GII.6 norovirus major capsid protein VP1 derived from distinct clusters induce cross-blocking effects.

Unlike pandemic GII.4 norovirus, GII.6 norovirus shows limited sequence variation in its major capsid protein VP1. In this study, we investigated the VP1 expression profiles, binding abilities, and cross-blocking effects of three GII.6 norovirus strains derived from three distinct variants. Norovirus VP1 was expressed using a recombinant baculovirus expression system and characterized by transmission electron microscopy, mass spectrometry, salivary histo-blood group antigen (HBGA)-virus like particles (VLPs) binding and binding blockade assays. Mass spectrometry revealed the expected molecular weight (MW) of full-length proteins and degraded or cleaved fragments of all three VP1 proteins. Peptide mapping showed loss of 2 and 3 amino acids from the N- and C-terminus, respectively. Further, the co-expression of VP1 and VP2 proteins did not lead to extra fragmentation during mass spectrometry. Salivary HBGA-VLP binding assay revealed similar binding patterns of the three GII.6 VP1 proteins. Salivary HBGA-VLP binding blockade assay induced cross-blocking effects. Our results demonstrate similar binding abilities against salivary HBGAs and specific cross-blocking effects for GII.6 norovirus strains derived from distinct variants, suggesting that fewer GII.6 strains from different evolutionary variants are needed for the development of norovirus vaccines.

Establishment of a Nucleic Acid Detection Method for Norovirus GII.2 Genotype Based on RT-RPA and CRISPR/Cas12a-LFS.

To establish a rapid detection method for norovirus GII.2 genotype, this study employed reverse transcription recombinase polymerase amplification (RT-RPA) combined with CRISPR/Cas12a and lateral flow strip (RT-RPA-Cas12a-LFS). Here, the genome of norovirus GII.2 genotype was compared to identify highly conserved sequences, facilitating the design of RT-RPA primers and crRNA specific to the conserved regions of norovirus GII.2. Subsequently, the reaction parameters of RT-RPA were optimized and evaluated using agar-gel electrophoresis and LFS. The results indicate that the conserved sequences of norovirus GII.2 were successfully amplified through RT-RPA at 37°C for 25 minutes. Additionally, CRISPR/Cas12a-mediated cleavage detection was achieved through LFS at 37°C within 10 minutes using the amplification products as templates. Including the isothermal amplification reaction time, the total time is 35 minutes. The established RT-RPA-Cas12a-LFS method demonstrated specific detection of norovirus GII.2, yielding negative results for other viral genomes, and exhibited an excellent detection limit of 10 copies/µl. The RT-RPA-Cas12a-LFS method was further compared with qRT-PCR by analyzing 60 food-contaminated samples. The positive conformity rate was 100%, the negative conformity rate was 95.45%, and the overall conformity rate reached 98.33%. This detection method for norovirus GII.2 genotype is cost-effective, highly sensitive, specific, and easy to operate, offering a promising technical solution for field-based detection of the norovirus GII.2 genotype.

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.

Epidemiology and multiple colonization of gastrointestinal pathogens in rural Tanzanian children with and without diarrhea: A case-control study.

Diarrheal diseases are important causes of morbidity and mortality, worldwide. The occurrence of multiple pathogens in stool samples of symptomatic and asymptomatic individuals in resource-limited countries have been repeatedly described. In this study, we assessed the differentiated effects of combined pathogen detections on recorded symptoms. A case-control study was conducted among 620 under-five-year-old children in rural northeastern Tanzania with emphasis of multiple detection. The median age of children was 11 months (IQR = 7, 20), and 52.1% were male. Cases (50.2%, n = 157) were less likely than controls (64.5%, n = 198) to have multiple colonization with gastrointestinal tract (GIT) pathogens. The children's age was positively associated with the likelihood of harboring multiple GIT pathogens [OR, 1.02, 95% CI = 1.01, 1.04]. Shigella spp./enteroinvasive Escherichia coli (EIEC) [OR = 2.80, 95% CI 1.62, 4.83] and norovirus [OR = 2.04, 95% CI 1.23, 3.39] were more common in cases and were strongly associated with diarrhea, while enteroaggregative E. coli (EAEC) [OR = 0.23, 95%CI 0.17-0.33] were more common in controls. Diarrheal diseases in under-five children from rural Tanzania are likely to be due to infections with Shigella spp./EIEC, and norovirus with strongly age-dependent associations.

Two risk assessments: Evaluating the use of indicator HF183 Bacteroides versus pathogen measurements for modelling recreational illness risks in an urban watershed.

The purpose of this study was to evaluate the performance of HF183 Bacteroides for estimating pathogen exposures during recreational water activities. We compared the use of Bacteroides-based exposure assessment to exposure assessment that relied on pathogen measurements. We considered two types of recreational water sites: those impacted by combined sewer overflows (CSOs) and those not impacted by CSOs. Samples from CSO-impacted and non-CSO-impacted urban creeks were analysed by quantitative polymerase chain reaction (qPCR) for HF183 Bacteroides and eight human gastrointestinal pathogens. Exposure assessment was conducted two ways for each type of site (CSO-impacted vs. non-CSO impacted): 1) by estimating pathogen concentrations from HF183 Bacteroides concentrations using published ratios of HF183 to pathogens in sewage and 2) by estimating pathogen concentrations from qPCR measurements. QMRA (quantitative microbial risk assessment) was then conducted for swimming, wading, and fishing exposures. Overall, mean risk estimates varied from 0.27 to 53 illnesses per 1,000 recreators depending on exposure assessment, site, activity, and norovirus dose-response model. HF183-based exposure assessment identified CSO-impacted sites as higher risk, and the recommended HF183 risk-based threshold of 525 genomic copies per 100 mL was generally protective of public health at the CSO-impacted sites but was not as protective at the non-CSO-impacted sites. In the context of our urban watershed, HF183-based exposure assessment over- and under-estimated risk relative to exposure assessment based on pathogen measurements, and the etiology of predicted pathogen-specific illnesses differed significantly. Across all sites, the HF183 model overestimated risk for norovirus, adenovirus, and Campylobacter jejuni, and it underestimated risk for E. coli and Cryptosporidium. To our knowledge, this study is the first to directly compare health risk estimates using HF183 and empirical pathogen measurements from the same waterways. Our work highlights the importance of site-specific hazard identification and exposure assessment to decide whether HF183 is applicable for monitoring risk.

Enteric viral pathogens and child growth among under-five children: findings from South Asia and sub-Saharan Africa.

Enteric viral pathogens are associated with a significant burden of childhood morbidity and mortality. We investigated the relationship between viral pathogens and child growth among under-5 children. We analyzed data from 5572/22,567 children enrolled in the Global Enteric Multicenter Study across seven study sites (2007-2011). Multiple linear regression was used to examine the association between the viral pathogens and changes of length/height-for-age (HAZ), weight-for-age (WAZ), and weight-for-length/height (WHZ) z-scores, stratified by diarrheal symptoms and adjusted for potential covariates. Rotavirus (18.51%) and norovirus (7.33%) were the most prevalent enteric viral pathogens among symptomatic and asymptomatic under-5 children, respectively. Infection with individual enteric viral pathogens hurts child growth in asymptomatic children. However, the relationship with HAZ was less clear and statistically non-significant. On the other hand, the combined viral pathogens demonstrated a strong negative influence on child growth [WAZ: ß coef.: - 0.10 (95%, CI - 0.15, - 0.05); P < 0.001 and WHZ: ß: - 0.12 (95% CI - 0.17, - 0.07); P < 0.001] among asymptomatic children. Infection with any viral pathogen was associated with growth shortfalls [HAZ: ß: - 0.05 (95% CI - 0.09, 0.00); P = 0.03 and WAZ: ß: - 0.11 (95% CI - 0.16, - 0.07); P < 0.001 and WHZ: ß: - 0.13 (95% CI - 0.18, - 0.09); P < 0.001], though the relationship with HAZ was less evident and became statistically non-significant in older children. Notably, among symptomatic children with moderate-to-severe diarrhea, individual enteric viral pathogens, as well as the combined effects of these pathogens [WHZ: ß: 0.07; (95% CI 0.01, 0.14); P = 0.03] and the presence of any virus [HAZ: ß: 0.09 (95% CI 0.05, 0.13) & WAZ: ß: 0.08 (95% CI 0.03, 0.12); P < 0.001], exhibited positive effects on child growth. While previous studies hypothesized that several viral pathogens had a conflicting controversial role in child growth, we find clear indications that enteric viral pathogens are associated with growth shortfalls, specifically among asymptomatic children. These findings highlight the need for preventive strategies targeting children with enteric viral pathogens, which could address the consequences of growth faltering.

Etiological surveillance of viral diarrhea from 2017 to 2019 in Zhangzhou city, Fujian province, China.

Background: Viral diarrhea is one of the major causes of morbidity and mortality in children. This study aimed to conduct etiological surveillance of viral diarrhea in Zhangzhou city, Fujian province, China, from 2017 to 2019 to identify the prevalence, distribution, and characteristics of viral pathogens causing gastrointestinal infections in the region. Methods: Stool samples were collected from patients with acute diarrhea in Zhangzhou city, Fujian province, China, from 2017 to 2019. Rotavirus, norovirus, astrovirus, and adenovirus were detected using fluorescence immunochromatography assay. Results: Of the total 5,627 samples that were collected, at least one of the viruses (rotavirus, norovirus, astrovirus and adenovirus) was found to be positive in 1,422 samples. Rotavirus, norovirus, astrovirus, and adenovirus, were detected in 53.73, 16.68, 15.52, and 14.97%, respectively. Mixed infections were determined in 17.65% of the positive samples. The predominant mixed infections observed were a combination of norovirus and astrovirus, followed by rotavirus and norovirus, and rotavirus and astrovirus. The highest positive rate was observed in the 12-23-month group for rotavirus and adenovirus, while a significantly higher positive rate was observed for norovirus and astrovirus in the 6-11-month group. Conclusion: These findings from this etiological surveillance highlight the significant burden of viral diarrhea in Zhangzhou city, with rotavirus being the predominant pathogen. The identification of common mixed infections provides insights into the complex nature of viral diarrhea transmission. Target interventions and public health strategies should be implemented, particularly during the winter and spring seasons, to prevent and control the spread of viral pathogens causing gastrointestinal infections in this region.

Measures for preventing norovirus outbreaks on campus in economically underdeveloped areas and countries: evidence from rural areas in Western China.

Background: The outbreak of norovirus represents a significant public health emergency within densely populated, impoverished, and underdeveloped areas and countries. Our objective is to conduct an epidemiology study of a norovirus outbreak that occurred in a kindergarten located in rural western China. We aim to raise awareness and garner increased attention towards the prevention and control of norovirus, particularly in economically underdeveloped regions. Methods: Retrospective on-site epidemiological investigation results, including data on school layout, case symptoms, onset time, disposal methods and sample testing results, questionnaire surveys, and case-control study were conducted in a kindergarten to analyze the underlying causes of the norovirus outbreak. Results: A total of 15 cases were identified, with an attack rate of 44.12% (15/34). Among them, 10 cases were diagnosed through laboratory tests, and 5 cases were diagnosed clinically. Vomiting (100%, 15/15) and diarrhea (93.33%, 14/15) were the most common symptoms in the outbreak. Case control study revealed that cases who had close contact (<1 m) with the patient's vomitus (OR = 5.500) and those who had close contact with similar patients (OR = 8.000) had significantly higher ORs compared to the control participants. The current study demonstrated that improper handling of vomitus is positively associated with norovirus outbreak. The absence of standardized disinfection protocols heightens the risk of norovirus outbreaks. Conclusion: To our knowledge, this study represents the first investigation into a norovirus outbreak in rural areas of western China. We aspire that amidst rapid economic development, a greater emphasis will be placed on the prevention and control of infectious diseases in economically underdeveloped areas and countries.

Production of norovirus-, rotavirus-, and enterovirus-like particles in insect cells is simplified by plasmid-based expression.

Insect cells have long been the main expression host of many virus-like particles (VLP). VLPs resemble the respective viruses but are non-infectious. They are important in vaccine development and serve as safe model systems in virus research. Commonly, baculovirus expression vector system (BEVS) is used for VLP production. Here, we present an alternative, plasmid-based system for VLP expression, which offers distinct advantages: in contrast to BEVS, it avoids contamination by baculoviral particles and proteins, can maintain cell viability over the whole process, production of alphanodaviral particles will not be induced, and optimization of expression vectors and their ratios is simple. We compared the production of noro-, rota- and entero-VLP in the plasmid-based system to the standard process in BEVS. For noro- and entero-VLPs, similar yields could be achieved, whereas production of rota-VLP requires some further optimization. Nevertheless, in all cases, particles were formed, the expression process was simplified compared to BEVS and potential for the plasmid-based system was validated. This study demonstrates that plasmid-based transfection offers a viable option for production of noro-, rota- and entero-VLPs in insect cells.

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.

Epidemiological Characteristics and Genotypic Features of Rotavirus and Norovirus in Jining City, 2021-2022.

Diarrhea, often caused by viruses like rotavirus (RV) and norovirus (NV), is a global health concern. This study focuses on RV and NV in Jining City from 2021 to 2022. Between 2021 and 2022, a total of 1052 diarrhea samples were collected. Real-Time Quantitative Fluorescent Reverse Transcriptase-PCR was used to detect RV-A, NV GI, and NV GII. For RV-A-positive samples, VP7 and VP4 genes were sequenced for genotype analysis, followed by the construction of evolutionary trees. Likewise, for NV-GII-positive samples, VP1 and RdRp genes were sequenced for genotypic analysis, and evolutionary trees were subsequently constructed. Between 2021 and 2022, Jining City showed varying detection ratios: RV-A alone (excluding co-infection of RV-A and NV GII) at 7.03%, NV GI at 0.10%, NV GII alone (excluding co-infection of RV-A and NV GII) at 5.42%, and co-infection of RV-A and NV GII at 1.14%. The highest RV-A ratios were shown in children ≤1 year and 2-5 years. Jining, Jinxiang County, and Liangshan County had notably high RV-A ratios at 24.37% (excluding co-infection of RV-A and NV GII) and 18.33% (excluding co-infection of RV-A and NV GII), respectively. Jining, Qufu, and Weishan had no RV-A positives. Weishan showed the highest NV GII ratios at 35.48% (excluding co-infection of RV-A and NV GII). Genotype analysis showed that, in 2021, G9P[8] and G2P[4] were dominant at 94.44% and 5.56%, respectively. In 2022, G8P[8], G9P[8], and G1P[8] were prominent at 75.86%, 13.79%, and 10.35%, respectively. In 2021, GII.3[P12], GII.4[P16], and GII.4[P31] constituted 71.42%, 14.29%, and 14.29%, respectively. In 2022, GII.3[P12] and GII.4[P16] accounted for 55.00% and 45.00%, respectively. RV-A and NV showed varying patterns for different time frames, age groups, and regions within Jining. Genotypic shifts were also observed in prevalent RV-A and NV GII strains in Jining City from 2021 to 2022. Ongoing monitoring of RV-A and NV is recommended for effective prevention and control.

Antiviral Activity of Ag5IO6, a Unique Silver Compound.

Pentasilver hexaoxoiodate (Ag5IO6) has broad-spectrum antimicrobial efficacy, including the long-term prevention of microbial adherence, the rapid killing of planktonic microorganisms, and the elimination of mature biofilms. This study's goal was to determine whether it may also have antiviral activity against structurally distinct viruses. Ag5IO6 was tested following ASTM E1052-20, Standard Practice to Assess the Activity of Microbicides Against Viruses in Suspension, against adenovirus type 5, murine norovirus, poliovirus type 1, SARS-CoV-2 (original), and SARS-CoV-2 (omicron) (host cells: H1HeLa, RAW 264.7, LLC-MK2, Vero E6, and Vero E6, respectively). A 0.1 g/mL Ag5IO6 suspension was prepared and the viruses were exposed for 30 min, 4 h, or 24 h. Exposure to Ag5IO6 resulted in complete kill of SARS-CoV-2 (omicron) within 30 min, as well as complete kill of both SARS-CoV-2 (original) and the murine norovirus within 4 h. Ag5IO6 showed increasing activity over time against the adenovirus, but did not achieve a 3-log reduction within 24 h, and showed no antiviral activity against the poliovirus. These results demonstrate that Ag5IO6 has antiviral activity against medically important viruses, in addition to its well-characterized antimicrobial activity, suggesting that it may be valuable in situations where the prevention or simultaneous treatment of microbes and viruses are necessary.

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

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Ten different viral agents infecting and co-infecting children with acute gastroenteritis in Southern Italy: Role of known pathogens and emerging viruses during and after COVID-19 pandemic.

Acute gastroenteritis (AGE) represents a world public health relevant problem especially in children. Enteric viruses are the pathogens mainly involved in the episodes of AGE, causing about 70.00% of the cases. Apart from well-known rotavirus (RVA), adenovirus (AdV) and norovirus (NoV), there are various emerging viral pathogens potentially associated with AGE episodes. In this study, the presence of ten different enteric viruses was investigated in 152 fecal samples collected from children hospitalized for gastroenteritis. Real time PCR results showed that 49.3% of them were positive for viral detection with the following prevalence: norovirus GII 19.7%, AdV 15.8%, RVA 10.5%, human parechovirus (HPeV) 5.3%, enterovirus (EV) 3.3%, sapovirus (SaV) 2.6%. Salivirus (SalV), norovirus GI and astrovirus (AstV) 1.3% each, aichivirus (AiV) found in only one patient. In 38.2% of feces only one virus was detected, while co-infections were identified in 11.8% of the cases. Among young patients, 105 were ≤5 years old and 56.0% tested positive for viral detection, while 47 were >5 years old with 40.0% of them infected. Results obtained confirm a complex plethora of viruses potentially implicated in gastroenteritis in children, with some of them previously known for other etiologies but detectable in fecal samples. Subsequent studies should investigate the role of these viruses in causing gastroenteritis and explore the possibility that other symptoms may be ascribed to multiple infections.

A Reliable Multifaceted Solution against Foodborne Viral Infections: The Case of RiLK1 Decapeptide.

Food-borne transmission is a recognized route for many viruses associated with gastrointestinal, hepatic, or neurological diseases. Therefore, it is essential to identify new bioactive compounds with broad-spectrum antiviral activity to exploit innovative solutions against these hazards. Recently, antimicrobial peptides (AMPs) have been recognized as promising antiviral agents. Indeed, while the antibacterial and antifungal effects of these molecules have been widely reported, their use as potential antiviral agents has not yet been fully investigated. Herein, the antiviral activity of previously identified or newly designed AMPs was evaluated against the non-enveloped RNA viruses, hepatitis A virus (HAV) and murine norovirus (MNV), a surrogate for human norovirus. Moreover, specific assays were performed to recognize at which stage of the viral infection cycle the peptides could function. The results showed that almost all peptides displayed virucidal effects, with about 90% of infectivity reduction in HAV or MNV. However, the decapeptide RiLK1 demonstrated, together with its antibacterial and antifungal properties, a notable reduction in viral infection for both HAV and MNV, possibly through direct interaction with viral particles causing their damage or hindering the recognition of cellular receptors. Hence, RiLK1 could represent a versatile antimicrobial agent effective against various foodborne pathogens including viruses, bacteria, and fungi.

Improving the Detection and Understanding of Infectious Human Norovirus in Food and Water Matrices: A Review of Methods and Emerging Models.

Human norovirus (HuNoV) is a leading global cause of viral gastroenteritis, contributing to numerous outbreaks and illnesses annually. However, conventional cell culture systems cannot support the cultivation of infectious HuNoV, making its detection and study in food and water matrices particularly challenging. Recent advancements in HuNoV research, including the emergence of models such as human intestinal enteroids (HIEs) and zebrafish larvae/embryo, have significantly enhanced our understanding of HuNoV pathogenesis. This review provides an overview of current methods employed for HuNoV detection in food and water, along with their associated limitations. Furthermore, it explores the potential applications of the HIE and zebrafish larvae/embryo models in detecting infectious HuNoV within food and water matrices. Finally, this review also highlights the need for further optimization and exploration of these models and detection methods to improve our understanding of HuNoV and its presence in different matrices, ultimately contributing to improved intervention strategies and public health outcomes.

Pepper mild mottle virus intended for use as a process indicator for drinking water treatment: Present forms and quantitative relations to norovirus and rotavirus in surface water.

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.

Seasonality of respiratory, enteric, and urinary viruses revealed by wastewater genomic surveillance.

Wastewater surveillance can reveal population-level infectious disease burden and emergent public health threats can be reliably assessed through wastewater surveillance. While molecular methods for wastewater monitoring of microorganisms have traditionally relied on PCR-based approaches, next-generation sequencing (NGS) can provide deeper insights via genomic analyses of multiple diverse pathogens. We conducted a year-long sequencing surveillance of 1,408 composite wastewater samples collected from 12 neighborhood-level access points in the greater Tempe area, Arizona, USA, and show that variation in wastewater viruses is driven by seasonal time and location. The temporal dynamics of viruses in wastewater were influenced cyclically, with the most dissimilarity between samples 23 weeks apart (i.e., winter vs summer, spring vs fall). We identified diverse urinary and enteric viruses including polyomaviruses, astroviruses, and noroviruses, and showed that their genotypes/subtypes shifted across seasons. We show that while wastewater data of certain respiratory viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strongly correlate with clinical case rates, laboratory-reported case incidences were discordant with surges of high viral load in wastewater for other viruses like human coronavirus 229E. These results demonstrate the utility of wastewater sequencing for informing decision-making in public health.IMPORTANCEWastewater surveillance can provide insights into the spread of pathogens in communities. Advances in next-generation sequencing (NGS) methodologies allow for more precise detection of viruses in wastewater. Long-term wastewater surveillance of viruses is an important tool for public health preparedness. This system can act as a public health observatory that gives real-time early warning for infectious disease outbreaks and improved response times.

Rotavirus vaccines in Africa and Norovirus genetic diversity in children aged 0 to 5 years old: a systematic review and meta-analysis : Rotavirus vaccines in Africa and Norovirus genetic diversity.

Noroviruses are the second leading cause of death in children under the age of 5 years old. They are responsible for 200 million cases of diarrhoea and 50,000 deaths in children through the word, mainly in low-income countries. The objective of this review was to assess how the prevalence and genetic diversity of noroviruses have been affected by the introduction of rotavirus vaccines in Africa. PubMed, Web of Science and Science Direct databases were searched for articles. All included studies were conducted in Africa in children aged 0 to 5 years old with gastroenteritis. STATA version 16.0 software was used to perform the meta-analysis. The method of Dersimonian and Laird, based on the random effects model, was used for the statistical analyses in order to estimate the pooled prevalence's at a 95% confidence interval (CI). Heterogeneity was assessed by Cochran's Q test using the I2 index. The funnel plot was used to assess study publication bias. A total of 521 studies were retrieved from the databases, and 19 were included in the meta-analysis. The pooled norovirus prevalence's for pre- and post-vaccination rotavirus studies were 15% (95 CI, 15-18) and 13% (95 CI, 09-17) respectively. GII was the predominant genogroup, with prevalence of 87.64% and 91.20% respectively for the pre- and post-vaccination studies. GII.4 was the most frequently detected genotype, with rates of 66.84% and 51.24% respectively for the pre- and post-vaccination studies. This meta-analysis indicates that rotavirus vaccination has not resulted in a decrease in norovirus infections in Africa.

Validation of one-step reverse transcription digital PCR assays for Norovirus GI.

Regular monitoring of Norovirus presence in environmental and food samples is crucial due to its high transmission rates and outbreak potential. For detecting Norovirus GI, reverse transcription qPCR method is commonly used, but its sensitivity can be affected by assay performance. This study shows significantly reduced assay performance in digital PCR or qPCR when using primers targeting Norovirus GI genome 5291-5319 (NC_001959), located on the hairpin of the predicted RNA structure. It is highly recommended to avoid this region in commercial kit development or diagnosis to minimizing potential risk of false negatives.