N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Self-Powered Immunoassay of Norovirus in Human Stools by π-Electron-Rich Homojunction for Enhanced Charge Transfer.

Norovirus (NoV) stands as a significant causative agent of nonbacterial acute gastroenteritis on a global scale, presenting a substantial threat to public health. Hence, the development of simple and rapid analytical techniques for NoV detection holds great importance in preventing and controlling the outbreak of the epidemic. In this work, a self-powered photoelectrochemical (PEC) immunosensor of NoV capsid protein (VP1) was proposed by the π-electron-rich carbon nitride homojunction (ER-CNH) as the photoanode. C4N2 ring derived from π-rich locust bean gum was introduced into the tri-s-triazine structure, creating a large π-delocalized conjugated carbon nitride homojunction. This strategy enhances the C/N atomic ratio, which widens light utilization, narrows the bandgap, and optimizes the electronic band structure of carbon nitride. By introduction of a π-rich conjugated structure, p-type domains were induced within n-type domains to build the internal electric field at the interface, thus forming a p-n homojunction to boost carrier separation and transfer. The ER-CNH photoanode exhibited excellent photoelectric performance and water oxidation capacity. Since VP1 inhibits the water oxidation of the ER-CNH photoanode, the open-circuit potential of the as-prepared PEC immunosensor system was reduced for detecting NoV VP1. The self-powered PEC immunosensor achieved a remarkably low detection limit (∼5 fg mL-1) and displayed high stability and applicability for actual stool samples. This research serves as a foundation concept for constructing immunosensors to detect other viruses and promotes the application of self-powered systems for life safety.

Foodborne Illnesses from Leafy Greens in the United States: Attribution, Burden, and Cost.

Leafy green vegetables are a major source of foodborne illnesses. Nevertheless, few studies have attempted to estimate attribution and burden of illness estimates for leafy greens. This study combines results from three outbreak-based attribution models with illness incidence and economic cost models to develop comprehensive pathogen-specific burden estimates for leafy greens and their subcategories in the United States. We find that up to 9.18% (90% CI: 5.81%-15.18%) of foodborne illnesses linked to identified pathogens are attributed to leafy greens. Including 'Unknown' illnesses not linked to specific pathogens, leafy greens account for as many as 2,307,558 (90% CI: 1,077,815-4,075,642) illnesses annually in the United States. The economic cost of these illnesses is estimated to be up to $5.278 billion (90% CI: $3.230-$8.221 billion) annually. Excluding the pathogens with small outbreak sizes, Norovirus, Shiga toxin-producingEscherichia coli (both non-O157 and O157:H7), Campylobacter spp., and nontyphoidal Salmonella, are associated with the highest number of illnesses and greatest costs from leafy greens. While lettuce (romaine, iceberg, "other lettuce") takes 60.8% of leafy green outbreaks, it accounts for up to 75.7% of leafy green foodborne illnesses and 70% of costs. Finally, we highlighted that 19.8% of Shiga toxin-producingEscherichia coli O157:H7 illnesses are associated with romaine among all food commodities, resulting in 12,496 estimated illnesses and $324.64 million annually in the United States.

CX-6258 hydrochloride hydrate: A potential non-nucleoside inhibitor targeting the RNA-dependent RNA polymerase of norovirus.

Human norovirus (HuNoV), a primary cause of non-bacterial gastroenteritis, currently lacks approved treatment. RdRp is vital for virus replication, making it an attractive target for therapeutic intervention. By application of structure-based virtual screening procedure, we present CX-6258 hydrochloride hydrate as a potent RdRp non-nucleoside inhibitor, effectively inhibiting HuNoV RdRp activity with an IC50 of 3.61 µM. Importantly, this compound inhibits viral replication in cell culture, with an EC50 of 0.88 µM. In vitro binding assay validate that CX-6258 hydrochloride hydrate binds to RdRp through interaction with the "B-site" binding pocket. Interestingly, CX-6258-contacting residues such as R392, Q439, and Q414 are highly conserved among major norovirus GI and GII variants, suggesting that it may be a general inhibitor of norovirus RdRp. Given that CX-6258 hydrochloride hydrate is already utilized as an orally efficacious pan-Pim kinase inhibitor, it may serve as a potential lead compound in the effort to control HuNoV infections.

Erratum: Establishment and application of a rapid assay for GII.4/GII.17 NoV detection based on the combination of CRISPR/Cas13a and isothermal amplification.

[This corrects the article DOI: 10.3389/fmicb.2024.1334387.].

Murine norovirus mutants adapted to replicate in human cells reveal a post-entry restriction.

RNA viruses lack proofreading in their RNA polymerases and therefore exist as genetically diverse populations. By exposing these diverse viral populations to selective pressures, viruses with mutations that confer fitness advantages can be enriched. To examine factors important for viral tropism and host restriction, we passaged murine norovirus (MNV) in a human cell line, HeLa cells, to select mutant viruses with increased fitness in non-murine cells. A major determinant of host range is expression of the MNV receptor CD300lf on mouse cells, but additional host factors may limit MNV replication in human cells. We found that viruses passaged six times in HeLa cells had enhanced replication compared with the parental virus. The passaged viruses had several mutations throughout the viral genome, which were primarily located in the viral non-structural coding regions. Although viral attachment was not altered for the passaged viruses, their replication was higher than the parental virus when the entry was bypassed, suggesting that the mutant viruses overcame a post-entry block in human cells. Three mutations in the viral NS1 protein were sufficient for enhanced post-entry replication in human cells. We found that the human cell-adapted MNV variants had reduced fitness in murine BV2 cells and infected mice, with reduced viral titers. These results suggest a fitness tradeoff, where increased fitness in a non-native host cell reduces fitness in a natural host environment. Overall, this work suggests that MNV tropism is determined by the presence of not only the viral receptor but also post-entry factors. IMPORTANCE: Viruses infect specific species and cell types, which is dictated by the expression of host factors required for viral entry as well as downstream replication steps. Murine norovirus (MNV) infects mouse cells, but not human cells. However, human cells expressing the murine CD300lf receptor support MNV replication, suggesting that receptor expression is a major determinant of MNV tropism. To determine whether other factors influence MNV tropism, we selected for variants with enhanced replication in human cells. We identified mutations that enhance MNV replication in human cells and demonstrated that these mutations enhance infection at a post-entry replication step. Therefore, MNV infection of human cells is restricted at both entry and post-entry stages. These results shed new light on factors that influence viral tropism and host range.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Health Care Utilization and Clinical Management of All-Cause and Norovirus-Associated Acute Gastroenteritis Within a US Integrated Health Care System.

Background: Norovirus-associated acute gastroenteritis (AGE) exacts a substantial disease burden, yet the health care utilization for and clinical management of norovirus-associated AGE are not well characterized. Methods: We describe the health care encounters and therapeutics used for patients with all-cause and norovirus-associated AGE in the Kaiser Permanente Northwest health system from 1 April 2014 through 30 September 2016. Medical encounters for patients with AGE were extracted from electronic health records, and encounters within 30 days of one another were grouped into single episodes. An age-stratified random sample of patients completed surveys and provided stool samples for norovirus testing. Results: In total, 40 348 individuals had 52 509 AGE episodes; 460 (14%) of 3310 participants in the substudy tested positive for norovirus. An overall 35% of all-cause AGE episodes and 29% of norovirus-associated AGE episodes had ≥2 encounters. While 80% of norovirus-associated AGE episodes had at least 1 encounter in the outpatient setting, all levels of the health care system were affected: 10%, 22%, 10%, and 2% of norovirus-associated AGE episodes had at least 1 encounter in virtual, urgent care, emergency department, and inpatient settings, respectively. Corresponding proportions of therapeutic use between norovirus-positive and norovirus-negative episodes were 13% and 10% for intravenous hydration (P = .07), 65% and 50% for oral rehydration (P < .001), 7% and 14% for empiric antibiotic therapy (P < .001), and 33% and 18% for antiemetics (P < .001). Conclusions: Increased health care utilization and therapeutics are likely needed for norovirus-associated AGE episodes during peak norovirus winter seasons, and these data illustrate that effective norovirus vaccines will likely result in less health care utilization.

Molecular Evolution of GII.P31/GII.4_Sydney_2012 Norovirus over a Decade in a Clinic in Japan.

Norovirus (NoV) genogroup II, polymerase type P31, capsid genotype 4, Sydney_2012 variant (GII.P31/GII.4_Sydney_2012) has been circulating at high levels for over a decade, raising the question of whether this strain is undergoing molecular alterations without demonstrating a substantial phylogenetic difference. Here, we applied next-generation sequencing to learn more about the genetic diversity of 14 GII.P31/GII.4_Sydney_2012 strains that caused epidemics in a specific region of Japan, with 12 from Kyoto and 2 from Shizuoka, between 2012 and 2022, with an emphasis on amino acid (aa) differences in all three ORFs. We found numerous notable aa alterations in antigenic locations in the capsid region (ORF2) as well as in other ORFs. In all three ORFs, earlier strains (2013-2016) remained phylogenetically distinct from later strains (2019-2022). This research is expected to shed light on the evolutionary properties of dominating GII.P31/GII.4_Sydney_2012 strains, which could provide useful information for viral diarrhea prevention and treatment.

Usefulness and Limitations of PFGE Diagnosis and Nucleotide Sequencing Method in the Analysis of Food Poisoning Pathogens Found in Cooking Employees.

In the case of a food poisoning outbreak, it is essential to understand the relationship between cooking workers and food poisoning. Many biological diagnostic methods have recently been developed to detect food poisoning pathogens. Among these diagnostic tools, this study presents PCR-based pulsed-field gel electrophoresis and nucleotide sequencing diagnostic analysis results for diagnosing food poisoning outbreaks associated with cooking employees in Chungcheongnam-do, Republic of Korea. Pulsed-field gel electrophoresis was useful in identifying the food poisoning outbreaks caused by Staphylococcus aureus and Enteropathogenic Escherichia coli. In the case of Norovirus, nucleotide sequencing was used to identify the relationship between cooking workers and the food poisoning outbreak. However, it is difficult to determine whether cooking employees directly caused the food poisoning outbreaks based on these molecular biological diagnostic results alone. A system is needed to integrate epidemiological and diagnostic information to identify a direct correlation between the food poisoning outbreak and cooking employees.

Evaluation of crAssphages as a potential marker of human viral contamination in environmental water and fresh leafy greens.

CrAssphages are human gut bacteriophages with potential use as an indicator of human fecal contamination in water and other environmental systems. We determined the prevalence and abundance of crAssphages in water, food, and fecal samples and compared these estimates with the prevalence of norovirus. Samples were tested using two crAssphage-specific qPCR assays (CPQ056 and TN201-203) and for norovirus using TaqMan realtime RT-PCR. CrAssphage was detected in 40% of human fecal specimens, 61% of irrigation water samples, 58.5% of stream water samples, and 68.5% of fresh leafy greens samples. Interestingly, across all sample categories, crAssphage concentrations were 2-3 log10 higher than norovirus concentrations. The correlation of detection of crAssphage and norovirus was significant for the irrigation water samples (r = 0.74, p = 7.4e-06). Sequences obtained from crAssphage positive samples from human fecal and stream water samples phylogenetically clustered with genotype I crAssphages, whereas sequences derived from irrigation water samples clustered differently from other genotypes. Our data show that crAssphages were prevalent in norovirus-positive water samples and in fresh leafy green samples, there was a strong correlation between the presence of crAssphage and norovirus. CrAssphage genomic copies were consistently higher than norovirus copies in all sample types. Overall, our findings suggest that crAssphages could be used as reliable indicators to monitor fecal-borne virus contamination within the food safety chain.

Scenario-based assessment of fecal pathogen sources affecting bathing water quality: novel treatment options to reduce norovirus and Campylobacter infection risks.

Wastewater discharge and runoff waters are significant sources of human and animal fecal microbes in surface waters. Human-derived fecal contamination of water is generally estimated to pose a greater risk to human health than animal fecal contamination, but animals may serve as reservoirs of zoonotic pathogens. In this study, quantitative microbial risk assessment (QMRA) tools were used to evaluate the hygienic impact of sewage effluents and runoff water from municipalities and animal farms on surface and bathing waters. The human-specific microbial source tracking (MST) marker HF183 was used to evaluate the dilution of fecal pathogens originating from the sewage effluent discharge to the downstream watershed. As novel risk management options, the efficiency of UV-LED disinfection and wetland treatment as well as biochar filtration was tested on-site for the contamination sources. According to the dilution pattern of the MST marker HF183, microbes from wastewater were diluted (2.3-3.7 log10) in the receiving waters. The scenario-based QMRA revealed, that the health risks posed by exposure to human-specific norovirus GII and zoonotic Campylobacter jejuni during the bathing events were evaluated. The risk for gastroenteritis was found to be elevated during wastewater contamination events, where especially norovirus GII infection risk increased (1-15 cases per day among 50 bathers) compared with the business as usual (BAU) situation (1 case per day). The noted C. jejuni infection risk was associated with animal farm contamination (1 case per day, versus 0.2-0.6 cases during BAU). Tertiary treatment of wastewater with wetland treatment and UV-LED disinfection effectively reduced the waterborne gastroenteritis risks associated with bathing. Based on the experiences from this study, a QMRA-based approach for health risk evaluations at bathing sites can be useful and is recommended for bathing site risk assessments in the future. In case of low pathogen numbers at the exposure sites, the MST marker HF183 could be used as a pathogen dilution coefficient for the watershed under evaluation. The full-scale implementation of novel tertiary treatment options at wastewater treatment plants (WWTPs) as well as on-site runoff water treatment options should be considered for infection risk management at locations where scenario-based QMRA implies elevated infection risks.

Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Dual-Mode Virus Detection: Combining Electrochemical and Fluorescence Modalities for Enhanced Sensitivity and Reliability.

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

Human Norovirus Surrogate Is Highly Stable in Berry Smoothies and under In Vitro Simulated Digestion.

Human noroviruses are major causes of foodborne outbreaks linked to berries. The overall goal of this study was to investigate the persistence of a human norovirus surrogate, Tulane virus (TV), in berry smoothies and under simulated digestion through the gastrointestinal track. Two types of smoothies were prepared from blueberries and strawberries. Tulane virus was spiked into each smoothie and incubated either at 37 or 4 °C for 2, 60, and 120 min. Furthermore, the virus-spiked smoothies were subjected to sequential oral (2 min), gastric (10 and 60 min), and intestinal (15 and 120 min) digestion according to the standardized INFOGEST model. Quantification of infectious TV was carried out using the TCID50 assay. At 4 °C, in both berry smoothies, TV infectivity did not show significant changes throughout the 120 min period. At 37 °C, TV infectivity showed significant reduction (~0.5 log TCID50/mL) only in blueberry smoothies starting at 60 min. During the oral, gastric, and intestinal digestion phases, the mean log reduction in TV infectivity in blueberry did not exceed ~0.5 log, while infectious TV in strawberry smoothies under all phases was stable. Given the notable stability of infectious viruses in berry smoothies and the gastrointestinal tract, prevention of norovirus contamination of berries is paramount to reduce virus outbreaks linked to berries.

Antigenic Characterization of Novel Human Norovirus GII.4 Variants San Francisco 2017 and Hong Kong 2019.

Norovirus is a major cause of acute gastroenteritis; GII.4 is the predominant strain in humans. Recently, 2 new GII.4 variants, Hong Kong 2019 and San Francisco 2017, were reported. Characterization using GII.4 monoclonal antibodies and serum demonstrated different antigenic profiles for the new variants compared with historical variants.

An Investigation of the Relationship between Henoch-Schönlein Purpura and Viral Infection in Korea Using the Health Insurance Database.

(1) Background: This study investigated the epidemiology and viral connections of Henoch-Schönlein purpura (HSP) using information from the Korea Disease Control and Prevention Agency and the Health Insurance Review and Assessment database. (2) Method: Between 2016 and 2019, a total of 25,443 patients with HSP were identified, with 51.3% of patients under the age of 20 years and the highest incidence in March. (3) Results: The autoregressive integrated moving average model and Granger causality test were used to analyze the association between the virus positivity detection rate and HSP incidence. (4) Conclusions: The incidence of HSP was associated with rotavirus, bocavirus, parainfluenza virus, and respiratory syncytial virus in individuals under 20 years of age, whereas adenovirus, respiratory syncytial virus, and norovirus were associated with individuals above that age.

Norovirus in children under 2 years of age: an epidemiological study in Panama during the COVID-19 pandemic.

Introduction: Norovirus infection is a common cause of acute gastroenteritis (AGE). Surveillance activities are important to aid investigation into effective norovirus control strategies, including vaccination. Here, we report ancillary findings related to the incidence, prevalence, and etiology of AGE caused by norovirus in Panama after adjustment of study methodology to comply with national coronavirus disease 2019 (COVID-19) mandates. Methods: In January 2020, children aged <2 years began enrolling into an epidemiological study in Panama to estimate the burden of norovirus in preparation for evaluating upcoming prevention strategies. This included an observational, longitudinal, community-based AGE surveillance study and a hospital-based AGE surveillance study. For the longitudinal study, healthy children aged 5-18 months were enrolled from January 6 through March 23, 2020, with a follow-up of approximately 6 months. The last participant was contacted on September 23, 2020. For the hospital-based study, starting on January 21, 2020, children aged <2 years who were admitted to the Hospital del Niño Dr. José Renán Esquivel in Panama City due to AGE were evaluated. The last sample was collected on September 29, 2020. Collected stool samples were tested for norovirus as well as astrovirus, sapovirus, and various enteropathogens. Unfortunately, this study was disrupted by the subsequent implementation of disease transmission control procedures for the COVID-19 pandemic, and the study methodology was revised to comply with COVID-19 mandates. Results: In the longitudinal surveillance cohort [N = 400 (Chiriquí, n = 239; Panama, n = 161)], a total of 185 AGE episodes were documented (Chiriquí, n = 85; Panama, n = 100) resulting in an overall AGE incidence of 11.6 (95% CI: 9.99-13.4) episodes per 100 child-months. The norovirus-related AGE incidence was 0.3 (95% CI: 0.10-0.73) episodes per 100 child-months (5/185 AGE episodes) and the prevalence of norovirus was 4.6% (13/282 stool samples collected). In the hospital-based surveillance cohort, at least one pathogen was detected in 50% of samples (44/88 stool samples collected) and norovirus prevalence was 6.8% (6/88 stool samples collected). Discussion: This report demonstrates how the occurrence of the COVID-19 pandemic hindered the conduct of clinical trials. However, this also created unique research opportunities to investigate the potential impact of pandemic control measures on the etiology of infectious diarrheal disease.

Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a.

Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 102 copies/µl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.

Evaluation of Extraction Methods to Detect Noroviruses in Ready-to-Eat Raw Milk Minas Artisanal Cheese.

This study aimed to assess two homogenization methods to recover norovirus from Minas artisanal cheese (MAC) made with raw bovine milk obtained from four microregions of the Minas Gerais state, Brazil, with different ripening times and geographical and abiotic characteristics. For this purpose, 33 fiscal samples were artificially contaminated with norovirus GI and GII, and Mengovirus (MgV), used as an internal process control (IPC). TRIzol® reagent and Proteinase K homogenization methods were evaluated for all samples were then subjected to RNA extraction using viral magnetic beads and RT-qPCR Taqman® for viral detection/quantification. Proteinase K method showed better efficiency results for both norovirus GI and GII, with means recovery efficiency of 45.7% (95% CI 34.3-57.2%) and 41.4% (95% CI 29.1-53.6%), respectively, when compared to TRIzol method (16.6% GI, 95% CI 8.4-24.9%, and 12.3% GII, 95% CI 7.0-17.6%). The limits of detection for norovirus GI and GII for this method were 101GC/g and 103GC/g, respectively, independent of cheese origin. MgV was detected and revealed in 100% success rate in all types of cheese, with mean recovery efficiency of 25.6% for Proteinase K, and 3.8% for the TRIzol method. According to cheese origin, Triangulo Mineiro MAC had the highest mean recovery rates for the three viral targets surveyed (89% GI, 87% GII, and 51% MgV), while Serro MAC showed the lowest rates (p < 0.001). Those results indicate that the proteinase K adapted method is suitable for norovirus GI and GII detection in MAC and corroborated MgV as an applicable IPC to be used during the process.