Human norovirus (HuNoV) and human astrovirus (HAstV) are viral enteric pathogens and known causative agents of acute gastroenteritis. Identifying the presence of these viruses in environmental samples such as irrigation water, or foods exposed to virus contaminated water (e.g., shellfish, agricultural crops), remains an important goal in the field of food safety. Determining if a virus species present in a sample is infectious is complicated by the recalcitrance of many enteric virus species to grow in culture, and/or the lack of a common cell culture system(s). Human intestinal enteroids (HIEs) can support the replication of HuNoV and HAstV, and thus hold promise as a platform for demonstrating the replication of multiple enteric virus species within a single sample. The objective of this study was to determine if HIEs can support co-replication of two genetically distinct human enteric viruses, HAstV3 and HuNoV GII.4[P16]. In single virus infections, HuNoV GII.4[P16] RNA levels were highest at 48-72 hpi (6.3-9.1 x 106 genome copy equivalents [gce]/well) and HAstV3 RNA levels were highest at 24 hpi (3.4 ×108 gce/well). HAstV3-infected cells stained positive for viral capsid protein at 24 hpi and induced the synthesis of RNA and protein expression of interferon (IFN)-beta, -lambda 1 and 2/3, peaking at 24 hpi and 48 hpi respectively. HuNoV GII.4[P16] replication was negatively impacted by HAstV3 co-infection, but HAstV3 was unaffected by HuNoV. A reduction in HuNoV GII.4[P16] RNA during co-infections was observed at 72 hpi, with partial restoration achieved using neutralizing anti-IFN-antibodies. Human intestinal enteroids can support the co-infection and replication of HuNoVGII.4[P16] and HAstV3, even at more than 100-fold excess in one virus over the other, and compounds (e.g., anti-IFN antibodies) that interfere with HIE antiviral mechanism(s) can aid in maximizing HuNoV replication during co-infection.

Multiplex digital PCR (dPCR) approaches are commonly employed in wastewater-based epidemiology (WBE) studies. However, optimizing the dPCR workflow is a critical step to ensure its reliability and accuracy before application. In this study, a 6-plex Crystal Digital PCR® (cdPCR) workflow was optimized for the simultaneous detection of six epidemiologically important pathogens, including three enteric viruses, noroviruses of genogroups I and II (NoV-GI and GII) and enteroviruses (EnV), and three respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), influenza A virus (InfA), and respiratory syncytial virus B (RSVB), in wastewater. Four cDNA input ratios (20 %-70 %) and two extraction kits were evaluated for optimization, with 30 % cDNA input and the AllPrep PowerViral DNA/RNA Kit (Qiagen) exhibiting optimal performance. The optimized 6-plex cdPCR assay was applied to a year-long wastewater surveillance study in Japan (n = 52), revealing distinct trends and prevalence ratios for enteric and respiratory viruses. NoV-GII was detected in 96 % of the samples with the highest mean concentration (6.1 ± 0.6 log10 copies/L), while SARS-CoV-2 and InfA were detected in 60 % and 50 % of the samples, respectively, which reflected the circulation of these pathogens within the community. Notably, RSVB was detected less frequently (25 %), in line with the fewer cases of RSVB reported during the study period. The wastewater concentrations of EnV and InfA showed significant positive correlations with hand foot and mouth disease and herpangina and influenza cases, respectively. However, no positive correlations were observed for RSV and COVID-19, possibly due to the testing of RSVB while RSVA was more prevalent and also due to cluster outbreaks. These findings demonstrated the utility of the 6-plex cdPCR assay in detecting pathogens and provided insights into community disease trends, representing an advancement in WBE.

This study aimed to estimate the incidence of norovirus (NoV)-associated diarrhea and asymptomatic infections in children under 4 years of age and identify the genotypes of multiple NoV infections.

A community-based cohort study was conducted in Tarlac, Philippines. Children aged 0-2 years were followed up for 2 years. The prevalence and incidence rates of NoV-associated diarrhea and asymptomatic infections were calculated. Risk factors were assessed using the Cox proportional hazards model. The genotypes and immunotypes of repeated infections were tabulated.

A total of 338 children aged 6208 child-months were analyzed. NoV was detected in 17.4% (84 of 527, 95% confidence interval [CI]: 12.7-19.7%) of diarrheal episodes and 10.8% (219 of 2031, 95% CI: 9.4-12.3%) of asymptomatic stool samples. The highest incidence of NoV-associated diarrhea occurred in children aged 6-11 months (2.31 per 100 child-months, 95% CI: 1.30-3.32) and 18-23 months (2.34 per 100 child-months, 95% CI: 1.57-3.12), whereas the highest incidence of asymptomatic NoV infection was observed in children aged 12-23 months (4.49 per 100 child-months, 95% CI: 3.41-5.56). Repeated NoV infections were detected between different genotypes, except in two children who had repeated NoV GI.3 and two children with GI.9 infections.

Children had the highest risk of NoV-associated diarrhea during their first year of life, whereas asymptomatic NoV infections persisted after the second year. Repeated NoV infections suggest genotype-specific immunity after NoV infection.

Globally, human norovirus (HuNoV) is the leading cause of foodborne illnesses. Norovirus transmission to fresh produce can occur via several sources, including contaminated irrigation water. HuNoV RNA has been detected in freshwater resources, but knowledge about virus infectivity is limited due to a historical lack of a HuNoV cell culture. Recently, HuNoV was shown to replicate in human intestinal enteroids (HIE). The objective of this study was to use HIE to evaluate the persistence of infectious HuNoV in raw (i.e. biologically active) surface freshwater. The virus was spiked into freshwater microcosms sampled from three freshwater ponds and then incubated inside an environmental chamber at 20-15 °C and 50-80 % relative humidity (day-night) and 12 h photoperiod. The water was tested for infectious HuNoV, intact HuNoV capsids, indigenous bacteria, and other water quality parameters over a period of 2 weeks. The persistence of infectious HuNoV in the three freshwater microcosms ranged from ≤1 day to ≥7 days. Decay rates for RNA from intact HuNoV capsids ranged from 0.04 to 0.54/day, predicting a 4.2 to 57.5 days, respectively for 1 log reduction. The intact virus showed a significant negative and positive linear relationship with indigenous bacteria and dissolved oxygen, respectively. Using multiple logistic regression, HuNoV RNA >4.4 log genomic equivalent/ml (Cycle threshold values <32) predicted higher probability of detecting infectious HuNoV in contaminated raw freshwater using HIE. Overall, our results provide valuable insights for enhancing quantitative microbial risk assessment models for pre-harvest agricultural water to understand the public health risks associated with the detection of HuNoV RNA in freshwater.

To detect viruses such as hepatitis A virus (HAV) and human norovirus (HuNoV) in foods, RT-qPCR or other molecular methods are used, which cannot distinguish between infectious and non-infectious virions. Samples can be pretreated to limit detection to intact and presumably infectious virions. We compared propidium monoazide (PMA or PMAxx), platinum (IV) chloride (PtCl4), magnetic silica beads and centrifugal filter using HAV or HuNoV inactivated by heat, pulsed light, or sodium hypochlorite (NaOCl). PMAxx completely or nearly eliminated (3.96 ± 1.24log gc) the RT-qPCR signal of HAV inactivated at 100°C for 10min. Pretreatments could not reduce significantly RT-qPCR signal of HAV after pulsed light (0.74 ± 0.36log gc) and NaOCl (0.24 ± 0.14log gc) inactivation. Enzymatic treatments did not improve the results obtained with PMAxx. The exudate of raspberry, strawberry or oyster used as food matrices needed dilution by at least tenfold for PMAxx to to yield results comparable to virions without a food matrix. Overall, PMAxx shows good potential to discriminate between infectious and non-infectious despite some remaining limitations.

Human enteric viruses can remain infective in surface waters for extended periods of time, posing a public health risk. Microbial activity contributes to the inactivation of waterborne enteric viruses, but while individual bacteria-virus interactions have been characterized, the importance of microbial diversity remains unknown. Here, we experimentally manipulated the diversity of bacterial communities from Lake Geneva across three seasons using a dilution-to-extinction approach and monitored the inactivation and genome decay of echovirus 11, a member of the Enterovirus genus. Long-read sequencing of the 16S rRNA gene revealed diversity gradients ranging between 373 and 2,722 bacterial species. Compared to sterile controls, echovirus 11 inactivation was enhanced by the presence of active bacteria and depended both on season and sample dilution. Throughout all seasons, the highest inactivation (between 3.0 and 7.9 log10 fold reduction in infectivity over 96 h) was observed in the least diluted incubations (i.e., the highest bacterial richness). Genome decay exhibited a 24-h lag and was less pronounced than the corresponding infectivity loss (ranging between 2.3 and 3.8 log10 fold over 96 h), indicating that microbial inactivation primarily targets the echovirus 11 capsid. We found a positive-saturating relationship between bacterial species richness and viral inactivation, suggesting functional redundancy and pointing toward the importance of rare species for viral inactivation. Biomarker analysis revealed several clades of bacteria, particularly members of Chitinophagaceae, to be significantly associated with echovirus 11 inactivation. Overall, these findings suggest that high microbial diversity enhances the capacity of surface waters to rid themselves of contamination by enteric viruses and hence protects public health.IMPORTANCEHuman enteric viruses in natural waterbodies pose a public health risk. Microorganisms, particularly bacteria, contribute to the inactivation of enteroviruses, thereby mitigating this risk. We use experimental manipulations of lake water bacterial diversity to unravel the importance of diversity for the inactivation of echovirus 11, a model human pathogen. Our findings suggest that bacterial diversity is important for echovirus 11 inactivation and that specific, but numerically rare, bacteria present in the surface water of Lake Geneva across different seasons contribute to viral inactivation. These findings contribute to our understanding of the inactivation of human enteric viruses in natural waterbodies-a hitherto understudied ecosystem service.

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

Noroviruses represent one of the leading causes of outbreaks and sporadic acute gastroenteritis (AGE) cases across all age groups. Although the GII.4 norovirus has been identified as the primary cause of most AGE outbreaks, the transient predominance of other genotypes has been reported globally. In this study, we describe a multi-province AGE outbreak caused by a potential new lineage of norovirus GII.17[P17], which has been recently detected at a high incidence in the United States and Europe. An amino acid analysis of the major viral capsid protein revealed several substitutions in the hypervariable region compared to strains circulating in the mid-2010s, which could play a key role in immune evasion. This is the first report of the detection of these viruses in the Southern Hemisphere, underscoring the importance of maintaining active genomic surveillance in the context of increasing numbers of acute gastroenteritis outbreaks.

This study examines at the prevalence and spread of Hepatitis A Virus (HAV) and norovirus GI/GII in local and imported food products in Greece over a five-year period (2019-2024). A total of two hundred sixty-six food samples were evaluated using obligatory inspections and virus detection procedures, including 202 for Hepatitis A and 64 for Norovirus. High-risk categories analyzed were vegetables [138 (HAV), 17 (NoV)], fruits [16 (HAV), 7 (NoV)], soft fruits/berries [37 (HAV), 31 (NoV)], processed meals [4 (HAV), 4 (NoV)], and animal-based products [1 (HAV), 5 (NoV)]. Viral RNA was isolated using QIAamp Viral RNA Mini Kit and detected using established RT-qPCR procedures that met ISO requirements for high sensitivity and reproducibility. The results demonstrated HAV contamination mostly in vegetables (4.35% positive rate), with sporadic findings in other categories. Norovirus GI/GII was detected primarily in soft fruits/berries, with a category-specific positive rate of 6.45%. A temporal study revealed that HAV peaks in 2020, while Norovirus contaminations were detected in 2021 and 2024. The findings highlight the important need to incorporate viral testing into routine food safety procedures, especially for high-risk product categories. This study establishes a basic framework for public health initiatives that address gaps in foodborne virus surveillance in Greece. The study's ramifications extend to global efforts to monitor and reduce foodborne virus contamination, pushing for higher regulatory requirements and targeted preventative actions.

There are limited biosecurity measures directed at preventing airborne transmission of viruses in swine. The effectiveness of dust mitigation strategies such as oil sprinkling, to decrease risk of airborne virus transmission are unknown. Metagenomics and qPCR for common fecal viruses were used to hunt for a ubiquitous virus to serve as a proxy when evaluating the efficiency of mitigation strategies against airborne viral infectious agents. Air particles were collected from swine buildings using high-volume air samplers. Extracted DNA and RNA were used to perform specific RT-qPCR and qPCR and analyzed by high-throughput sequencing. Porcine astroviruses group 2 were common (from 102 to 105 genomic copies per cubic meter of air or gc/m3, 93% positivity) while no norovirus genogroup II was recovered from air samples. Porcine torque teno sus virus were detected by qPCR in low concentrations (from 101 to 102 gc/m3, 47% positivity). Among the identified viral families by metagenomics analysis, Herelleviridae, Microviridae, Myoviridae, Podoviridae, and Siphoviridae were dominant. The phage vB_AviM_AVP of Aerococcus was present in all air samples and a newly designed qPCR revealed between 101 and 105 gc/m3 among the samples taken for the present study (97% positivity) and banked samples from 5- and 15-year old studies (89% positivity). According to the present study, both the porcine astrovirus group 2 and the phage vB_AviM_AVP of Aerococcus could be proxy for airborne viruses of swine buildings.

Norovirus is a highly virulent pathogen that causes enteritis in all age groups worldwide. Owing to the diversity of noroviruses, the development of vaccines and treatments is challenging, and an early and accurate diagnosis is crucial. Reference materials (RMs) developed previously for norovirus genotypes I (GI) and II (GII) were quantified using reverse transcription quantitative PCR. In this study, we developed norovirus GI and GII RMs as in vitro transcribed RNA forms. These RMs were then assigned reference values for the RNA copy number concentration. The concentrations of GI and GII RMs determined using in-house reverse transcription digital PCR assays were (1.92±0.37)×107 and (1.20±0.27)×107 copy/mL, respectively. The homogeneity and stability of the RMs were evaluated, and their compatibility with commercial diagnostic kits was validated. These RMs can be used for the development of detection assays, as calibrants for various molecular measurement techniques, and as test materials for internal and external quality assurance.

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

Human noroviruses (HNoVs) are a leading cause of acute gastroenteritis worldwide, with significant public health implications. In this study, wastewater-based epidemiology (WBE) was used to monitor the circulation and genetic diversity of HNoVs in Rome over an eight-year period (2017-2024). A total of 337 wastewater samples were analyzed using RT-nested PCR and next-generation sequencing (NGS) to identify genogroups GI and GII and their respective genotypes. The results showed that GII had higher detection rates (66.5%) compared to GI (50.7%), with significant variation between years. Detection rates peaked in 2019 before declining sharply in 2020, coinciding with the COVID-19 pandemic and rebounding after the pandemic in 2023. A total of 24 genotypes were identified (8 GI and 17 GII), including persistent variants GII.2, GII.3 and GII.4 and emerging genotypes such as GII.8, GII.10 and GII.14. Only two GII.4 variants, Sydney_2016 and Sydney_2012, were detected in the study. These results demonstrate the utility of WBE in tracking HNoVs circulation, identifying genotype diversity and capturing shifts in transmission dynamics. WBE provides a cost-effective and comprehensive tool for public health surveillance, particularly in regions with limited clinical surveillance. Sustained investment in WBE is crucial for advancing our understanding of HNoVs epidemiology and its long-term trends.

Despite regulations set up to monitor the microbiological quality of shellfish in producing areas, shellfish-borne gastroenteritis outbreaks still occur. Indeed, oyster depuration practices that are efficient to eliminate bacteria, fail to eliminate human norovirus from oyster flesh. In order to evaluate the impact of seawater temperature on the elimination of norovirus particles from oysters, large batches of oysters were contaminated using raw sewage containing norovirus and subjected to depuration at 8 °C or 18 °C. Over the experiment, quantitative RT-qPCR showed a one-log decrease of norovirus (both genogroups combined) genome copies per gram of digestive tissue after 41 days for oysters depurated at 8 °C and 24 days at 18 °C. The decrease of norovirus (both genogroups combined) in two batches of field-contaminated oysters depurated for two weeks at 18 °C was in the same range (21 and 23 days, respectively). All experiments showed a difference in genomic decay between the two norovirus genogroups, with norovirus genogroup I being more persistent in March/April compared to April/May.

Bivalve molluscs are filter-feeding organisms, capable of concentrating pathogenic microorganisms from the surrounding environment, thus contributing to the spread of viral pathogens, which they can transmit to humans, especially if eaten raw or undercooked. Although norovirus (NoV) and the hepatitis A virus (HAV) are considered the most common causes of foodborne infections, in recent years, other viruses with a zoonotic potential have been identified in shellfish, such as the hepatitis E virus (HEV), astrovirus (AsV), and aichi virus (AiV). The aim of the study was to investigate the presence of classical and emerging pathogenic enteric viruses in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis) from a mollusc farming area in the northwest of Italy, between April 2022 and March 2023. In the period considered, a total of 168 samples (84 oysters and 84 mussels) were analysed. The prevalence of NoV was highest, with 32.7% (55/168) positive samples, followed by 18.4% (31/168) for AsV and 19.6% (33/168) for AiV. This study revealed, for the first time, the presence of AsV and AiV in molluscs farmed in this sea area. All the samples tested were negative for HAV and HEV. The emergence of new enteric viruses like AiV and AsV in bivalve molluscs underscores the importance of improving surveillance and environmental monitoring methods, particularly in shellfish production areas.

Evidence on unnecessary antibiotic use in children with acute viral gastroenteritis (AGE) is scarce. We characterized the extent and correlates of antibiotic use among children hospitalized with viral AGE. A single-center study enrolled children aged 0-59 months hospitalized for AGE between 2008 and 2015 in Israel. Information was collected on laboratory tests, diagnoses, antibiotic treatment, and rotavirus vaccination. Stool samples were tested for rotavirus antigen, GII-norovirus, and stool cultures were performed for bacterial enteropathogens. Data from 2240 children were analyzed. Rotavirus vaccine was given to 79% of eligible children. Rotavirus test was performed on 1419 (63.3%) children. Before the introduction of universal rotavirus vaccination (2008-2010), rotavirus positivity in stool samples was 37.0%, which declined to 17.3% during the universal vaccination years (2011-2015). Overall, 1395 participants had viral AGE. Of those, 253 (18.1% [95% CI 16.1-20.2]) had unnecessary antibiotic treatment, mostly penicillin 46.6%, ceftriaxone 34.0% and azithromycin 21.7%. A multivariable analysis showed an inverse association between rotavirus vaccination and unnecessary antibiotic treatment (odds ratio = 0.53 [95% CI 0.31-0.91]), while positive associations were found with performing chest-X-ray test (3.00 [1.73-5.23]), blood (3.29 [95% CI 1.85-5.86]) and urine cultures (7.12 [3.77-13.43]), levels of C-reactive protein (1.02 [1.01-1.02]) and leukocytes (1.05 [1.01-1.09]). The results were consistent in an analysis of children with laboratory-confirmed rotavirus or norovirus AGE, or after excluding children with CRP > 50 mg/L. In conclusion, antibiotic prescription was common among hospitalized children with viral AGE, which was inversely related to rotavirus vaccination, possibly due to less severe illness in the vaccinated children.

Noroviruses, belonging to the family Caliciviridae, are classified into at least ten genogroups (G) based on their major capsid protein (VP1). The common genogroup to be identified in both humans and pigs is GII, although porcine noroviruses (PoNoVs) belong to genotypes of their own (GII.11, GII.18, and GII.19). So far, PoNoVs have not been studied much in Finland, possibly due to their rather symptomless nature in pigs. In the present study, we enrolled a total of 189 fecal samples collected from pigs from Finnish farms. Samples were taken from 12 farms in 2010, 2019 and 2020. We analyzed feces from growing pigs ranging from 2.1 to 6 months of age. RNA was extracted from fecal suspensions using a commercial viral RNA extraction kit, followed by RT (reverse transcription)-qPCR. The genotypes were determined by Sanger sequencing of the PCR fragments amplified by conventional PCR. Of the 12 farms, 6 (50%) had at least one PoNoV-infected pig. Altogether 18 (9.5%) of the 189 pigs tested positive for PoNoVs. Pigs mostly aged over 4 months were infected with PoNoVs. Eventually, 12 positive samples were determined as genotype GII.18. We could demonstrate the presence of PoNoVs in Finnish pigs. In future, more studies in which longer sequences from PoNoV genome can be obtained, are required.

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.

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.

Suspected false positive norovirus results occurred after introduction of the BioFire® FilmArray® Gastrointestinal panel (BF-GIP) into 6 laboratory sites, in British Columbia, Canada.

The primary objective was to investigate suspected false positive results by performing additional molecular assays and whole genome sequencing (WGS). The second objective was to determine if melting curve review could predict false positive results.

From February 2023 to May 2024, the proportion of potential false positive norovirus results from the BF-GIP was calculated based on confirmatory testing using alternate molecular method. A subset of 65 norovirus BF-GIP positive specimens, including 35 negatives and 30 positives on additional molecular assays, underwent WGS. Melting curves appearances from 150 specimens were reviewed.

Overall, 215/784 (27.4 %) BF-GIP norovirus results were suspected to be false positives. When using WGS, 64/65 results were in agreement with confirmatory testing. Notably, 35 specimens negative on confirmatory testing and suspected to be BF-GIP norovirus false positive results were undetectable by WGS. Melting curves did not accurately predict false positives, since 20/72 (27.8 %) had typical appearances upon review.

BF-GIP produces false positive results for norovirus, which cannot be predicted by melting curve review.

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.

Recent increases in zoonotic diseases underscore the integration of companion animals into urban environments, posing complex transmission risks and highlighting the necessity of One Health approaches. Respiratory and enteric viruses have been consistently linked to interspecies transmission between humans and animals. This study aimed to assess the circulation of human noroviruses (NoV), human adenoviruses (HAdV), enteroviruses (EV), parechoviruses (PeV-A), human bocaviruses (HBoV), hepatitis A (HAV) and E viruses (HEV), Influenza A and B viruses (Flu A/B), respiratory syncytial virus (RSV), and SARS-CoV-2 in domestic dogs and cats in Brazil to understand potential zooanthroponosis risks. Between 2012 and 2021, 600 fecal samples from dogs and cats (516 and 84, respectively) were collected at small animal clinics in São Paulo state, Brazil. The specimens underwent in-house qPCR screening for HBoV and HAdV, while EV, PeV-A, NoV, and HEV were tested using in-house RT-qPCR. SARS-CoV-2, Flu A/B, and RSV were investigated with a commercial RT-qPCR kit assay. HAV detection utilized conventional nested (RT)-PCR. Positive samples were sequenced for molecular characterization and phylogenetic analysis. NoV was detected in 0.2 % (1/600) of the animals, while all other investigated viruses tested negative. The NoV-positive sample, collected in 2012 from a pet dog, was identified as genotype GII.4_Sydney[P31]. The Dog/BRA/2012/GII.4_Sydney[P31]/IAL-M21 strain exhibited a close genetic relationship to Brazilian human and environmental NoV GII.4_Sydney[P31] strains, with 98.1-99.2 % nucleotide similarity in ORF1 and 99.2-99.6 % in ORF2 sequences, suggesting interspecies transmission. Pet dogs are frequently exposed to human fecal-borne viruses, highlighting the potential for zooanthroponotic transmission due to their close interaction with humans in shared environments. There is an urgent need to enhance surveillance studies in companion animals to better understand the implications of detecting human NoV strains in pets, as NoV could potentially act as a reverse zoonotic disease in households, animal hospitals, or shelters worldwide.

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.

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.

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.

The present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses, explicitly focusing on norovirus genogroup Ⅱ (GⅡ), astrovirus, rotavirus, Aichi virus, sapovirus, hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR (ddPCR). Enteric viruses are of significant public health concern, as they are the leading cause of diseases like gastroenteritis. Regular monitoring of environmental samples, particularly from wastewater treatment plants, is crucial for early detection and control of these viruses. This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions. Our protocol's objective is to establish a novel ddPCR-based methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal, India. Our assay is capable of accurately quantifying virus concentrations without standard curves, minimizing extensive optimization, and enhancing sensitivity and precision, especially for low-abundance targets.

The study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years, ensuring comprehensive coverage and consistent data. Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater, a more advanced technique. Additionally, we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses.

This study will aid in understanding these viruses' genetic diversity and mutation rates, which is crucial for developing tailored intervention strategies. The findings will be instrumental in shaping public health responses and improving epidemiological surveillance, especially in localities heaving sewage networks.

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.

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.

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.

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).

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.

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 (CT 35.2) was tested negative in the AIOS. Two additional sapovirus positive samples, CT 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 CT values over 35 and therefore unlikely of clinical relevance.

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.

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.

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.

Comprehensive data on bacterial and viral pathogens of diarrhea and studies applying culture-independent methods for examining antibiotic resistance in wastewater are lacking. This study aimed to simultaneously quantify antibiotic resistance genes (ARGs), class 1 integron-integrase (int1), bacterial and viral pathogens of diarrhea, 16S rRNA, and other indicators using a high-throughput quantitative PCR (HT-qPCR) system. Thirty-six grab wastewater samples from a wastewater treatment plant in Japan, collected three times a month between August 2022 and July 2023, were centrifuged, followed by nucleic acid extraction, reverse transcription, and HT-qPCR. Fourteen targets were included, and HT-qPCR was performed on the Biomark X9™ System (Standard BioTools). For all qPCR assays, R2 was ≥0.978 and the efficiencies ranged from 90.5% to 117.7%, exhibiting high performance. Of the 36 samples, 20 (56%) were positive for Norovirus genogroup II (NoV-GII), whereas Salmonella spp. and Campylobacter jejuni were detected in 24 (67%) and Campylobacter coli in 13 (36%) samples, with mean concentrations ranging from 3.2 ± 0.8 to 4.7 ± 0.3 log10 copies/L. NoV-GII detection ratios and concentrations were higher in winter and spring. None of the pathogens of diarrhea correlated with acute gastroenteritis cases, except for NoV-GII, suggesting the need for data on specific bacterial infections to validate bacterial wastewater-based epidemiology (WBE). All samples tested positive for sul1, int1, and blaCTX-M, irrespective of season. The less explored blaNDM-1 showed a wide prevalence (>83%) and consistent abundance ranging from 4.3 ± 1.0 to 4.9 ± 0.2 log10 copies/L in all seasons. sul1 was the predominant ARG, whereas absolute abundances of 16S rRNA, int1, and blaCTX-M varied seasonally. int1 was significantly correlated with blaCTX-M in autumn and spring, whereas it showed no correlation with blaNDM-1, questioning the applicability of int1 as a sole indicator of overall resistance determinants. This study exhibited that the HT-qPCR system is pivotal for WBE.

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.

In June 2022, a gastroenteritis outbreak occurred in a town in Northern Italy, possibly associated with the ingestion of norovirus from public drinking water. Noroviruses are highly infectious RNA viruses, with high stability in the environment. They are the primary cause of non-bacterial gastroenteritis worldwide, and despite the fact that the disease is mainly self-limiting, norovirus infection can lead to severe illness in the immunocompromised, the elderly and children. Immediately after the notification of the suspected norovirus outbreak, faecal specimens were collected from hospitalised patients, and water samples were collected from public drinking fountains in the affected area, to confirm the presence of norovirus. Norovirus was detected in 80 % (95 % CI 0.58-0.91) of the faecal specimens, and in 50 % (95 % CI 0.28-0.72) of the water samples using RT (reverse transcription) Real-time PCR. The identification of GII genotype in all samples confirmed public drinking water as the source of norovirus contamination. In addition, in one faeces and one water sample, the co-presence of genotypes GI and GII was detected. The strains were typed by sequencing, with most of them belonging to the genotype GII.3. Immediately after the confirmation of norovirus contamination in public drinking water, the local competent authorities applied safety measures, resulting in a decline in number of cases. Moreover, after the application of disinfection protocols in the water plant, the sampling was repeated with negative results for norovirus in the affected area. However, positive samples were found in the neighbouring area (prevalence 10.00 %, 95 % CI 0.02-0.40) and in the water spring (prevalence 50.00 %, 95 % CI 0.21-0.78), suggesting norovirus persistence and spread from the water source. The prompt identification of the source of contamination, and collaboration with the local authorities guided the implementation of proper procedures to control viral spread, resulting in the successful control of the outbreak.

In recent years, the transmission of viruses from wildlife to humans has raised significant public health concerns, exemplified by the COVID-19 pandemic caused by the betacoronavirus SARS-CoV-2. Human activities play a substantial role in increasing the risk of zoonotic virus transmission from wildlife to humans. Rats and mice are prevalent in urban environments and may act as reservoirs for various pathogens. This study aimed to evaluate the presence of zoonotic viruses in wild rats and mice in both urban and rural areas, focusing on well-known zoonotic viruses such as betacoronavirus, hantavirus, arenavirus, kobuvirus, and monkeypox virus, along with other viruses occasionally detected in rats and mice, including rotavirus, norovirus, and astrovirus, which are known to infect humans at a high rate. A total of 128 animals were captured, including 70 brown rats (Rattus norvegicus), 45 black rats (Rattus rattus), and 13 house mice (Mus musculus), and feces, lung, and liver were collected. Among brown rats, one fecal sample tested positive for astrovirus RNA. Nucleotide sequencing revealed high sequence similarity to both human and rat astrovirus, suggesting co-presence of these viruses in the feces. Murine kobuvirus (MuKV) was detected in fecal samples from both black (n = 7) and brown (n = 6) rats, primarily from urban areas, as confirmed by sequence analysis. These findings highlight the importance of surveillance and research to understand and mitigate the risks associated with the potential transmission of pathogens by rodents.

Pepper mild mottle virus (PMMoV) has been proposed as a potential indicator of human enteric viruses in environmental water and for viral removal during drinking water treatment. To investigate the occurrence and present forms of PMMoV and quantitative relations to norovirus GII and rotavirus A (RVA) in surface waters, 147 source water samples were collected from 21 drinking water treatment plants (DWTPs) in Japan between January 2018 and January 2021, and the concentrations of viruses in suspended and dissolved fractions were measured using real-time RT-PCR. PMMoV was detected in 81-100 % of samples in each sample month and observed concentrations ranged from 3.0 to 7.0 log10 copies/L. The concentrations of PMMoV were higher in dissolved fraction compared to suspended fractions, while different partitioning was observed for NoV GII depending on seasons. The concentrations of PMMoV were basically higher than those of norovirus GII (1.9-5.3 log10 copies/L) and RVA (1.9-6.6 log10 copies/L), while in 18 samples, RVA presented higher concentrations than PMMoV. Partial regions of VP7, VP4, and VP6 of the RVA in the 18 samples were amplified using nested PCR, and the genotypes were determined using an amplicon-based next-generation sequencing approach. We found that these source water samples included not only human RVA but also various animal RVA and high genetic diversity due to the existence of animal RVA was associated with a higher RVA concentration than PMMoV. Our findings suggest that PMMoV can be used as an indicator of norovirus GII and human RVA in drinking water sources and that the indicator performance should be evaluated by comparing to zoonotic viruses as well as human viruses.

An optimized digital RT-PCR (RT-dPCR) assay for the detection of human norovirus GI and GII RNA was compared with ISO 15216-conform quantitative real-time RT-PCR (RT-qPCR) assays in an interlaboratory study (ILS) among eight laboratories. A duplex GI/GII RT-dPCR assay, based on the ISO 15216-oligonucleotides, was used on a Bio-Rad QX200 platform by six laboratories. Adapted assays for Qiagen Qiacuity or ThermoFisher QuantStudio 3D were used by one laboratory each. The ILS comprised quantification of norovirus RNA in the absence of matrix and in oyster tissue samples. On average, results of the RT-dPCR assays were very similar to those obtained by RT-qPCR assays. The coefficient of variation (CV%) of norovirus GI results was, however, much lower for RT-dPCR than for RT-qPCR in intra-laboratory replicates (eight runs) and between the eight laboratories. The CV% of norovirus GII results was in the same range for both detection formats. Had in-house prepared dsDNA standards been used, the CV% of norovirus GII could have been in favor of the RT-dPCR assay. The ratio between RT-dPCR and RT-qPCR results varied per laboratory, despite using the distributed RT-qPCR dsDNA standards. The study indicates that the RT-dPCR assay is likely to increase uniformity of quantitative results between laboratories.

Growing global concerns over water scarcity, worsened by climate change, drive wastewater reclamation efforts. Inadequately treated wastewater presents significant public health risks. Previous studies in South Africa (SA) have reported high norovirus levels in final effluent and sewage-polluted surface water, indicating pathogen removal inefficiency. However, the viability of these virions was not explored. This study assessed human norovirus viability in final effluent from wastewater treatment works (WWTWs) in Pretoria, SA. Between June 2018 and August 2020, 200 samples were collected from two WWTWs, including raw sewage and final effluent. Norovirus concentrations were determined using in-house RNA standards. Viability of noroviruses in final effluent was assessed using viability RT-qPCR (vPCR) with PMAxx™-Triton X-100. There was no significant difference in GI concentrations between raw sewage (p = 0.5663) and final effluent (p = 0.4035) samples at WWTW1 and WWTW2. WWTW1 had significantly higher GII concentrations in raw sewage (p < 0.001) compared to WWTW2. No clear seasonal pattern was observed in norovirus concentrations. At WWTW1, 50% (7/14) of GI- and 64.9% (24/37) of GII-positive final effluent samples had no quantifiable RNA after vPCR. At WWTW2, the majority (92.6%, 25/27) of GII-positive final effluent samples showed a 100% RNA reduction post vPCR. PMAxx™-Triton X-100 vPCR provides a more accurate reflection of discharge of potentially viable noroviruses in the environment than standard RT-qPCR. Despite significant reductions in potentially viable noroviruses after wastewater treatment, the levels of potentially viable viruses in final effluent are still of concern due to the high initial load and low infectious dose of noroviruses.