Norovirus Genogroup Research Articles

Seasonal Variability of Human Enteric Viruses Discovered in Food Production Mussels (Mytilus galloprovincialis) Farmed in the Central Adriatic Sea (Italy).

Among the different naked and quasi-enveloped viruses, the hepatitis A virus (HAV), hepatitis E virus (HEV), and norovirus genogroups I and II (NoV GI and NoV GII) are considered the main microbiological noxae involved in foodborne outbreaks. Mussels can harbor pathogens in their tissues. In addition to epidemiological attention, marine water temperature changes are considered a crucial variable influencing viral loads. This study aimed to biomolecularly screen 1775 farmed mussels (Mytilus galloprovincialis) for viral ribonucleic acid (RNA) sequence detection (belonging to the HAV, HEV, and NoV GI and GII genogroups) in three different sampling periods (spring, summer, and winter), with the mussels collected from three farms located in the Central Adriatic Sea (Italy). The results showed that 10.42% of the screened animals harbored at least one type of pathogen RNA, more specifically, 5.35% HEV, 4.51% NoV GI, and 0.56% HAV. The highest genetic equivalent (GE) amounts were majorly observed in the winter season (NoV GI 1.0 × 103 GE/g and HEV 1.0 × 102 GE/g), resulting in statistical differences when compared to summer and spring (p-value: <0.001). The original data obtained serve to bring scientific attention to the possible influence of environmental and climatic aspects on viral loads, highlighting the crucial role played by biomolecular assays as preventive medicine tools.

Multisite community-scale monitoring of respiratory and enteric viruses in the effluent of a nursing home and in the inlet of the local wastewater treatment plant.

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.

Protocol for detection of pathogenic enteric RNA viruses by regular monitoring of environmental samples from wastewater treatment plants using droplet digital PCR

BackgroundThe 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 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. MethodsThe 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. ConclusionThis 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.

Surveillance of norovirus, SARS-CoV-2, and bocavirus in air samples collected from a tertiary care hospital in Thailand

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 × 102 genome copies/m3. The norovirus GII RNA concentrations ranged from 3.4 × 101 to 5.0 × 103 genome copies/m3. 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.

Virus Shedding and Diarrhea: A Review of Human Norovirus Genogroup II Infection in Gnotobiotic Pigs.

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

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

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

Droplet Digital PCR for Precise Quantification of Human Norovirus in Shellfish Associated with Gastroenteritis Illness

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-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 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.

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

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

Assessment of the viral contamination of fecal origin over a wide geographical area using an active approach with Dreissena polymorpha

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.

Prevalence of Norovirus Coinfection in Clostridioides difficile Toxin Positive Patients

Objective: In this study we aimed to evaluate the prevalence of norovirus genogroups I and II and C.difficile coinfection among patients with gastroenteritis symptoms. Method: A total of 76 patients with diarrhea were included in the study. Of these, 40 are children (

Piloting wastewater-based surveillance of norovirus in England

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.

VP1 of human and murine noroviruses recognizes glycolipid sulfatide via the P domain.

Noroviruses are a prevalent cause of human viral gastroenteritis, yet the precise mechanisms underlying their infection cycle, particularly their interactions with and entry into cells, remain poorly understood. Human norovirus (HuNoV) primarily targets human small intestinal epithelial cells, within which 3-O-sulfogalactosylceramide (sulfatide) ranks among the most abundant glycosphingolipids (GSLs). While sulfatide involvement in the binding and infection mechanism of several viruses has been documented, its interaction with noroviruses remains underexplored. This study investigated whether noroviruses interact with sulfatide. We found that the recombinant viral capsid protein VP1 of HuNoV (genogroups I and II) and murine norovirus (genogroup V) exhibited robust binding to sulfatide compared with other tested GSLs using enzyme-linked immunosorbent assay, thin-layer chromatography binding assay and real-time quantitative reverse transcription polymerase chain reaction binding assay. VP1 also bound 3-O-sulfated lactosylceramide, which shares the 3-O-sulfated galactose moiety with sulfatide. However, both VP1 and its P domain, identified as the sulfatide-binding domain, exhibited limited binding to structural analogues of sulfatide and other sulfated compounds. These findings suggest a specific recognition of the 3-O-sulfated galactose moiety. Notably, we found that sulfatide is a novel binding target for norovirus particles. Overall, our findings reveal a previously unknown norovirus-sulfatide interaction, proposing sulfatide as a potential candidate for norovirus infection receptors.

Exploring viral contamination in urban groundwater and runoff

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.

Epidemiology of norovirus infection in Nigeria: a systematic review and meta-analysis.

Human norovirus (HuNoV) is responsible for most cases of gastroenteritis worldwide, but information about the prevalence and diversity of HuNoV infections in lower-income settings is lacking. In order to provide more information about the burden and distribution of norovirus in Nigeria, we systematically reviewed original published research articles on the prevalence of HuNoV in Nigeria by accessing databases, including PubMed, Web of Science, ScienceDirect, Google Scholar, and African Journals Online (AJOL). The protocol for the review was registered on PROSPERO (registration number CRD42022308857). Thirteen relevant articles were included in the review, and 10 of them were used for meta-analysis. The pooled prevalence of HuNoV-associated gastroenteritis among children below 5 years of age in Nigeria, determined using the random-effects model, was 10.9% (95% CI, 6.7-16.7%). Among children below the age of 5 presenting with HuNoV infections, the highest prevalence was in children ≤2 years old (n = 127, 83%). The prevalence of HuNoV infections was seen to decrease with increasing age. In addition, HuNoV was detected in asymptomatic food handlers, bats, and seafoods. A total of 85 sequences of HuNoV isolates from Nigeria have been determined, and based on those sequences, the most prevalent norovirus genogroup was GII (84%). Genotypes GII.4 and GI.3 were the most frequently identified genotypes, with GII.4 constituting 46% of all of the HuNoVs identified in Nigeria. These results suggest a risk associated with cocirculation of emerging variants with known genotypes because of their recombination potential. Larger molecular epidemiological studies are still needed to fully understand the extent and pattern of circulation of HuNoVs in Nigeria.

Diverse genotypes of norovirus genogroup I and II contamination in environmental water in Thailand during the COVID-19 outbreak from 2020 to 2022

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.

Genetic diversity and distribution of noroviruses among all age groups of patients with diarrhea in Amhara National Regional State, Ethiopia.

Norovirus (NoV) is the leading cause of diarrheal disease worldwide and the impact is high in developing countries, including Ethiopia. Moreover, there is a significant and fluctuating global genetic diversity that varies across diverse environments over time. Nevertheless, there is a scarcity of data on the genetic diversity of NoV in Ethiopia. This study was aimed to assess the genetic diversity and distribution of NoVs circulating in the Amhara National Regional State, Ethiopia, by considering all age groups. A total of 519 fecal samples were collected from diarrheal patients from May 01/2021 to November 30/ 2021. The fecal samples were screened for the presence of NoVs using real-time RT-PCR by targeting a portion of the major capsid protein coding region. The positive samples were further amplified using conventional RT-PCR, and sequenced. The positivity rate of NoV was (8.9%; 46/519). The detection rate of NoV genogroup II (GII) and genogroup I (GI) was 38 (82.6%) and 8 (17.4%), respectively. Overall, five distinct GII (GII.3, GII.6, GII.10, GII.17, and GII.21) and two GI (GI.3 and GI.5) genotypes were detected. Within the GII types, GII.3 was the predominant (34.2%) followed by GII.21 (15.8%), GII.17 (10.5%), GII.6 and GII.10 each (2.6%). Norovirus GII.21 is reported for the first time in Ethiopia. The genetic diversity and distribution of NoVs were significantly different across the four sampling sits and age groups. The phylogenetic analysis revealed close relatedness of the current strains with published strains from Ethiopia and elsewhere. The distribution and genetic diversity of NoV was considerably high, with predominance of non-GII.4 genotypes. The GII.21 genotype is a new add on the growing evidences on the genetic diversity of NoVs in Ethiopia. Future nationwide surveillance studies are necessary to gain comprehensive data in Ethiopia.

Capsid Integrity Detection of Enteric Viruses in Reclaimed Waters.

Climate change, unpredictable weather patterns, and droughts are depleting water resources in some parts of the globe, where recycling and reusing wastewater is a strategy for different purposes. To counteract this, the EU regulation for water reuse sets minimum requirements for the use of reclaimed water for agricultural irrigation, including a reduction in human enteric viruses. In the present study, the occurrence of several human enteric viruses, including the human norovirus genogroup I (HuNoV GI), HuNoV GII, and rotavirus (RV), along with viral fecal contamination indicator crAssphage was monitored by using (RT)-qPCR methods on influent wastewater and reclaimed water samples. Moreover, the level of somatic coliphages was also determined as a culturable viral indicator. To assess the potential viral infectivity, an optimization of a capsid integrity PMAxx-RT-qPCR method was performed on sewage samples. Somatic coliphages were present in 60% of the reclaimed water samples, indicating inefficient virus inactivation. Following PMAxx-RT-qPCR optimization, 66% of the samples tested positive for at least one of the analyzed enteric viruses, with concentrations ranging from 2.79 to 7.30 Log10 genome copies (gc)/L. Overall, most of the analyzed reclaimed water samples did not comply with current EU legislation and contained potential infectious viral particles.

Human enteric viruses’ detection in mussels (&lt;i&gt;Mytilus galloprovincialis&lt;/i&gt;) farmed in the central Adriatic Sea

Human enteric viruses, such as hepatitis A virus (HAV), hepatitis E virus (HEV), and norovirus genogroups I and II (NoVGI and NoVGII), cause infections, and it has been largely demonstrated that mussels play an important role if consumed as raw or undercooked food matrices. This study aimed to investigate, through qualitative and quantitative biomolecular assays, the detection of partial genomic regions belonging to the most relevant enteropathogenic viruses for humans (HAV, HEV, NoVGI and NoVGII) in mussels (Mytilus galloprovincialis) farmed along the coasts of two Italian regions on the central Adriatic Sea: Abruzzo (Casalbordino, Chieti) and Molise (Termoli, Campobasso). A total of 425 animals were sampled, and the respective georeferentiations were registered. A total of 85 pools, each composed of five subjects/aliquot, were formed (22 from Abruzzo and 63 from Molise regions). This step was followed by homogenization and RNA extraction, and then the biomolecular assays [nested reverse transcription polymerase chain reaction (PCR) and real-time reverse transcription-quantitative PCR] were performed. 1.17% of the pool was positive for HAV RNA detection (102 copies/mL), 9.41% for HEV (102-103 copies/mL), 2.35% for NoVGI (101 copies/mL), and no pool was positive for NoVGII. This study demonstrated the human enteric viruses’ presence in mussels farmed in a low-investigated marine area. Based on a one-health point of view, this paper wants to enforce the importance of biomolecular and epidemiological screenings as surveillance systems to guarantee human, animal, and environmental health.

High-throughput microfluidic quantitative PCR system for the simultaneous detection of antibiotic resistance genes and bacterial and viral pathogens in wastewater

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.

Acute Gastroenteritis Outbreak Among Colorado River Rafters and Backpackers in the Grand Canyon, 2022.

From April 1 to May 31, 2022, Grand Canyon National Park received increased acute gastroenteritis reports. Pooled portable toilet specimens identified norovirus genogroups I and II. We sought to determine outbreak transmission contributors and individual risk factors while rafting or backpacking in the park. Grand Canyon rafters and backpackers were surveyed online from June 13-July 8, 2022, and a Cox proportional hazards model was used to identify predictors associated with illness and adjusted for potential confounding factors. Among 762 surveys, 119 cases and 505 well persons submitted complete survey data. Illness among rafters was associated with interaction with ill persons during the trip (adjusted hazard ratio [adjHR] = 3.4 [95%CI 2.3-5.0]) and lack of any hand hygiene (1.2 [0.7-1.9]) or use of only sanitizer or water (1.6 [1.04-2.6]) before snacks. Younger rafters had higher illness rates compared to those ≥60 y (1.5 [1.2-1.8] for ages 40-59 and 2.2 [1.4-3.5] for ages <40 y). Person-to-person transmission likely accounted for the widespread outbreak. Future outbreak mitigation efforts on river trips could focus on symptom screening before the trip starts, prompt separation of ill and well passengers, strict adherence to hand hygiene with soap and water, minimizing social interactions among rafting groups, and widespread outbreak notices and education to all park users.