. The objective was to investigate the impact of external clinical, epidemiological, and climatic factors on norovirus detection rates in samples from children under 5 years of age from Roraima, the Amazon region of Brazil. A total of 941 samples were included. According to climatic factors, we observed correlations between external climatic factors and weekly positivity rates, where temperature (P = 0.002), relative humidity (P = 0.0005), absolute humidity (P < 0.0001) and wind speed had the strongest effect (P = 0.0006). The Brazilian Amazon region presents a typical and favorable scenario for the persistence, expansion, and distribution of viral gastroenteritis. Importance: This study is important as it will serve as a basis for studies carried out in Brazil and Latin American countries on the epidemiological importance, seasonality, climate change, antigenic diversity, among other factors in the circulation of gastroenteric virus.

Reactive species of plasma-activated water for murine norovirus 1 inactivation.

Human norovirus (HuNoV), the leading cause of foodborne acute gastroenteritis, poses a serious threat to public health. Traditional disinfection methods lead to destructions of food properties and functions, and/or environmental contaminations. Green and efficient approaches are urgently needed to disinfect HuNoV. Plasma-activated water (PAW) containing amounts of reactive species is an emerging nonthermal and eco-friendly disinfectant towards the pathogenic microorganisms. However, the disinfection efficacy and mechanism of PAW on HuNoV has not yet been studied. Murine norovirus 1 (MNV-1) is one of the most commonly used HuNoV surrogates to evaluate the efficacy of disinfectants. In the current study, the inactivation efficacy of MNV-1 by PAW was investigated. The results demonstrated that PAW significantly inactivated MNV-1, reducing the viral titer from approximately 6 log10 TCID50/mL to non-detectable level. The decreased pH, increased oxidation-reduction potential (ORP) and conductivity of PAW were observed compared with that of deionized water. Compositional analysis revealed that hydrogen peroxide (H2O2), nitrate (NO3-) and hydroxyl radical (OH) were the functional reactive species in MNV-1 inactivation. L-histidine could scavenge most of the inactivation effect in a concentration-dependent manner. Moreover, PAW could induce damage to viral proteins. Part of MNV-1 particles was destroyed, while others were structurally intact without infectiousness. After 45 days of storage at 4 °C, PAW generated with 80 % O2 and 100 % O2 could still reduce over 4 log10 TCID50/mL of the viral titer. In addition, PAW prepared using hard water induced approximately 6 log10 TCID50/mL reduction of MNV-1. PAW treatment of MNV-1-inoculated blueberries reduced the viral titer from 3.79 log10 TCID50/mL to non-detectable level. Together, findings of the current study uncovered the crucial reactive species in PAW inactivate MNV-1 and provided a potential disinfection strategy to combat HuNoV in foods, water, and environment.

Long Amplicon Nanopore Sequencing for Dual-Typing RdRp and VP1 Genes of Norovirus Genogroups I and II in Wastewater.

Noroviruses (NoVs) are the leading cause of non-bacterial gastroenteritis with societal costs of US$60.3 billion per annum. Development of a long amplicon nanopore-based method for dual-typing the RNA-dependent RNA polymerase (RdRp) and major structural protein (VP1) regions from a single RNA fragment could improve existing norovirus typing methods. Application to wastewater-based epidemiology (WBE) and environmental testing could enable the discovery of novel types and improve outbreak tracking and source apportionment. Here, we have developed such a method with a consensus-based bioinformatics pipeline and optimised reverse transcription (RT) and PCR procedures. Inhibitor removal and LunaScript® RT gave robust amplification of the ≈ 1000 bp RdRP + VP1 amplicon for both the GI and GII PCR assays. Platinum™ Taq polymerase showed good sensitivity and reduced levels non-specific amplification (NSA) when compared to other polymerases. Optimised PCR annealing temperatures significantly reduced NSA (51.3 and 42.4% for GI and GII), increased yield (86.5% for GII) and increased taxa richness (57.7%) for GII. Analysis of three NoV positive faecal samples showed 100% nucleotide similarity with Sanger sequencing. Eight GI genotypes, 11 polymerase types (p-types) and 13 combinations were detected in wastewater along with 4 GII genotypes, 4 p-types and 8 combinations; highlighting the diversity of norovirus taxa present in wastewater in England. The most common genotypes detected in clinical samples were all detected in wastewater while we also frequently detected several GI genotypes not reported in the clinical data. Application of this method into a WBE scheme, therefore, may allow for more accurate measurement of norovirus diversity within the population.

Detection of Gastrointestinal Pathogens with Zoonotic Potential in Horses Used in Free-Riding Activities during a Countrywide Study in Greece.

The objectives of this study were (a) to detect zoonotic gastrointestinal pathogens in faecal samples of horses using the FilmArray® GI Panel and (b) to identify variables potentially associated with their presence. Faecal samples collected from 224 horses obtained during a countrywide study in Greece were tested by means of the BioFire® FilmArray® Gastrointestinal (GI) Panel, which uses multiplex-PCR technology for the detection of 22 pathogens. Gastrointestinal pathogens were detected in the faecal samples obtained from 97 horses (43.3%). Zoonotic pathogens were detected more frequently in samples from horses in courtyard housing (56.0%) than in samples from horses in other housing types (39.7%) (p = 0.040). The most frequently detected zoonotic pathogens were enteropathogenic Escherichia coli (19.2% of horses) and Shiga-like toxin-producing E. coli stx1/stx2 (13.8%). During multivariable analysis, two variables emerged as significant predictors for the outcome 'detection of at least one zoonotic pathogen in the faecal sample from an animal': (a) the decreasing age of horses (p = 0.0001) and (b) the presence of livestock at the same premises as the horses (p = 0.013). As a significant predictor for the outcome 'detection of two zoonotic pathogens concurrently in the faecal sample from an animal', only the season of sampling of animals (autumn) emerged as significant in the multivariable analysis (p = 0.049). The results indicated a diversity of gastrointestinal pathogens with zoonotic potential in horses and provided evidence for predictors for the infections; also, they can serve to inform horse owners and handlers regarding the possible risk of transmission of pathogens with zoonotic potential. In addition, our findings highlight the importance of continuous surveillance for zoonotic pathogens in domestic animals.

Sensitive Electrochemical and Thermal Detection of Human Noroviruses Using Molecularly Imprinted Polymer Nanoparticles Generated against a Viral Target.

Norovirus (NoV) is the predominant cause of foodborne illness globally; current detection methods are typically expensive, have inadequate sensitivities, and utilize biological receptors with poor stability. Therefore, accurate, cost-effective, and highly stable detection methods are needed to screen for NoV in foods. We developed molecularly imprinted polymer nanoparticles (nanoMIPs) to detect NoV using a small target epitope (12 amino acids) with a solid-phase synthesis approach. The performance of three batches of nanoMIPs with varying monomer compositions (nanoMIP-1, -2, and -3) were compared both experimentally and computationally. Surface plasmon resonance examined nanoMIP binding affinity to norovirus virus-like particles (NoV-LPs), whereby nanoMIP-1 had the lowest KD value of 0.512 µM. This is significant, as traditional targets for generation of norovirus ligands previously reported were generated against drastically larger norovirus capsid segments that have limitations in ease of production. Further, an electrochemical sensor was developed by covalently attaching the nanoMIPs to glassy carbon electrodes. In agreement with our predictions from density functional theory simulations, electrochemical impedance spectroscopy showed a sensitive response toward NoV-LPs for nanoMIP batches tested; however, nanoMIP-1 was optimal, with an excellent detection limit of 3.4 pg/mL (1.9 × 105 particles/mL). Due to its exceptional performance, nanoMIP-1 was immobilized to screen-printed electrodes and utilized within a thermal sensor, where it exhibited a low detection limit of 6.5 pg/mL (3.7 × 105 particles/mL). Crucially, we demonstrated that nanoMIP-1 could detect NoV in real food samples (romaine lettuce) by using electrochemical and thermal sensors. Consequently, the study highlights the exceptional potential of nanoMIPs to replace traditional biological materials (e.g., antibodies) as sensitive, versatile, and highly stable receptors within NoV sensors.

Machine Learning and Imputation to Characterize Human Norovirus Genotype Susceptibility to Sodium Hypochlorite.

Human norovirus (HuNoV) is the leading cause of foodborne illness in the developed world and a major contributor to gastroenteritis globally. Its low infectious dose and environmental persistence necessitate effective disinfection protocols. Sodium hypochlorite (NaOCl) bleach is a widely used disinfectant for controlling HuNoV transmission via contaminated fomites. This study aimed to evaluate the susceptibility of HuNoV genotypes (n = 11) from genogroups I, II, and IV to NaOCl in suspension. HuNoV was incubated for 1 and 5 min in diethyl pyrocarbonate (DEPC) treated water containing 50 ppm, 100 ppm, or 150 ppm NaOCl, buffered to maintain a pH between 7.0 and 7.5. Neutralization was achieved by a tenfold dilution into 100% fetal bovine serum. RNase pre-treatment followed by RT-qPCR was used to distinguish between infectious and non-infectious HuNoV. Statistical methods, including imputation, machine learning, and generalized linear models, were applied to process and analyze the data. Results showed that NaOCl reduced viral loads across all genotypes, though efficacy varied. Genotypes GI.1, GII.4 New Orleans, and GII.4 Sydney were the least susceptible, while GII.6 and GII.13 were the most susceptible. All NaOCl concentrations above 0 ppm were statistically indistinguishable, and exposure duration did not significantly affect HuNoV reduction, suggesting rapid inactivation at effective concentrations. For instance, some genotypes were completely inactivated within 1 min, rendering extended exposure unnecessary, while other genotypes maintained the initial concentration at both 1 and 5 min, indicating a need for longer contact times. These findings underscore the critical role of HuNoV genotype selection in testing disinfection protocols and optimizing NaOCl concentrations. Understanding HuNoV susceptibility to NaOCl bleach informs better disinfection strategies, aiding public health and food safety authorities in reducing HuNoV transmission and outbreaks.

Replication-deficient Sendai virus expressing human norovirus capsid protein elicits robust NoV-specific antibody and T-cell responses in mice.

Human norovirus (HuNoV) is a major global cause of acute gastroenteritis, with vaccine development facing several challenges. Despite years of research, there are currently no licensed vaccines available for controlling HuNoVs. Here, we describe the construction and testing of a replication-deficient Sendai virus (SeV) vector as a potential vaccine candidate against the HuNoV GII.4 genotype. SeV was chosen as the vaccine backbone due to its non-pathogenic nature in humans, its capability for long-term antigen expression in mammalian cells, and its suitability for mucosal administration. By inserting the HuNoV GII.4 capsid gene, VP1, into the SeV genome, we generated a replication-deficient SeV (SeV/dP.VP1) vector. The resultant SeV/dP.VP1 virus were observed to successfully express the inserted NoV VP1 gene upon infection. Inoculating the vaccine into wild-type mice elicited NoV-specific IgG antibodies, along with INF-γ and IL-2-producing T cells, through both intranasal (i.n.) and intramuscular (i.m.) immunization. Furthermore, a significant level of NoV-specific IgA was detected in lung homogenates after i.n. immunization, particularly using a high dose of the viral vector. Additionally, a synergistic effect was observed with heterologous prime-boost regimens using SeV/dP.VP1 and MVA.VP1 vectors, indicating the potential for more robust immune responses when the vaccine design is optimized. Our study demonstrates the potential of a SeV vaccine candidate in eliciting a broad immune response and lays the foundation for further exploration of the SeV vector platform's potential as a HuNoV vaccine. Additionally, the results emphasize the importance of vaccine dosage and administration route, highlighting the need for tailored immunization strategies.

Outbreak investigation of norovirus gastroenteritis in a childcare facility in Central Queensland, Australia: a household level case series analysis.

Introduction: Noroviruses are one of the most common causes of gastroenteritis in all age groups, including children. However, little has been reported on the transmission of norovirus within childcare facilities and the subsequent impact at the household level. Methods: We conducted an outbreak investigation of norovirus gastroenteritis in Central Queensland, Australia during May 2021, in a childcare facility and the associated exposed households. Case definitions and outbreak management were employed as per the Communicable Disease Network Australia guidelines for norovirus and suspected viral gastroenteritis. Each case or carer and respective household member was interviewed to determine the date and time of symptom onset, health outcomes, and infector-infectee pairs. We estimated attack rates within the childcare facility and households, and basic reproductive number (R0) for norovirus using time-dependent methods. Results: A total of 41 people developed gastrointestinal symptoms as a result of this outbreak, with 25 cases (61%) acquiring the infection in the centre and 16 cases (39%) occurring at households. Serial intervals were estimated as a mean 2.4 days (standard deviation 1.7 days), with a majority of cases (73%) in children under two years of age within the centre. Three faecal specimens were obtained, all detecting norovirus genotype II. The time-dependent R0 was 1.5 (95% confidence interval [95% CI]: 1.0-2.2). Discussion: The attack rate within the childcare facility was highest amongst children aged less than 2 years, highlighting the risk of infection for this age group. We recommend the exclusion of asymptomatic household contacts from childcare facilities to reduce the length and severity of norovirus outbreaks. Further investigation into childcare facility risk factors and associated households are required to optimise public health interventions.

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Lifting COVID-19-associated non-pharmaceutical interventions: potential impact on notifications of infectious diseases transmitted from person to person in 2022 in Bavaria, Germany.

Background: The COVID-19 pandemic and associated non-pharmaceutical interventions (NPIs) have led to substantial decreases in case numbers of infectious diseases in several countries worldwide. As NPIs were gradually lifted, intense or out-of-season outbreaks of respiratory and gastrointestinal diseases were reported, raising the hypothesis of a potential catch-up effect of infections. By analysing surveillance data from the federal reporting system for notifiable infectious diseases, we aimed to assess the potential impact of lifting COVID-19 associated NPIs on notifications of selected infectious diseases in Bavaria, 2022. Methods: We compared influenza, chickenpox, norovirus gastroenteritis, rotavirus gastroenteritis weekly case numbers in a pre-pandemic period (2016-2019) and 2022 using two time series analyses approaches: (i) a predictive model forecasting weekly case numbers for the pandemic years 2020-2022, based on 2016-2019 data, (ii) interrupted time series model, based on 2016-2022 data, including a term per pandemic period. Results: In 2022, incidence rates were higher compared to pre-pandemic period for influenza (IRR = 3.47, 95%CI: 1.49-7.94) and rotavirus gastroenteritis (IRR = 1.36, 95%CI: 0.95-1.93), though not significant for rotavirus gastroenteritis. Conversely, case numbers remained significantly below pre-pandemic levels for chickenpox (IRR = 0.52, 95%CI: 0.41-0.65) and norovirus gastroenteritis (IRR = 0.59, 95%CI: 0.42-0.82). Seasonality changed notably for influenza, showing an earlier influenza wave compared to pre-pandemic periods. Conclusion: The lifting of NPIs was associated with heterogenic epidemiological patterns depending on the selected disease. The full impact of NPIs and their discontinuation may only become clear with continued monitoring and assessment of potential additional contributing factors.

A Pseudovirus Nanoparticle Displaying the Vaccinia Virus L1 Protein Elicited High Neutralizing Antibody Titers and Provided Complete Protection to Mice against Mortality Caused by a Vaccinia Virus Challenge.

The recent worldwide incidence of mpox infection and concerns about future emerging variants of mpox viruses highlight the need for the development of a new generation of mpox vaccines. To achieve this goal, we utilized our norovirus S nanoparticle vaccine platform to produce and evaluate two pseudovirus nanoparticles (PVNPs), S-L1 and S-J1. These PVNPs displayed the L1 neutralizing antigen target of the vaccinia virus and a yet-untested J1 antigen of the mpox virus, respectively, with the aim of creating an effective nanoparticle-based mpox vaccine. Each self-assembled PVNP consists of an inner shell resembling the interior layer of the norovirus capsid and multiple L1 or J1 antigens on the surface. The PVNPs improved the antibody responses toward the displayed L1 or J1 antigens in mice, resulting in significantly greater L1/J1-specific IgG and IgA titers than those elicited by the corresponding free L1 or J1 antigens. After immunization with the S-L1 PVNPs, the mouse sera exhibited high neutralizing antibody titers against the vaccinia virus, and the S-L1 PVNPs provided mice with 100% protection against mortality caused by vaccinia virus challenge. In contrast, the S-J1 PVNPs induced low neutralizing antibody titers and conferred mice weak protective immunity. These data confirm that the L1 protein is an excellent vaccine target and that the readily available S-L1 PVNPs are a promising mpox vaccine candidate worthy of further development.

A Decade of Chronic Norovirus Infection Surveillance at the NIH Clinical Research Center: Clinical Characteristics, Molecular Epidemiology, and Replication.

BACKGROUND: Noroviruses are an important viral cause of chronic diarrhea in immunocompromised individuals. METHOD: We collected norovirus-positive stool samples (n=448) from immunocompromised patients (n=88) at the National Institutes of Health Clinical Research Center, U.S. from 2010-2022. We assessed clinical characteristics of the cohort, norovirus molecular epidemiology, and infectivity of norovirus specimens in human intestinal enteroids (HIEs) monolayers. RESULTS: Thirty-nine of the 88 patients had sequential stool samples that allowed documentation of chronic norovirus infection with shedding levels ranging from 104 to 1011 genome copies/g of stool. The majority with confirmed chronic norovirus infection in this cohort (32/39, 82%) had clinical evidence of an inborn error of immunity (13 identified monogenic diseases), most with combined immunodeficiency (15 of 32) or common variable immunodeficiency (11 of 32). Noroviruses detected in the cohort were genetically diverse: both Genogroup I (GI.2, GI.3, GI.5, and GI.6) and Genogroup II (GII.1-GII.4, GII.6, GII.7, GII.12, GII.14, and GII.17) genotypes were detected, with GII.4 variants (Osaka, Apeldoorn, Den Haag, New Orleans, and Sydney) predominant (51 of 88, 57.9%). Viruses belonging to the GII.4 Sydney variant group that replicated in HIEs (n=9) showed a higher fold-increase in RNA genome copies during infection compared to others that replicated. CONCLUSIONS: Genetically and biologically diverse noroviruses established chronic infection in individuals with both inborn and acquired immunologic defects enrolled in an NIH surveillance study spanning 12 years, demonstrating the unique nature of each virus and host interaction.

Exploring waterborne viruses in groundwater: Quantification and Virome characterization via passive sampling and targeted enrichment sequencing.

Aquifers, which provide drinking water for nearly half the world's population, face significant challenges from microbial contamination, particularly from waterborne viruses such as human adenovirus (HAdV), norovirus (NoV) and enterovirus (EV). This study, conducted as part of the UPWATER project, investigates the sources of urban groundwater contamination using viral passive sampling (VPS) and target enrichment sequencing (TES). We assessed the abundance of eight viral pathogens (HAdV, EV, NoV genogroup I and II, rotavirus, influenza A virus, hepatitis E virus and SARS-CoV-2) and investigated the virome diversity of groundwater in the aquifer of the Besòs River Delta in Catalonia. Over a period of 7 months, we collected 114 samples from the aquifer using nylon and nitrocellulose membranes to adsorb viruses over a 10-day period. Human faecal contamination was detected in nearly 50 % of the groundwater samples, with mean HAdV total counts ranging from 1.23E+02 to 3.66E+03 GC, and occasional detections of EV and NoV GI and GII. In addition, deep sequencing revealed a diverse virome in the aquifer, with detection of human pathogens, including adenovirus, astrovirus, calicivirus, enterovirus, herpesvirus, papillomavirus and rotavirus. Time-integrated sampling using VPS increases the likelihood of virus detection and, when combined with TES, can provide a deeper understanding of virus prevalence in this important water compartment. This approach is expected to streamline long-term monitoring efforts and enable small communities or water managers with limited resources to effectively manage their groundwater reservoirs.

Risk Factors for Norovirus Outbreaks in Schools and Kindergartens - Beijing Municipality, China, July 2017-June 2022.

What is already known on this topic?: Norovirus is the leading cause of global acute gastroenteritis outbreaks. Norovirus outbreaks mainly occur in schools and kindergartens in China, always causing public health issues. What is added by this report?: Conditional logistic regression method was used to analyze the risk factors for norovirus outbreaks in schools and kindergartens, and found that students vomiting at school or kindergarten, case activity in public areas, and the first case's classroom less than 5 meters from toilets were risk factors. What are the implications for public health practice?: Effective measures to address these factors can help reduce the risk of norovirus outbreaks in schools and kindergartens.

Development of an intestinal mucosa ex vivo co-culture model to study viral infections.

Studying viral infections necessitates well-designed cell culture models to deepen our understanding of diseases and develop effective treatments. In this study, we present a readily available ex vivo 3D co-culture model replicating the human intestinal mucosa. The model combines fully differentiated human intestinal epithelium (HIE) with human monocyte-derived macrophages (hMDMs) and faithfully mirrors the in vivo structural and organizational properties of intestinal mucosal tissues. Specifically, it mimics the lamina propria, basement membrane, and the air-exposed epithelial layer, enabling the pioneering observation of macrophage migration through the tissue to the site of viral infection. In this study, we applied the HIE-hMDMs model for the first time in viral infection studies, infecting the model with two globally significant viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human norovirus GII.4. The results demonstrate the model's capability to support the replication of both viruses and show the antiviral role of macrophages, determined by their migration to the infection site and subsequent direct contact with infected epithelial cells. In addition, we evaluated the production of cytokines and chemokines in the intestinal niche, observing an increased interleukin-8 production during infection. A parallel comparison using a classical in vitro cell line model comprising Caco-2 and THP-1 cells for SARS-CoV-2 experiments confirmed the utility of the HIE-hMDMs model in viral infection studies. Our data show that the ex vivo tissue models hold important implications for advances in virology research.IMPORTANCEThe fabrication of intricate ex vivo tissue models holds important implications for advances in virology research. The co-culture model presented here provides distinct spatial and functional attributes not found in simplified models, enabling the evaluation of macrophage dynamics under severe acute respiratory syndrome coronavirus 2 and human norovirus (HuNoV) infections in the intestine. Moreover, these models, comprised solely of primary cells, facilitate the study of difficult-to-replicate viruses such as HuNoV, which cannot be studied in cell line models, and offer the opportunity for personalized treatment evaluations using patient cells. Similar co-cultures have been established for the study of bacterial infections and different characteristics of the intestinal tissue. However, to the best of our knowledge, a similar intestinal model for the study of viral infections has not been published before.

Nonstructural protein 4 of human norovirus self-assembles into various membrane-bridging multimers.

Single-stranded, positive-sense RNA ((+)RNA) viruses replicate their genomes in virus-induced intracellular membrane compartments. (+)RNA viruses dedicate a significant part of their small genomes (a few thousands to a few tens of thousands of bases) to the generation of these compartments by encoding membrane-interacting proteins and/or protein domains. Noroviruses are a very diverse genus of (+)RNA viruses including human and animal pathogens. Human noroviruses are the major cause of acute gastroenteritis worldwide, with genogroup II genotype 4 (GII.4) noroviruses accounting for the vast majority of infections. Three viral proteins encoded in the N-terminus of the viral replication polyprotein direct intracellular membrane rearrangements associated with norovirus replication. Of these three, nonstructural protein 4 (NS4) seems to be the most important, although its exact functions in replication organelle formation are unknown. Here we produce, purify and characterize GII.4 NS4. AlphaFold modeling combined with experimental data refine and correct our previous crude structural model of NS4. Using simple artificial liposomes, we report an extensive characterization of the membrane properties of NS4. We find that NS4 self-assembles and thereby bridges liposomes together. Cryo-EM, NMR and membrane flotation show formation of several distinct NS4 assemblies, at least two of them bridging pairs of membranes together in different fashions. Noroviruses belong to (+)RNA viruses whose replication compartment is extruded from the target endomembrane and generates double-membrane vesicles. Our data establish that the 21-kDa GII.4 human norovirus NS4 can, in the absence of any other factor, recapitulate in tubo several features, including membrane apposition, that occur in such processes.

Comparison of four concentration methods of adenovirus, norovirus and rotavirus in tap water.

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 MgCl2 with or without acid rinse by H2SO4 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 MgCl2 using electronegative filters with acid rinse by H2SO4 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.