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The aim of this study was to evaluate whether community-level monitoring of respiratory and enteric viruses in wastewater can provide a comprehensive picture of local virus circulation. Wastewater samples were collected weekly at the wastewater treatment plant (WWTP) inlet and at the outlet of a nearby nursing home (NH) in Burgundy, France, during the winter period of 2022/2023. We searched for the pepper mild mottle virus as an indicator of fecal content as well as for the main respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus] and enteric viruses (rotavirus, sapovirus, norovirus, astrovirus, and adenovirus). Samples were analyzed using real-time reverse transcription PCR-based methods. SARS-CoV-2 was the most frequently detected respiratory virus, with 66.7% of positive samples from the WWTP and 28.6% from the NH. Peaks of SARS-CoV-2 were consistent with the chronological incidence of infections recorded in the sentinel surveillance and the nearby hospital databases. The number of positive samples was lower in the NH than in WWTP for the three respiratory viruses. Enteric viruses were frequently detected, most often sapovirus and norovirus genogroup II, accounting both for 77.8% of positive samples in the WWTP and 57.1% and 37%, respectively, in the NH. The large circulation of sapovirus was unexpected in particular in the NH. Combined wastewater surveillance using simple optimized methods can be a valuable tool for monitoring viral circulation and may serve as a suitable early warning system for identifying both local outbreaks and the onset of epidemics. These results encourage the application of wastewater-based surveillance (WBS) to SARS-CoV2, norovirus, and sapovirus.IMPORTANCEWBS provides valuable information on the spread of epidemic viruses in the environment using appropriate and sensitive detection methods. By monitoring the circulation of viruses using reverse transcription PCR methods in wastewater from the inlet of a wastewater treatment plant and the outlet of a nearby retirement home (connected to the same collective sewer network), we aimed to demonstrate that implementing combined WBS at key community sites allows effective detection of the occurrence of respiratory (influenza, respiratory syncytial virus, and SARS-CoV-2) and enteric (norovirus, rotavirus, and sapovirus) virus infections within a given population. This analysis on a localized scale provided new information on the viral circulation in the two different sites. Implementing WBS to monitor the circulation or the emergence of infectious diseases is an important means of alerting the authorities and improving public health management. WBS could participate actively to the health of humans, animals, and the environment., Competing Interests: Floriane Pez was an employee of BioSellal S.A. until October 2023.

Human norovirus (HuNoV) is recognized as the leading causative agent of foodborne outbreaks of epidemic gastroenteritis. Consequently, there is a high demand for developing point-of-care testing for HuNoV. We developed an origami microfluidic device that facilitates rapid detection of murine norovirus 1 (MNV-1), a surrogate for HuNoV, encompassing the entire process from sample preparation to result visualization. This process includes RNA absorption via a paper strip, RNA amplification using recombinase polymerase amplification (RPA), and a lateral flow assay for signal readout. The on-chip detection of MNV-1 was completed within 35 min, demonstrating 100% specificity to MNV-1 in our settings. The detection limit of this microfluidic device for MNV-1 was 200 PFU/mL, comparable to the in-tube RPA reaction. It also successfully detected MNV-1 in lettuce and raspberries at concentrations of 170 PFU/g and 230 PFU/g, respectively, without requiring extra concentration steps. This device demonstrates high compatibility with isothermal nucleic acid amplification and holds significant potential for detecting foodborne viruses in agri-food products in remote and resource-limited settings., Importance: HuNoV belongs to the family of Caliciviridae and is a leading cause of acute gastroenteritis that can be transmitted through contaminated foods. HuNoV causes around one out of five cases of acute gastroenteritis that lead to diarrhea and vomiting, placing a substantial burden on the healthcare system worldwide. HuNoV outbreaks can occur when food is contaminated at the source (e.g., wild mussels exposed to polluted water), on farms (e.g., during crop cultivation, harvesting, or livestock handling), during packaging, or at catered events. The research outcomes of this study expand the approaches of HuNoV testing, adding value to the framework for routine testing of food products. This microfluidic device can facilitate the monitoring of HuNoV outbreaks, reduce the economic loss of the agri-food industry, and enhance food safety., Competing Interests: The authors declare no conflict of interest.

Noroviruses (family Caliciviridae) are common causes of acute gastroenteritis worldwide. Multiple polymerase/capsid combinations have been identified among members of norovirus genogroup GII, at least 10 of which contain GII.P16 polymerase. During hospital-based surveillance (2003-2013) in Russia, we identified eight noroviruses with GII.P16 polymerase - five GII.3[P16], two GII.16[P16], and one GII.5[P16]. This is the first report of the nearly complete genome sequence of a rare recombinant GII.5[P.16] norovirus, which was found in the feces of a child in 2010. Phylogenetic analysis revealed that ORF1 and ORF2/3 of the strain GII.5[P.16]/RUS/Novosibirsk/Nsk-N490/2010 formed separate branches in clusters GII.P16 and GII.5, respectively., Competing Interests: Declarations Conflict of interest The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The prevalence of foodborne illnesses is a significant global concern, resulting in numerous illnesses, deaths, and substantial economic losses annually. Traditional detection methods for foodborne pathogens are often slow, limited, and impractical for field use, underscoring the need for rapid, sensitive, and portable assays. Microfluidic technology has emerged as a promising solution for sample preparation, reaction, and detection on a small scale. Our study introduces a novel microfluidic digital loop-mediated isothermal amplification (LAMP) assay platform, which employs digital microfluidic chips for absolute quantitative analysis of nucleic acids. This portable chip utilizes LAMP technology to achieve ultrasensitive detection of target nucleic acids within 30 min and reduces the detection limit to 1 fM without the need for complex instrumentation. By digitizing amplification signals directly from the target sample, our platform offers simplicity, affordability, portability, and quantitative molecular readouts. This innovation represents a crucial step toward the on-site detection of foodborne pathogens, thereby enhancing food safety and mitigating disease outbreaks.

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marta Rusinol reports financial support was provided by European Commission. Marta Rusinol reports administrative support was provided by Government of Catalonia Agency for Administration of University and Research Grants. If there are other authors, they 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 Ltd.)

Rapid and accurate diagnostic methods are crucial for managing viral gastroenteritis in children, a leading cause of global childhood morbidity and mortality. This study introduces a novel microfluidic-Flap endonuclease 1 (FEN1)-assisted isothermal amplification (MFIA) method for simultaneously detecting major viral pathogens associated with childhood diarrhea-rotavirus, norovirus, and adenovirus. Leveraging the specificity-enhancing properties of FEN1 with a universal dspacer-modified flap probe and the adaptability of microfluidic technology, MFIA demonstrated an exceptional detection limit (5 copies/μL) and specificity in the simultaneous detection of common diarrhea pathogens in clinical samples. Our approach addresses the limitations of current diagnostic techniques by offering a rapid (turn around time <1 h), cost-effective, easy design steps (universal flap design), and excellent detection performance method suitable for multiple applications. The validation of MFIA against the gold-standard PCR method using 150 actual clinical samples showed no statistical difference in the detection performance of the two methods, positioning it as a potential detection tool in pediatric diagnostic virology and public health surveillance. In conclusion, the MFIA method promises to transform pediatric infectious disease diagnostics and contribute significantly to global health efforts combating viral gastroenteritis., 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 Elsevier B.V. All rights reserved.)

Background: Quantitative molecular assays are increasingly used for detection of enteric viruses., Methods: We compared the clinical severity using the modified Vesikari score (mVS) of enteric viruses detected by conventional assays (enzyme immunoassays [EIAs] for rotavirus and adenovirus 40/41 and conventional polymerase chain reaction for astrovirus, sapovirus, and norovirus) and a quantitative molecular assay (TaqMan Array Card [TAC]) among children aged 0-59 months in the Global Enteric Multicenter Study. For rotavirus and adenovirus 40/41, we compared severity between EIA-positive and TAC-positive cases assigned etiologies using different cycle threshold (Ct) cutoffs., Results: Using conventional assays, the median mVS (interquartile range) was 10 (8-11) for rotavirus, 9 (7-11) for adenovirus 40/41, 8 (6-10) for astrovirus, sapovirus, and norovirus GII, and 7 (6-9) for norovirus GI. Compared with rotavirus EIA-positive cases, the median mVS was 2 and 3 points lower for EIA-negative/TAC-positive cases with Ct <32.6 or Ct ≥32.6 and <35, respectively (P < .001). Adenovirus 40/41 EIA-positive and EIA-negative/TAC-positive cases were similar, regardless of Ct cutoff., Conclusions: Quantitative molecular assays compared with conventional assays, such as EIA, may influence the severity of identified cases, especially for rotavirus. Cutoffs to assign etiology for quantitative assays should be considered in the design and interpretation of enteric virus studies., Competing Interests: Potential conflicts of interest. D. S. reports employment and shares with GSK Vaccines, outside the submitted work. A. K. M. Z. was involved in this work as the site principial investigator of the GEMS study at the Aga Khan University during 2008–2012; she is currently the President of Gender Equality at the Bill & Melinda Gates Foundation and has not influenced this analysis from her position there. K. L. K. received funding from the Bill & Melinda Gates Foundation for part of the submitted work and outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2024.)

Concerns are rising about the contamination of recreational waters from human and animal waste, along with associated risks to public health. However, existing guidelines for managing pathogens in these environments have not yet fully integrated risk-based pathogen-specific criteria, which, along with recent advancements in indicators and markers, are essential to improve the protection of public health. This study aimed to establish risk-based critical concentration benchmarks for significant enteric pathogens, i.e., norovirus, rotavirus, adenovirus, Cryptosporidium spp., Giardia lamblia, Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7. Applying a 0.036 risk benchmark to both marine and freshwater environments, the study identified the lowest critical concentrations for children, who are the most susceptible group. Norovirus, C. jejuni, and Cryptosporidium presented lowest median critical concentrations for virus, bacteria, and protozoa, respectively: 0.74 GC, 1.73 CFU, and 0.39 viable oocysts per 100 mL in freshwater for children. These values were then used to determine minimum sample volumes corresponding to different recovery rates for culture method, digital polymerase chain reaction and quantitative PCR methods. The results indicate that for children, norovirus required the largest sample volumes of freshwater and marine water (52.08 to 178.57 L, based on the 5th percentile with a 10 % recovery rate), reflecting its low critical concentration and high potential for causing illness. In contrast, adenovirus and rotavirus required significantly smaller volumes (approximately 0.24 to 1.33 L). C. jejuni and Cryptosporidium, which required the highest sampling volumes for bacteria and protozoa, needed 1.72 to 11.09 L and 4.17 to 25.51 L, respectively. Additionally, the presented risk-based framework could provide a model for establishing pathogen thresholds, potentially guiding the creation of extensive risk-based criteria for various pathogens in recreational waters, thus aiding public health authorities in decision-making, strengthening pathogen monitoring, and improving water quality testing accuracy for enhanced health protection., 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 Elsevier B.V. All rights reserved.)

Background: Viral enteric infections are illnesses caused by several types of viruses that primarily affect the gastrointestinal system. Globally, three major viruses associated with gastroenteritis, including norovirus (NoV), rotavirus A (RVA), and human adenovirus (HAdV), have been widely recognized. Accordingly, a rapid and sensitive diagnostic tool, such as immunochromatographic (IC) test, for diarrheal virus detection is required and helpful for rapid diagnosis., Methods: The IP-Triple I IC test kit for simultaneous triple virus detections of NoV, RVA, and HAdV was evaluated for its efficacy by using stool specimens that were known positive for these viruses by real-time RT-PCR or PCR, which was used as the gold standard method., Results: The results revealed that the IP-Triple I IC test kit exhibited a very high level of specificity (100%) for NoV, RVA, and HAdV detections, while the sensitivity was slightly different for these three viruses. The sensitivity of detection for RVA was 86.7%, whereas those for NoV and HAdV were 70.6% and 76.2%, respectively. This IP-Triple I IC kit could detect common antigens of a wide range of NoV, RVA, and HAdV genotypes (NoV GII.2, GII.4, GII.6, GII.7, GII.10, GII.17, RVA G1P[8], G2P[4], G3P[8], G8P[8], G9P[8], HAdV-C1, -C2, -C5, -A12, -F40, and -F41)., Conclusions: Our results revealed that the IP-Triple I IC test kit is highly effective for simultaneous, direct detection of common antigens of several genotypes of NoV, RVA, and HAdV in stool specimens and useful for screening and rapid diagnosis of diarrheal viruses during the seasonal outbreak.

Norovirus is the predominant cause of viral gastroenteritis globally with foodborne outbreaks commonly reported. Filter-feeding bivalve molluscan shellfish can become contaminated with norovirus when grown in waters impacted by inadequately treated effluent wastewater, overflows, or other human fecal sources. Contaminated shellfish pose a significant risk to consumers, because combined with a low norovirus infectious dose, oysters and mussels are often eaten raw or lightly cooked resulting in no or minimal virus inactivation, respectively. In addition, shellfish contamination has significant economic impacts on the seafood industry. To improve risk assessments, reverse transcription (RT)-digital droplet PCR (ddPCR) was used to determine the precise norovirus concentrations in 20 shellfish samples, all positive for norovirus genogroup I and/or II (GI or GII) by RT-quantitative PCR (qPCR), and associated with reported norovirus illness in New Zealand. Using RT-ddPCR, total norovirus GI and/or GII concentrations in shellfish ranged between 44 and 4,630 genome copies (GC)/g digestive tissue. Importantly, 40% (8/20) of shellfish samples contained a total norovirus concentration less than 200 GC/g digestive tissue. In parallel, RNase treatment was applied, prior to viral extraction to remove free viral RNA, which subsequently led to average reductions in norovirus GC/g concentration of 37.1% and 19.4% for GI and GII, respectively. These RT-ddPCR data provide valuable evidence for risk assessment of contaminated shellfish and evaluation of safety guidelines and highlight issues associated with setting a safe threshold of norovirus in shellfish., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Wastewater-based monitoring has been widely implemented worldwide for the tracking of SARS-CoV-2 outbreaks and other viral diseases. In many surveillance programmes, unprocessed and processed wastewater samples are often frozen and stored for long periods of time in case the identification and tracing of an emerging health threat becomes necessary. However, extensive sample bioarchives may be difficult to maintain due to limitations in ultra-freezer capacity and associated cost. Furthermore, the stability of viruses in such samples has not been systematically investigated and hence the usefulness of bioarchives is unknown. In this study, we assessed the stability of SARS-CoV-2, influenza viruses, noroviruses and the faecal indicator virus, crAssphage, in raw wastewater and purified nucleic aacid extracts stored at -80 °C for 6-24 months. We found that the isolated viral RNA and DNA showed little signs of degradation in storage over 8-24 months, whereas extensive decay viral and loss of qPCR signal was observed during the storage of raw unprocessed wastewater. The most stable viruses were noroviruses and crAssphage, followed by SARS-CoV-2 and influenza A virus. Based on our findings, we conclude that bioarchives comprised of nucleic acid extracts derived from concentrated wastewater samples may be archived long-term, for at least two years, whereas raw wastewater samples may be discarded after one year., 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 Ltd.)

Highly sensitive viral analytical techniques are essential tools for preventing the spread of infections. In this study, we established a digital enzyme-linked immunosorbent assay (ELISA) system to quantify norovirus proteins with high sensitivity. We used norovirus-like particles (noro-VLPs) as a surrogate for norovirus and constructed two digital ELISA systems using two different antibody pairs. The quantitative performance of the noro-VLP measurement using each digital ELISA system was evaluated. Both assay systems exhibited high sensitivity, good linearity, and high stability. The first system exhibited a limit of detection (LOD) of 87 pg mL -1 and correlation coefficient ( R 2 ) of 0.9984. Analysis of samples containing 5 ng per mL noro-VLP confirmed inter-assay variation of 5.5%, and intra-assay variation of 5.2%. The second system exhibited an LOD of 19 pg mL -1 and R 2 of 0.9984. Analysis of samples containing 5 ng per mL noro-VLP confirmed inter-assay variation of 4.5%, and intra-assay variation of 2.5%. Comparison of the two systems using the same calibrant for unpurified and fractionated noro-VLPs revealed that the quantitative values for unpurified noro-VLPs were the same, whereas those for fractionated noro-VLPs were dramatically different. Our findings indicate that the reactivity to various components in the noro-VLP solution was altered depending on the different antibodies. Furthermore, our study highlights the importance of using appropriate calibrants, which contain the same ratio of components as the noro-VLP analyte, to afford accurate measurements.

Background: Acute gastroenteritis (AGE) is a major medical condition for travellers worldwide, particularly travellers to low- and middle-income countries. Norovirus (NoV) is the most common cause of viral AGE in older children and adults, but data on prevalence and impact amongst travellers is limited., Methods: Prospective, multi-site, observational cohort study conducted 2015-2017, amongst adult international travellers from the US and Europe to areas of moderate to high risk of travel-acquired AGE. Participants provided self-collected pre-travel stool samples and self-reported AGE symptoms whilst travelling. Post-travel stool samples were requested from symptomatic subjects and a sample of asymptomatic travellers within 14 days of return. Samples were tested for NoV by RT-qPCR, genotyped if positive and tested for other common enteric pathogens by Luminex xTAG GPP., Results: Of the 1109 participants included, 437 (39.4%) developed AGE symptoms resulting in an overall AGE incidence of 24.7 per 100 person-weeks [95% confidence interval (CI): 22.4; 27.1]. In total, 20 NoV-positive AGE cases (5.2% of those tested) were identified at an incidence of 1.1 per 100 person-weeks (95% CI: 0.7; 1.7). NoV-positive samples belonged mostly to genogroup GII (18, 85.7%); None of the 13 samples sequenced belonged to genotype GII.4. Clinical severity of AGE was higher for NoV-positive than for NoV-negative cases (mean modified Vesikari Score 6.8 vs 4.9) with more cases classified as severe or moderate (25% vs 6.8%). In total, 80% of NoV-positive participants (vs 38.9% in NoV-negative) reported at least moderate impact on travel plans., Conclusions: AGE is a prevalent disease amongst travellers with a small proportion associated with NoV. Post-travel stool sample collection timing might have influenced the low number of NoV cases detected; however, NoV infections resulted in high clinical severity and impact on travel plans. These results may contribute to targeted vaccine development and the design of future studies on NoV epidemiology., (© International Society of Travel Medicine 2023. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Wastewater serves as a valuable source of information as it contains biological markers that have been shed by infected individuals and from other biological organisms such as plants and animals. Wastewater has been proven to indicate the presence of emerging pathogens in a community before the manifestation of clinical symptoms. Several methods of concentration and nucleic acid extraction have been employed all around the world without a unified method. One such method involves the use of the adsorption extraction method (AE-method), which involves the use of electronegative membrane filters of different pore sizes. The membrane filters also differ by diameter, but no study has been reported on the effect of diameter on capture efficiency. This study was aimed at evaluating the comparative capture efficiency of two different membrane filter diameters of 45 and 90 mm with pore sizes of 0.45 μm for the detection of indicator and pathogenic viruses. Primary influent samples were obtained from two wastewater treatment plants in Baltimore, Maryland, between April 27 and June 29, 2023. A total of twenty samples were processed using 45- and 90-mm membrane filters. Nucleic acids were extracted from the filters using the QIAmp Viral RNA Mini Kit and assayed for four different targets: PMMoV, Norovirus (GI and GII), and CrAssphage by RT-qPCR. The result showed that 45 mm membrane filters had a higher combined mean capture efficiency in log10 gene copies per liter (gc/l) for crAssphage (7.40) than 90 mm membrane filters (7.10). Similarly, the 45-mm filter had higher mean capture efficiency for Norovirus GI (4.67) than the 90-mm filter (1.84) and likewise for Norovirus GII (2.14, 1.04). On the contrary, 90-mm membrane filters were observed to have better capture of PMMoV (6.84) compared to 45-mm membrane filters (6.69). This result therefore implies that 45-mm membrane filters could be more efficient for wastewater surveillance studies through the AE method for indicator viruses like CrAssphage and human disease-causing viruses like Norovirus., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The reliance of the global population on urban aquifers is steadily increasing, and urban aquifers are susceptible to pathogenic contamination through sources such as sewer leakage or urban runoff. However, there is insufficient monitoring of groundwater quality in urban areas. In this study, quantitative polymerase chain reaction (qPCR) was employed to evaluate the presence of human fecal viral indicators and viral pathogens in urban wastewater (n = 13) and groundwater (n = 12) samples from four locations in Barcelona with different degrees of urbanization, as well as in runoff samples (n = 2). Additionally, a target enrichment sequencing (TES) approach was utilized to explore the viral diversity within groundwater and runoff samples, offering insights into viral contamination and potential virus transmission routes in urban areas. Human adenovirus (HAdV) was identified in all wastewater samples, 67 % (8/12) of groundwater samples, and one runoff sample by qPCR indicating human viral fecal contamination. The viral pathogen Norovirus genogroup GI (NoV GI) was detected in wastewater and two winter groundwater samples from highly and medium urbanized areas. NoV genogroup GII (NoV GII), Enterovirus (EV) and SARS-CoV-2 were exclusively detected in wastewater. Human and other vertebrate viruses were detected in groundwater and runoff samples using TES. This study gives insights about the virome present in urban water sources, emphasizing the need for thorough monitoring and deeper understanding to address emerging public health concerns., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) is a critical tool for monitoring community health. Although much attention has focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a causative agent of coronavirus disease 2019 (COVID-19), other pathogens also pose significant health risks. This study quantified the presence of SARS-CoV-2, influenza A virus (Inf-A), and noroviruses of genogroups I (NoV-GI) and II (NoV-GII) in wastewater samples collected weekly (n = 170) from July 2023 to February 2024 from five wastewater treatment plants (WWTPs) in Yamanashi Prefecture, Japan, by quantitative PCR. Inf-A RNA exhibited localized prevalence with positive ratios of 59 %-82 % in different WWTPs, suggesting regional outbreaks within specific areas. NoV-GI (94 %, 160/170) and NoV-GII (100 %, 170/170) RNA were highly prevalent, with NoV-GII (6.1 ± 0.8 log 10 copies/L) consistently exceeding NoV-GI (5.4 ± 0.7 log 10 copies/L) RNA concentrations. SARS-CoV-2 RNA was detected in 100 % of the samples, with mean concentrations of 5.3 ± 0.5 log 10 copies/L in WWTP E and 5.8 ± 0.4 log 10 copies/L each in other WWTPs. Seasonal variability was evident, with higher concentrations of all pathogenic viruses during winter. Non-normalized and normalized virus concentrations by fecal indicator bacteria (Escherichia coli and total coliforms), an indicator virus (pepper mild mottle virus (PMMoV)), and turbidity revealed significant positive associations with the reported disease cases. Inf-A and NoV-GI + GII RNA concentrations showed strong correlations with influenza and acute gastroenteritis cases, particularly when normalized to E. coli (Spearman's ρ = 0.70-0.81) and total coliforms (ρ = 0.70-0.81), respectively. For SARS-CoV-2, non-normalized concentrations showed a correlation of 0.61, decreasing to 0.31 when normalized to PMMoV, suggesting that PMMoV is unsuitable. Turbidity normalization also yielded suboptimal results. This study underscored the importance of selecting suitable normalization parameters tailored to specific pathogens for accurate disease trend monitoring using WBE, demonstrating its utility beyond COVID-19 surveillance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eiji Haramoto received research funding from Takara Bio Inc. The other authors declared 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 The Authors. Published by Elsevier B.V. All rights reserved.)

A new method has been developed to improve the detection of norovirus (NoV) in complex fecal samples using nanocatalyst-based immunoassays. The method involves using multifunctional trimetallic nanoparticles, known as Ag@Fe 3 O 4 @Au NPs. These nanoparticles consist of a core of silver (Ag) and a shell of iron oxide (Fe 3 O 4 ) decorated with isolated gold nanoparticles (Au NPs). The nanoparticles have enhanced catalytic activity, making them an ideal nanocatalyst for reducing 4-nitrophenol (4-NP, yellow) to 4-aminophenol (4-AP, colorless). The developed Ag@Fe 3 O 4 @Au NPs-based immunoassay achieved a limit of detection (LOD) of 1.9 pg/mL for norovirus-like particles (NoV-LP) and 6.97 RNA copy number/mL for fecal NoV. In fecal sample analysis for NoV, a heat treatment at 65°C was necessary to prevent degradation of the target protein, ensuring sensitive detection. This work successfully combined multifunctional nanocatalysts for advanced immunoassays, which could contribute to developing nano-biosensing platforms., (© 2024 Wiley‐VCH GmbH.)

Purpose of Review: Viruses are the most common etiological agents of diarrhea in children. Despite rotavirus vaccine introduction, rotavirus remains as the leading cause of death globally, followed by norovirus, which represents a diagnostic challenge. Here, we describe new advances in the diagnosis and management of viral diarrheas., Recent Findings: Although immunoassays are widely used for their fast turnaround time and low cost, molecular techniques have become the most reliable diagnostic method due to their high sensitivity and capacity to analyze multiple pathogens in gastrointestinal panels. Isothermal nucleic acid amplification assays (LAMP and RPA) are promising techniques since they do not require sophisticated equipment and can be used as point-of-care testing. CRISPR/Cas nucleic acid detection systems are new diagnostic methods with great potential. Several recent published articles describe the role of human intestinal enteroids to characterize norovirus infection, to test new drugs, and for vaccine development. The interaction between the human gut microbiota and gastrointestinal viral infections has been extensively reviewed and offers some innovative mechanisms for therapeutic and preventive measures., Summary: Although important advances have been made, more research is needed to address remaining challenges and further improve diagnostic capabilities and better management strategies for this critical infectious disease., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The demonstration of enteric virus removal for indirect potable reuse of advanced purified water is necessary to ensure safe water reclamation practices. This study evaluated the efficacy of soil treatment in reducing concentrations of Pepper Mild Mottle Virus (PMMoV), Hepatitis A (HAV), and Norovirus (NoV) gene markers through bench scale unsaturated soil columns. Three different infiltration rates were evaluated to determine their impact on viral gene marker removal. The concentrations of viral markers in the column influent and effluent samples were measured through RNA extraction and then RT-qPCR, and the log reduction values (LRVs) were calculated to quantify the effectiveness of removal across the columns. The LRVs achieved for PMMoV were 2.80 ± 0.36, 2.91 ± 0.48, and 2.72 ± 0.32 for infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h, respectively. A one-way ANOVA indicated no statistically significant differences in LRVs among the various infiltration rates (p-value = 0.329). All samples measured for HAV were below the detection limit both in the influent and effluent of the soil columns. While NoV GI and GII markers were measurable in the soil column influent, they were removed to below the detection limit in the effluent. The use of half the Limit-of-Detection (LoD) for effluent values enabled the estimation of log removals, which were calculated as 1.42 ± 0.07, 1.64 ± 0.29, and 1.74 ± 0.18 for NoV GI and 1.14 ± 0.19, 1.58 ± 0.21, and 1.87 ± 0.41 for NoV GII at infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h. This highlights the efficacy of soil treatment in reducing virus gene marker concentrations at various infiltration rates, and that spreading basins employed for reclaimed water recharge to ground water aquifers are an effective method for reducing the presence of viral contaminants in indirect potable reuse systems., 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 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) gained widespread use as a tool for supporting clinical disease surveillance during the COVID-19 pandemic. There is now significant interest in the continued development of WBE for other pathogens of clinical significance. In this study, approximately 3,200 samples of wastewater from across England, previously collected for quantification of SARS-CoV-2, were re-analysed for the quantification of norovirus genogroup I (GI) and II (GII). Overall, GI and GII were detected in 93% and 98% of samples respectively, and at least one of the genogroups was detected in 99% of samples. GI was found at significantly lower concentrations than GII, but the proportion of each genogroup varied over time, with GI becoming more prevalent than GII in some areas towards the end of the study period (May 2021 - March 2022). Using relative strength indices (RSI), it was possible to study the trends of each genogroup, and total norovirus over time. Increases in norovirus levels appeared to coincide with the removal of COVID-19 related lockdown restrictions within England. Local Moran's I analyses indicated several localised outbreaks of both GI and GII across England, notably the possible GI outbreak in the north of England in early 2022. Comparisons of national average norovirus concentrations in wastewater against concomitant norovirus reported case numbers showed a significant linear relationship. This highlights the potential for wastewater-based monitoring of norovirus as a valuable approach to support surveillance of norovirus in communities., 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 Ltd.)

Background: Noroviruses (NoVs) are the leading cause of diarrheal disease among all age groups worldwide, with an increased burden in developing countries. As there is no surveillance, epidemiological data is limited in Ethiopia. Hence, this study aimed to investigate the prevalence and associated factors of NoV infection among patients with diarrhea in the Amhara National Regional State, Ethiopia., Methods: A prospective health facility-based cross-sectional study was conducted from May 2021 to November 2021. A total of 550 study participants of all age groups with symptoms of diarrhea were proportionately assigned to the four study areas, area with three health facilities. Study participants were systematically sampled in each health facility. A fecal sample from each case was collected. The RNA was extracted and tested for NoV by one-step RT-PCR. Sociodemographic and other variables were gathered using a pre-tested questionnaire. A descriptive analysis was performed. Both binary and multiple logistic regressions were utilized to identify factors associated with NoV infection. Variables with a p-value < 0.05 in the final model were considered statistically significant., Results: Five hundred nineteen out of 550 samples were analyzed (94.4% response rate). The overall prevalence of NoV was 8.9% (46/519). The positivity rates were higher among the elderly (33.3%) and under-5 children (12.5%). Both genogroup I and genogroup II (GII) were identified, with GII being the predominant, at 82.6% (38/46). Of all participants, only 20% reported a history of vomiting. Norovirus infection was more prevalent among participants from Debre Tabor (AOR = 4, 95%CI: 1.2-14) and Bahir Dar areas (AOR = 3.6, 95%CI: 1.04-11) compared to Debre Markos. Additionally, older adults (AOR = 7, 95% CI: 2-24) and under-5 children (AOR = 3.5, 95% CI: 2.8-12) were disproportionately affected compared to adults. The previous history of diarrhea (AOR = 3.6, 95% CI: 1.7-7) was a significant factor contributing to NoV infections. Moreover, the odds of NoV infection were higher among individuals with a high frequency of diarrhea (AOR = 15.3, 95%CI: 7.6-43) and vomiting (AOR = 3.5, 95%CI: 1.5-8)., Conclusions: The prevalence of NoV was considerably high, with the predominance of NoV-GII. The positivity rate was higher among the extreme age groups and varied across the study areas. To obtain a comprehensive understanding of the virus`s epidemiology and its genetic diversity, further research is warranted., (© 2024. The Author(s).)

The norovirus (NoV) genome is diverse. Therefore, this study explored the epidemiological characteristics and genetic features of NoV in Ningxia Hui Autonomous Region, China, from 2011 to 2022 to clarify the genetic diversity in this region. Stool samples were screened for NoV and then sequenced and genotyped. In total, 1,788 of 13,083 specimens were NoV -positive (13.67%); 204 (1.56%) and 1,584 (12.11%) cases were GI and GII, respectively. Additionally, 559 were NoV infection with other viruses (4.27%), primarily with rotavirus (277/559, 49.55%). The NoV incidence rate was the highest among children aged 0-2 years (18.09%, 1054/5,828) and lowest among adults aged 45-64 years (110/1,495, 7.36%); it was also higher in the winter and spring than in the other seasons. GI.3[P3] was the dominant GI genotype. The dominant GII genotype changed roughly every two years. In the GII group, GII.4 was the most common genotype (46.79%), followed by GII.3 (21.34%), GII.2 (12.34%), and GII.17 (9.77%). There were three variants of GII.4 Den Haag, GII.4 New Orleans and GII.4 Sydney identified in the detected GII.4 strains, with GII.4 Sydney dominating. The GII.4 (87.36%), GII.3 (86.35%), and GII.2 (72.92%) strains were primarily detected in children, whereas it was the GII.17 (52.63%) strain in adults. Overall, the NoV genotypes in the Ningxia Hui Autonomous Region were diverse. Primarily, GII groups were dominant, but this changed over time., (© 2024. The Author(s).)

This study aims to determine the presence of norovirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and bocavirus in air samples from a tertiary care hospital in Bangkok, Thailand. Air samples were collected in water using the BioSampler and concentrated using speedVac centrifugation. Based on RT-qPCR, norovirus RNA and SARS-CoV-2 RNA were detected in 13/60 (21.7%) and 3/60 (5.0%) of samples, respectively. One air sample had a weak positivity for both norovirus and SARS-CoV-2 RNAs. Detection rate of norovirus genogroup (G) II (13.3%) was higher than norovirus GI (6.7%). One air sample (1.7%) tested positive for GI and GII. The norovirus GI RNA concentration was 6.0 × 10 2 genome copies/m 3 . The norovirus GII RNA concentrations ranged from 3.4 × 10 1 to 5.0 × 10 3 genome copies/m 3 . Based on RT-nested PCR, norovirus GII was detected in two (3.3%) samples. All samples tested negative for GI RNA and bocavirus DNA. By phylogenetic analysis, GII.17, which is closely related to the outbreak Kawasaki308/JPN/2015 strain, was found in the RT-nested PCR-positive samples. This study highlights the potential of aerosols for norovirus and SARS-CoV-2 transmission and probably cause gastrointestinal and respiratory illnesses, respectively., (© 2024. The Author(s).)

Noroviruses are the most common cause of acute gastroenteritis (AGE) outbreaks in childcare centers and kindergartens. Their high transmissibility is partly due to their genetic diversity. AGE outbreaks that occurred in Seoul childcare centers and kindergartens from 2019 to 2022 were investigated, and 68 epidemiological reports prepared by public health centers in Seoul were used for data collection. In the three quarters of 2022, there were 40 outbreaks of AGE in Seoul childcare centers and kindergartens, which exceeded the 35 total outbreaks that occurred during the previous three years. The proportion of childcare centers and kindergartens with AGE outbreaks among all facilities in Seoul increased from 12.6% in 2019 to 58.8% in 2022. Noroviruses were the most common pathogens responsible for AGE outbreaks in these cases. From 2019 to 2021, norovirus GII.2 was the predominant genotype, and GII.4 was detected in about 25% of cases. However, in 2022, GII.4 became predominant and was detected in about 79% of cases. The attack rate and infection source of AGE outbreaks from 2019 to 2021 were not significantly different from those in 2022. In conclusion, the number of AGE outbreaks in Seoul childcare centers and kindergartens increased significantly, primarily because of increased norovirus GII.4 transmission in 2022., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

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 K D 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 × 10 5 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 × 10 5 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.

Feline norovirus (FNoV) is a potential pathogen of feline gastroenteritis and has two genogroups (GIV and GVI). Few epidemiological studies have been conducted on FNoV. We designed two enzyme-linked immunosorbent assays (ELISAs) to identify genogroup-specific FNoV antibodies for serological surveillance. Analysis of sera from cats experimentally infected with FNoV GIV or GVI and from specific-pathogen-free (SPF) cats confirmed that the two recombinant proteins used in the assay react in a genogroup-specific manner. Of the 183 samples tested, 6.6% were positive for GIV and 26.2% were positive for GVI. Antibodies to both FNoV genogroups were detected in sera collected in 2005, seven years before FNoV was first reported., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

We report an increase in GII.17 norovirus outbreaks and sporadic infections of acute gastroenteritis in Austria, Germany, France, Ireland, the Netherlands, England and the United States during the 2023/24 season. A decrease in GII.4 coincided with GII.17 prevalence increasing to between 17% and 64% of all GII detections. Overall, 84% of the GII.17 strains clustered closely with strains first reported in Romania in 2021 and two new sub-lineages were identified. Norovirus surveillance and molecular characterisation should be prioritised this winter.

Background: Acute gastroenteritis (AGE) is a common cause of pediatric morbidity and mortality worldwide. AGE can cause an imbalance in the intestinal microbiota. This study aimed to investigate the diversity of the gut microbiome in Korean children hospitalized for infectious AGE at a university hospital., Methods: A total of 23 stool samples from patients aged 5 months to 11 years with AGE were analyzed. Thirteen convalescent stool samples were collected 1 month after discharge. Multiplex polymerase chain reaction (PCR) for the five viruses and 16 bacteria-specific AGE pathogens (PowerChek Multiplex Real time PCR Kit, Seoul, Korea), and 16s rRNA sequencing (Illumina MiSeq Sequencing system, Illumina, USA) were performed., Results: According to the results of multiplex PCR for causative pathogens, the microbiome taxonomic profile (MTP) of the gut microbiome in three groups of AGE, norovirus AGE (n = 11), Campylobacter AGE (n = 7) and Salmonella AGE (n = 5) was compared. The phylum Actinobacteria was significantly more abundant in the norovirus AGE (P = 0.011), whereas the phylum Proteobacteria was significantly more abundant in Salmonella AGE (P = 0.012). Alpha diversity, which indicates species richness and diversity, showed no statistical differences. However, beta diversity, representing the similarity in MTP between norovirus, Campylobacter, and Salmonella AGE, was significantly different (P = 0.007). In convalescence, compared with their corresponding AGE samples, the phylum Firmicutes; and the lower taxa Christensenellaceae (P = 0.0152) and Lachnospiraceae (P = 0.0327) were significantly increased., Conclusions: In pediatric AGE, the type of infectious agent can affect the diversity and dominance of gut microbiota in pediatric patients. Furthermore, healthy gut bacteria increased during the period of 1 month after infection, allowing a return to a healthy state without causing long-term dysbiosis., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2024 Taiwan Pediatric Association. Published by Elsevier B.V. All rights reserved.)

Norovirus is an important etiologic agent of acute gastroenteritis and has become even more relevant in Brazil after the implementation of the monovalent rotavirus vaccine in 2006 through the public health system, now representing a significant portion of the etiology of acute diarrheal diseases. Although diagnosing acute gastroenteritis caused by norovirus is a relatively simple process, and the infection tends to be self-limited, the virus can be considerably harmful to vulnerable populations, such as children, the elderly, and immunocompromised individuals. The spread of norovirus is also particularly favorable among such groups due to its mode of transmission, favored by cluttered environments such as in hospitals and densely populated regions. Additionally, norovirus' ability to spread through water and food creates the need for measures to ensure adequate sanitation and the development of effective measures to prevent outbreaks and severe manifestations of the disease. This review aims to address the main reports of human norovirus detected in Brazil over the years, focusing on clinical-hospital, food-related, and urban conglomerate contexts, including the circulating strains., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The STARlet All-In-One system is a modular platform that integrates the complete molecular diagnostic workflow from nucleic acid extraction of clinical samples to PCR set-up and amplification. The platform was evaluated in comparison with laboratory developed tests (LDT) on fecal samples from patients with suspected viral gastro-enteritis. In a retrospective study, 72 positive samples were analysed, including all pathogens detected by the Seegene Allplex™ GI-virus assay, adenovirus, astrovirus, norovirus GI and GII, sapovirus, and rotavirus. Concordant results were obtained for 69 samples (96 %). Three discordant results were observed, one norovirus GII positive that gave an invalid result in the AIOS and two samples that were negative in the AIOS. One adenovirus positive that was subtyped as a genotype 2 virus, which is not associated with gastro-enteritis, and a sapovirus. In the prospective part of the study, 661 fecal samples were included. A total of 61 positive samples were detected, of which 60 were also detected by the AIOS. One norovirus GII positive sample (C T 35.2) was tested negative in the AIOS. Two additional sapovirus positive samples, C T 37 and 38, were detected by the AIOS but not by the LDT. The STARlet All-In-One platforms results in an automated molecular workflow with reduced hands-on time and enables running assays during out of office hours. Application of the Seegene Allplex™ GI-virus assay showed excellent concordance to the current diagnostic LDT. In a prospective comparison, only three discordant results were observed, all with C T values over 35 and therefore unlikely of clinical relevance., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Regular monitoring of Norovirus presence in environmental and food samples is crucial due to its high transmission rates and outbreak potential. For detecting Norovirus GI, reverse transcription qPCR method is commonly used, but its sensitivity can be affected by assay performance. This study shows significantly reduced assay performance in digital PCR or qPCR when using primers targeting Norovirus GI genome 5291-5319 (NC&#95;001959), located on the hairpin of the predicted RNA structure. It is highly recommended to avoid this region in commercial kit development or diagnosis to minimizing potential risk of false negatives., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Viral gastroenteritis is commonly reported in dogs and involves a great diversity of enteric viruses. In this research, viral diversity was investigated in dogs with diarrhea in Northern Brazil using shotgun metagenomics. Furthermore, the presence of norovirus (NoV) was investigated in 282 stool/rectal swabs of young/adult dogs with or without diarrhea from two public kennels, based on one-step reverse transcription polymerase chain reaction (RT-PCR) for genogroup VI and VII (GVI and GVII) and real-time RT-PCR for GI, GII, and GIV. Thirty-one viral families were identified, including bacteriophages. Phylogenetic analyses showed twelve complete or nearly complete genomes belonging to the species of Protoparvovirus carnivoran1, Mamastrovirus 5, Aichivirus A2, Alphacoronavirus 1, and Chipapillomavirus 1. This is the first description of the intestinal virome of dogs in Northern Brazil and the first detection of canine norovirus GVII in the country. These results are important for helping to understand the viral groups that circulate in the canine population., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Evandro Chagas Institute. Published by Elsevier B.V. All rights reserved.)

Biomonitoring appears to be a key approach to assess chemical or microbiological contaminations. The freshwater mussel, Dreissena polymorpha (D. polymorpha), is a suitable tool already used to monitor chemical and, more recently, microbiological pollution. In the present study, we used this sentinel species to monitor viral contamination of fecal origin over a wide geographical distribution. An active approach was implemented based on caging of calibrated and pathogen-free organisms with the same exposure conditions, allowing spatio-temporal comparisons between different water bodies. In addition, different types of sites were selected to investigate the range of environmental concentrations that D. polymorpha are able to translate. Different viral genome targets were measured: norovirus genogroup I and II (NoV GI and GII) and F-specific RNA bacteriophages belonging to the genogroup -I and -II (FRNAPH-I and -II). Total infectious FRNAPH were also monitored. D. polymorpha was able to translate a wide range of concentrations for all the viral targets studied, meaning that this sentinel species can be used for both low and highly anthropised sites. Moreover, D. polymorpha caging proved effective in achieving gradients of viral contamination of fecal origin pressure and to highlight the contribution of tributaries to the main rivers. D. polymorpha provided spatial and temporal variations of the viral contamination. It allowed to highlight the prevalence of the FRNAPH-I and -II genogroups according to the caging site. FRNAPH-II was found to be dominant in urban areas and FRNAPH-I in rural areas. This strategy uses the caging of the sentinel species D. polymorpha on selected sites with standardised analysis methods has proven to be a promising tool for characterizing viral contamination at both large and very fine scales., 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 Ltd.)

Background: Norovirus is the predominant pathogen causing foodborne illnesses and acute gastroenteritis (AGE) outbreaks worldwide, imposing a significant disease burden. This study aimed to investigate the epidemiological characteristics and genotypic diversity of norovirus outbreaks in Hongshan District, Wuhan City., Methods: A total of 463 AGE cases from 39 AGE-related outbreaks in Hongshan District between January 1, 2021, and June 30, 2023, were included in the study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to identify norovirus types GI and GII in anal swab samples from all cases. Norovirus-positive samples were sequenced and analyzed for the open reading frame (ORF) 1/ORF2 hinge region., Results: 26 norovirus infectious outbreaks were reported among 39 acute diarrheal outbreaks, including 14 outbreaks in kindergartens, 8 in elementary schools, and 4 in universities. Based on clinical symptoms and epidemiological investigations, a total of 1295 individuals were identified as having been exposed to norovirus, yielding an attack rate of 35.75 %. A higher proportion of outbreaks was observed during the winter and spring seasons (38.46 %). Additionally, norovirus-positive samples were subjected to sequencing and analysis of the open reading frame (ORF) 1/ORF2 hinge region. Genotypic data for norovirus was successfully obtained from 18 (69.23 %) of the infectious outbreaks, revealing 10 distinct recombinant genotypes. GII.4 Sydney 2012 [P31] and GII.17[P17] were the predominant strains in 2021 and 2022, GII.3 [P12] emerged as the dominant strain in 2023., Conclusion: Norovirus outbreaks in Hongshan District predominantly occurred in crowded educational institutions, with peaks in the cold season and a high attack rate in universities. GII.3 [P12] has become the locally predominant strain., 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Acute gastrointestinal infections (AGIs) and diarrheal disease disproportionately impact children. The purpose of this study was to characterize the clinical and epidemiological characteristics of pediatric patients who had stool samples tested using the BIOFIRE® FILMARRAY® Gastrointestinal Panel at Nationwide Children's Hospital in Columbus, Ohio from January 2016 to December 2020. Detection rates were estimated by pathogen. Poisson and logistic regression were used to assess trends and identify risk factors for detection. A total of 12,783 patients had 17,611 stool samples tested during the study period. Nearly half of stool samples (47.3 %) tested positive for at least one pathogen. Enteropathogenic Escherichia coli, Clostridioides difficile, and norovirus were the most commonly detected. Overall annual detection rates for most pathogens increased from 2016 to 2019 and declined in 2020. This paper presents an analysis of five years of data that characterizes detection, hospitalizations, and deaths of children tested for AGI pathogens by sociodemographics., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Norovirus is the leading cause of sporadic viral gastroenteritis cases and outbreaks. Gut microbiota plays a key role in maintaining immune homeostasis. We aimed to investigate the composition and functional effects of gut microbiota in children infected with norovirus., Methods: Stool samples were collected from 31 children infected with norovirus and 25 healthy children. The gut microbiota was analyzed by 16S rRNA gene sequencing, followed by composition, correlation network, functional and phenotype prediction analyses., Results: Gut microbiota in children infected with norovirus was characterized by lower species richness and diversity. Veillonella is the dominant gut microbiota specie in norovirus infection. Blautia was significantly lower in norovirus infection. There was a positive correlation between Faecalibacterium , Blautia , Subdoligranulum , Eubacterium&#95;hallii&#95;group , Fusicatenibacter , Agathobacter , Roseburia and Dorea . Functionally, secondary metabolites biosynthesis, transport and catabolism, selenocysteine lyase and peroxiredoxin were the most significantly higher functional compositions of gut microbiota in norovirus infection. However, sn-glycerol-1-phosphate dehydrogenase and fermentation were the most significantly lower functional compositions in norovirus infection group. Phenotype analysis showed that Contains&#95;Mobile&#95;Elements had the highest level of phenotypes in the gut microbiota of norovirus infection., Conclusion: Norovirus infection may lead to dysregulation of the gut microbiome in children.

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., (© 2024. Crown.)

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., Competing Interests: Declaration of Competing Interest All authors declare that there is no conflict of interest in this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human norovirus (HuNoV) is a leading cause of foodborne diseases worldwide, making rapid and accurate detection crucial for prevention and control. In recent years, the CRISPR/Cas13a system, known for its single-base resolution in RNA recognition and unique collateral cleavage activity, is particularly suitable for sensitive and rapid RNA detection. However, isothermal amplification-based CRISPR/Cas13 assays often require an external transcription step, complicating the detection process. In our study, an efficient diagnostic technique based on the NASBA/Cas13a system was established to identify conserved regions at the ORF1-ORF2 junction of norovirus. The RNA amplification techniques [Nucleic Acid Sequence-Based Amplification (NASBA)] integrates reverse transcription and transcription steps, enabling sensitive, accurate, and rapid enrichment of low-abundance RNA. Furthermore, the CRISPR/Cas13a system provides secondary precise recognition of the amplified products, generating a fluorescence signal through its activated accessory collateral cleavage activity. We optimized the reaction kinetics parameters of Cas13a and achieved a detection limit as low as 51pM. The conditions for the cascade reaction involving CRISPR analysis and RNA amplification were optimized. Finally, we validated the reliability and accuracy of the NASBA/Cas13a method by detecting norovirus in shellfish, achieving results comparable to qRT-PCR in a shorter time and detecting viral loads as low as 10 copies/μL., 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 Elsevier B.V. All rights reserved.)

The development of an innovative approach is explored to amplify the signal of a surface-enhanced Raman scattering (SERS)-based detection system using a novel nanotag: Au@Ag NPs covered by satellite AuNPs and conjugated by 4-mercaptbenzoic acid (4-MBA) as a Raman tag (Au@Ag-MBA-AuNPs). The Au@Ag-MBA-AuNPs nanotags showed strong SERS activities with an enhancement factor in the 10 8 order of magnitude. This indicates the formation of many hot spots due to the combination of core-shell nanoparticles and satellite AuNPs on the surface of Au@Ag-MBA NPs. The newly fabricated nanotags were employed in a small-sized Palmtop Raman spectrometer. A concentration-dependent increase in SERS intensity was observed in the norovirus-like particle (NoV-LP) concentration range 10 fg/mL to 100 pg/mL with a detection limit of 0.76 fg/mL. Even in the severe interfering matrices, this detection method's coefficient of variation was less than 10%. This detection system was approximately 10 7 times more sensitive than commercially available ELISA kits. Norovirus in clinical samples was detected over a wide concentration range of 1.0 × 10 1 - 1.0 × 10 6 RNA copy number/mL with a detection limit of 7.8 RNA copy number/mL, indicating sensitivity comparable to real-time PCR. These results suggest that this detection system is stable in a complex matrix and has the potential for detecting norovirus in clinical samples with a small Palmtop Raman spectrometer., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The main capsid protein (CP) of norovirus, the leading cause of gastroenteritis, is expected to self-assemble into virus-like particles with the same structure as the wild-type virus, a capsid with 180 CPs in a T = 3 icosahedron. Using charge detection mass spectrometry (CD-MS), we find that the norovirus GI.1 variant is structurally promiscuous, forming a wide variety of well-defined structures, some that are icosahedral capsids and others that are not. The structures that are present evolve with time and vary with solution conditions. The presence of icosahedral T = 3 and T = 4 capsids (240 CPs) under some conditions was confirmed by cryo-electron microscopy (cryo-EM). The cryo-EM studies also confirmed the presence of an unexpected prolate geometry based on an elongated T = 4 capsid with 300 CPs. In addition, CD-MS measurements indicate the presence of well-defined peaks with masses corresponding to 420, 480, 600, and 700 CPs. The peak corresponding to 420 CPs is probably due to an icosahedral T = 7 capsid, but this could not be confirmed by cryo-EM. It is possible that the T = 7 particles are too fragile to survive vitrification. There are no mass peaks associated with the T = 9 and T = 12 icosahedra with 540 and 720 CPs. The larger structures with 480, 600, and 700 CPs are not icosahedral; however, their measured charges suggest that they are hollow shells. The use of CD-MS to monitor virus-like particles assembly may have important applications in vaccine development and quality control.

Background: Molecular syndromic panels can improve rapidity of results and ease clinical laboratory workflow, although caution has been raised for potential false-positive results. Upon implementation of a new panel for infectious diarrhea (BioFire® FilmArray® Gastrointestinal [GI] Panel, bioMérieux) in our clinical laboratory, a higher than expected number of stool samples with norovirus were detected., Objectives: The goal of this study was to investigate positive percent agreement and the false-positive rate of norovirus detected by the multiplex BioFire GI panel compared to a singleplex commercial assay., Study Design: From October 2023 to January 2024, all prospective stool samples with a positive norovirus result by BioFire had melting curves reviewed manually using the BioFire FilmArray Torch System. Stool samples further underwent testing by a supplementary real-time RT-PCR assay (Xpert® Norovirus, Cepheid) for comparative analysis., Results: Of the 50 stool samples with norovirus detected by BioFire, 18 (36 %) tested negative by Xpert (deemed "false-positives"). Furthermore, melting curve analysis revealed nearly all of these samples had atypical melting curve morphologies for the "Noro-1" target on BioFire (16/18, 89 %), which was statistically significant (Odds Ratio 173.2, 95 % CI [22.2, 5326.9], p < 0.0001). Stool samples with multiple pathogens detected by BioFire including norovirus were not more likely to produce false-positive norovirus results (Odds Ratio 1, 95 % CI [0.3, 3.3], p = 1)., Conclusions: Although not described in the manufacturer's Instructions for Use, we propose routine manual review of melting curves for the BioFire GI panel prior to reporting, to mitigate potential false-positive norovirus results., 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 B.V.)

Noroviruses (NoVs) are the most significant viral pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. This study aimed to investigate the prevalence and diversity of NoVs contaminated in the environmental water in Chiang Mai, Thailand. A total of 600 environmental water samples were collected from ten sampling sites in Chiang Mai from July 2020 to December 2022. The presence of NoV genogroups I (GI), GII, and GIV were examined using real-time RT-PCR assay. The genotype of the virus was determined by nucleotide sequencing and phylogenetic analysis. The results showed that NoV GI and GII were detected at 8.5% (51/600) and 11.7% (70/600) of the samples tested, respectively. However, NoV GIV was not detected in this study. NoV circulated throughout the year, with a higher detection rate during the winter season. Six NoV GI genotypes (GI.1-GI.6) and eight NoV GII genotypes (GII.2, GII.3, GII.7, GII.8, GII.10, GII.13, GII.17, and GII.21) were identified. Among 121 NoV strains detected, GII.17 was the most predominant genotype (24.8%, 30 strains), followed by GII.2 (21.5%, 26 strains), GI.3 (17.4%, 21 strains), and GI.4 (16.5%, 20 strains). Notably, NoV GII.3, GII.7, GII.8, and GII.10 were detected for the first time in water samples in this area. This study provides insight into the occurrence and seasonal pattern of NoV along with novel findings of NoV strains in environmental water in Thailand during the COVID-19 outbreak. Our findings emphasize the importance of further surveillance studies to monitor viral contamination in environmental water., Competing Interests: Declaration of competing interest The authors declare no conflict of interest relevant to this article., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Noroviruses are a group of non-enveloped single-stranded positive-sense RNA virus belonging to Caliciviridae family. They can be transmitted by the fecal-oral route from contaminated food and water and cause mainly acute gastroenteritis. Outbreaks of norovirus infections could be difficult to detect and investigate. In this study, we developed a simple threshold detection approach based on variations of the P2 domain of the capsid protein. We obtained sequences from the norovirus hypervariable P2 region using Sanger sequencing, including 582 pairs of epidemiologically-related strains from 35 norovirus outbreaks and 6402 pairs of epidemiologically-unrelated strains during the four epidemic seasons. Genetic distances were calculated and a threshold was performed by adopting ROC (Receiver Operating Characteristic) curve which identified transmission clusters in all tested outbreaks with 80 % sensitivity. In average, nucleotide diversity between outbreaks was 67.5 times greater than the diversity within outbreaks. Simple and accurate thresholds for detecting norovirus transmissions of three genotypes obtained here streamlines molecular investigation of norovirus outbreaks, thus enabling rapid and efficient responses for the control of norovirus., 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 B.V.)

The gut of healthy neonates is devoid of viruses at birth, but rapidly becomes colonised by normal viral commensals that aid in important physiological functions like metabolism but can, in some instances, result in gastrointestinal illnesses. However, little is known about how this colonisation begins, its variability and factors shaping the gut virome composition. Thus, understanding the development, assembly, and progression of enteric viral communities over time is key. To explore early-life virome development, metagenomic sequencing was employed in faecal samples collected longitudinally from a cohort of 17 infants during their first six months of life. The gut virome analysis revealed a diverse and dynamic viral community, formed by a richness of different viruses infecting humans, non-human mammals, bacteria, and plants. Eukaryotic viruses were detected as early as one week of life, increasing in abundance and diversity over time. Most of the viruses detected are commonly associated with gastroenteritis and include members of the Caliciviridae, Picornaviridae, Astroviridae, Adenoviridae, and Sedoreoviridae families. The most common co-occurrences involved asymptomatic norovirus-parechovirus, norovirus-sapovirus, sapovirus-parechovirus, observed in at least 40 % of the samples. Majority of the plant-derived viruses detected in the infants' gut were from the Virgaviridae family. This study demonstrates the first longitudinal characterisation of the gastrointestinal virome in infants, from birth up to 6 months of age, in sub-Saharan Africa. Overall, the findings from this study delineate the composition and variability of the healthy infants' gut virome over time, which is a significant step towards understanding the dynamics and biogeography of viral communities in the infant gut., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Enteric viruses are the leading cause of diarrhoea in children <5 years. Despite existing studies describing rotavirus diarrhoea in Mozambique, data on other enteric viruses remains scarce, especially after rotavirus vaccine introduction. We explored the prevalence of norovirus GI and GII, adenovirus 40/41, astrovirus, and sapovirus in children <5 years with moderate-to-severe (MSD), less severe (LSD) diarrhoea and community healthy controls, before (2008-2012) and after (2016-2019) rotavirus vaccine introduction in Manhiça District, Mozambique. The viruses were detected using ELISA and conventional reverse transcription PCR from stool samples. Overall, all of the viruses except norovirus GI were significantly more detected after rotavirus vaccine introduction compared to the period before vaccine introduction: norovirus GII in MSD (13/195, 6.7% vs. 24/886, 2.7%, respectively; p = 0.006) and LSD (25/268, 9.3% vs. 9/430, 2.1%, p < 0.001); adenovirus 40/41 in MSD (7.2% vs. 1.8%, p < 0.001); astrovirus in LSD (7.5% vs. 2.6%, p = 0.002); and sapovirus in MSD (7.1% vs. 1.4%, p = 0.047) and controls (21/475, 4.4% vs. 51/2380, 2.1%, p = 0.004). Norovirus GII, adenovirus 40/41, astrovirus, and sapovirus detection increased in MSD and LSD cases after rotavirus vaccine introduction, supporting the need for continued molecular surveillance for the implementation of appropriate control and prevention measures.

This study introduces a dual-mode biosensor specifically designed for the quantitative detection of viruses in rapid analysis. The biosensor is unique in its use of both optical (fluorescence) and electrochemical (impedance) detection methods using the same nanocomposites, providing a dual confirmation system for virus (norovirus-like particles) quantification. The system is based on using two antibody-conjugated nanocomposites: CdSeS quantum dots and Au-N,S-GQD nanocomposites. For optical detection, the principle relies on the fluorescence quenching of CdSeS by Au-N,S-GQD in a sandwich structure with the target. Conversely, electrochemical detection is based on the change in impedance caused by the formation of the same sandwich structure. The biosensor demonstrated exceptional sensitivity, capable of detecting norovirus at concentrations of as low as femtomolar in the electrochemical method and picomolar in the optical method. In the dual-responsive concentration range from 10 -13 to 10 -10 M, the sensor is highly sensitive in both methods, creating significant changes in fluorescence intensity and impedance in the presence of virus. Furthermore, the biosensor exhibits a high degree of specificity, with a negligible response to nontarget proteins, even within complex test solutions. This work represents a significant advancement in the field of biosensor technology, offering a fast, accurate, and reliable method for diagnosing viral infections and diseases.

How to address public health priorities after COVID-19 is becoming a critical task. To this end, we conducted wastewater surveillance for six leading pathogens, namely, SARS-CoV-2, norovirus, rotavirus, influenza A virus (IAV), enteroviruses and respiratory syncytial virus (RSV), in Nanchang city from January to April 2023. Metaviromic sequencing was conducted at the 1st, 4th, 7th, 9th, 12th and 14th weeks to reveal the dynamics of viral pathogens that were not covered by qPCR. Amplicon sequencing of the conserved region of norovirus GI and GII and the rotavirus and region encoding nonstructural protein of RSV was also conducted weekly. The results showed that after a rapid decrease in SARS-CoV-2 sewage concentrations occurred in January 2023, surges of norovirus, rotavirus, IAV and RSV started at the 6th, 7th, 8th and 11th weeks, respectively. The dynamics of the sewage concentrations of norovirus, rotavirus, IAV and RSV were consistent with the off-season resurgence of the above infectious diseases. Notably, peak sewage concentrations of norovirus GI, GII, rotavirus, IAV and RSV were found at the 6th, 3rd, 7th, 7th and 8th weeks, respectively. Astroviruses also resurge after the 7th week, as revealed by metaviromic data, suggesting that wastewater surveillance together with metaviromic data provides an essential early warning tool for revealing patterns of infectious disease resurgence., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Viral gastrointestinal infections remain a major public health concern in developing countries. In Burkina Faso, there are very limited updated data on the circulating viruses and their genetic diversity., Objectives: This study investigates the detection rates and characteristics of rotavirus A (RVA), norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) in patients of all ages with acute gastrointestinal infection in urban and rural areas., Study Design & Methods: From 2018 to 2021, stool samples from 1,295 patients with acute gastroenteritis were collected and screened for RVA, NoV, SaV and HAstV. Genotyping and phylogenetic analyses were performed on a subset of samples., Results: At least one virus was detected in 34.1% of samples. NoV and SaV were predominant with detection rates of respectively 10.5 and 8.8%. We identified rare genotypes of NoV GII, RVA and HAstV, recombinant HAstV strains and a potential zoonotic RVA transmission event., Conclusions: We give an up-to-date epidemiological picture of enteric viruses in Burkina Faso, showing a decrease in prevalence but a high diversity of circulating strains. However, viral gastroenteritis remains a public health burden, particularly in pediatric settings. Our data advocate for the implementation of routine viral surveillance and updated management algorithms for diarrheal disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Badjo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human noroviruses (HuNoVs) are highly contagious pathogens responsible of norovirus-associated acute gastroenteritis (AGE). GII.4 is the prevailing HuNoV genotype worldwide. Currently there are no studies on the molecular monitoring and phylogenetic analysis of HuNoVs in the territory of the Sverdlovsk region; therefore, it is not possible to objectively assess their genetic diversity. The aim of the study is to carry out genotyping and phylogenetic analysis of HuNoVs in the Sverdlovsk region from 2022 to 2023. Fecal samples ( n = 510) were collected from children suffering from HuNoV-AGE in municipalities of the Sverdlovsk region and the capsid genotype was determined by amplifying the ORF1/ORF2 junction. Of the 196 HuNoVs typed, which represent 38% of the studied samples, the largest share of HuNoV genotypes belong to the GII genogroup-86%, followed by the GI genogroup-14%. Noroviruses GII.4 and GII.17 were the co-dominant capsid genotypes (33.2% each). Phylogenetic analysis demonstrates that the identified sequences on the territory of the Sverdlovsk region have the smallest genetic distance, which gives grounds for their unification into a common cluster. Routine monitoring and phylogenetic analysis of circulating norovirus pathogens spectrum will enable timely tracking of HuNoVs genetic diversity and evolutionary events. This will lead to the development of more effective anti-epidemic measures, ultimately reducing the burden of infectious diseases.