Norovirus GII Research Articles

Multiple Novel Human Norovirus Recombinants Identified in Wastewater in Pretoria, South Africa by Next-Generation Sequencing.

The genogroup II genotype 4 (GII.4) noroviruses are a major cause of viral gastroenteritis. Since the emergence of the Sydney_2012 variant, no novel norovirus GII.4 variants have been reported. The high diversity of noroviruses and periodic emergence of novel strains necessitates continuous global surveillance. The aim of this study was to assess the diversity of noroviruses in selected wastewater samples from Pretoria, South Africa (SA) using amplicon-based next-generation sequencing (NGS). Between June 2018 and August 2020, 200 raw sewage and final effluent samples were collected fortnightly from two wastewater treatment plants in Pretoria. Viruses were recovered using skimmed milk flocculation and glass wool adsorption-elution virus recovery methods and screened for noroviruses using a one-step real-time reverse-transcription PCR (RT-PCR). The norovirus BC genotyping region (570-579 bp) was amplified from detected norovirus strains and subjected to Illumina MiSeq NGS. Noroviruses were detected in 81% (162/200) of samples. The majority (89%, 89/100) of raw sewage samples were positive for at least one norovirus, compared with 73% (73/100) of final effluent samples. Overall, a total of 89 different GI and GII RdRp-capsid combinations were identified, including 51 putative novel recombinants, 34 previously reported RdRp-capsid combinations, one emerging novel recombinant and three Sanger-sequencing confirmed novel recombinants.

Clinical and virological characteristics of viral shedding in children with norovirus gastroenteritis.

The correlation between the clinical manifestations and fecal viral load of norovirus (NoV) infection remains unknown. We established a SYBR® Green-based real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) method to quantify NoV and then sequenced its genomes from the feces of patients admitted at the Chang Gung Memorial Hospital from 2017 to 2018. NoV GII.4 Sydney (n=21, 36.2%) and GII.P16-GII.2 (n=19, 32.8%), the two predominant genotypes found among 58 isolates, were closely related to the Taiwan variant 2012a cluster in the VP1 region and genotypes of China strain. An increase in viral load could be observed on Day 3 following the onset of NoV infection. The viral load then declined rapidly from days 10-15 but remained high for >1 month in a severe combined immunodeficiency patient. Significantly longer shedding was found in patients with fever (p=0.03) or infected by the GII.4 Sydney strain (p<0.01). The qRT-PCR-mediated method proposed in this work could quantify the viral load in patients with NoV infection. Significant viral shedding over a period of 2 weeks in children with acute gastroenteritis and >1 month in an immunodeficient patient was observed. Significantly longer shedding could be correlated with infection by the GII.4 Sydney strain and febrile patients.

The emergence of recombinant norovirus GII.12[P16] and predominance of GII.3[P12] strains in pediatric patients with acute gastroenteritis in Thailand, 2019-2020.

Norovirus (NoV) and sapovirus (SaV) are important pathogens that cause acute gastroenteritis (AGE) in all age groups, commonly in children worldwide. Recently, a number of studies have reported a wide variety of NoV recombinant strains. This study aimed to investigate the distribution of NoV and SaV recombinant strains circulating in Chiang Mai, Thailand, during 2019-2020. One hundred and twenty-four NoV and seven SaV strains detected in children admitted to the hospital with AGE were included in this study. The partialRNA-dependent RNA-polymerase(RdRp)/VP1 regions of these NoV and SaV strains were analyzed by phylogenetic analysis, Simplot, and RDP software. Overall, eight recombination patterns of NoV were detected. NoV GII.4[P16] was the most common strain detected (39.1%), followed by GII.3[P12] (25.0%), GII.4[P31] (17.2%), and other recombinant strains were detected at a lower rate. NoV GII.12[P16] strains were detected for the first time in Thailand. For SaV, none of the recombinant strains was detected. All SaV strains, GI.1/GI.1, GI.2/GI.2, and GII.5/GII.5, exhibited VP1 genotype corresponded to RdRp genotype. In conclusion, this study demonstrates the distribution and diversity of NoV and SaV recombinant strains circulating in pediatric patients with AGE in Chiang Mai, during 2019-2020 with the emergence of NoV GII.3[P12] and GII.12[P16].

Prevalence of foodborne and zoonotic viral pathogens in raw cow milk samples.

Foodborne and zoonotic viral pathogens are responsible for substantial morbidity and mortality worldwide. These viruses can be transmitted through foods such as dairy products to humans and cause several acute and chronic diseases. This study aimed to investigate the prevalence and profile of different foodborne and zoonotic viruses in raw cow milk samples. We collected 492 raw cow milk samples from local dairy markets in Qazvin, Iran. Then we evaluated the presence of hepatitis A virus, noroviruses, rotavirus, astrovirus, bovine leukaemia virus (BLV) and tick-borne encephalitis virus (TBEV) in samples using conventional and nested reverse transcription-polymerase chain reaction methods. We found that 34.95, 7.72, 25.81, 14.63, 66.86, 12.80 and 21.34% of raw milk samples were contaminated with norovirus GI, norovirus GII, hepatitis A virus, rotavirus, astrovirus, BLV and TBEV viruses, respectively. Interestingly, the samples collected from the city's south area revealed a higher prevalence of foodborne and zoonotic viruses. Astrovirus and its combination with norovirus GI were the most prevalent virus profiles. Also, the highest correlations were observed among the presence of rotavirus and hepatitis A viruses (0.36) and TBEV and norovirus GII (0.31). Considering the prevalence rate and virus profiles of different foodborne and zoonotic viruses in raw milk samples, hygiene practices and the pasteurization process are strongly suggested to be conducted throughout the cow milk production chain and in dairy industries to prevent infections with these pathogens.

Lack of molecular evidence of fecal-borne viruses in capybaras from São Paulo state, Brazil, 2018-2020: a minor public health issue.

Capybara (Hydrochoerus hydrochaeris) is the world's largest rodent species distributed throughout South America. These animals are incredibly tolerant to anthropogenic environments and are occupying large urban centers. Capybaras are known to carry potentially zoonotic agents, including R. rickettsia, Leishmania spp., Leptospira spp., Trypanosoma spp., Salmonella spp., Toxoplasma gondii, and rabies virus. Focusing on the importance of monitoring potential sources of emerging zoonotic viruses and new viral reservoirs, the aim of the present study was to assess the presence of fecal-borne viruses in the feces of capybaras living in urban parks in São Paulo state, Brazil. A total of 337 fecal samples were collected between 2018 and 2020 and screened for the following: (i) Rotavirus group A (RVA) by ELISA; (ii) non-RVA species and Picobirnavirus (PBV) using PAGE; (iii) Human Bocaparvovirus (HBoV), Bufavirus (BuV), Tusavirus (TuV), and Cutavirus (CuV) qPCR; (iv) Human Enterovirus (EV), Norovirus GII (NoV), and Hantavirus by in houses RT-qPCR; (v) SARS-CoV-2 via commercial RT-qPCR kit assay; and (vi) Astrovirus (AstV) and Adenovirus (AdV) using conventional nested (RT)-PCRs. All fecal samples tested were negative for fecal-borne viruses. This study adds further evidence that the fecal-borne viruses is a minor public health issue in Brazilian capybaras, at least during the surveillance period and surveyed areas. Continuous monitoring of sylvatic animals is essential to prevent and control the emergence or re-emergence of newly discovered virus as well as viruses with known zoonotic potential.

Investigation of genotype diversity of 7,804 norovirus sequences in humans and animals of China.

Norovirus is a prominent enteric virus responsible for severe acute gastroenteritis disease burden worldwide. In our current study, we analyzed 7,804 norovirus sequences of human and animals in China which were detected from 1980 to 2020 from GenBank. The GenBank database was searched up to May 2021 with the following search terms: "norovirus" or "norwalk virus" and "China." The 7,804 norovirus sequences were collected and evaluated by phylogenetic analysis using MEGA X software package. The online typing tool (https://www.rivm.nl/mpf/typingtool/norovirus/) was used to confirm the genotypes. There were 36 norovirus genotypes prevailing in China. GII.4 was the most prevalent genotype, and GII.2, GII.3 and GII.17 also emerged during different time periods. Most sequences were detected in East China (41.72%, 3,256/7,804), but different norovirus genotypes were distributed widely across the country. A variety of norovirus genotypes, including GI, GII, GIII, GIV, GV, GVI, GVII and GX, were reported in different animals. Furthermore, a GI.3 sequence detected from animal had high identity with norovirus detected in human from the same region, indicating the potential norovirus zoonotic transmission in China. In conclusion, these results indicated that norovirus sequences with considerable genetic diversity distributed widely in China, with potential reverse zoonotic transmission from human to animals.

Qualitative and quantitative analysis of wastewater viral contamination as a control element for the circulation of enteric viruses in the Republic of Belarus

Wastewater epidemiology is a new field of research that has great potential in the system of enteric virus surveillance. The purpose of the presented study was to analyze the results of wastewater monitoring of enteric viruses in 2020– 2021 in the Republic of Belarus. Detection and quantification of nucleic acids of rotaviruses A, noroviruses 1 and 2, enteroviruses and adenoviruses F were carried out using a quantitative real time RT-PCR followed by nucleic acid sequence genotyping. In 2020–2021, 487 (46.9 %) of 1038 wastewater samples contained nucleic acid of one or more viruses. The results indicated that there was a decrease in the circulating activity of the most enteric viruses in 2021 compared to 2020, likely due to stricter hygiene practices during the COVID-19 pandemic. Predominance of adenoviruses F in wastewater could be associated with their wide asymptomatic spread, on the one hand, and with greater resistance of pathogen DNA in the external environment, on the other. The quantitative analysis showed that the highest viral nucleic acid concentrations were found in hospital wastewater samples. According to the results of molecular typing, wastewater contained norovirus GII.4 [P16], adenovirus type 41, Coxsackievirus B3, B5 and Echovirus 13, the most common genotypes of enteric viruses in human population. Quantitative analysis and genotyping of nucleic acids of viruses from wastewater can be a source of valuable information for tracking their circulation in the human population to assess the current epidemiological situation and to predict its development in the short term.

Use of bentonite-coated activated carbon for improving the sensitivity of RT-qPCR detection of norovirus from vegetables and fruits: The ISO 15216-1:2017 standard method extension

Produce-related foodborne outbreaks are becoming increasingly prevalent worldwide. In plant tissues, various compounds, including polysaccharides, phenolic compounds, and chlorophyll, can inhibit RT-PCR detection of viruses. In this study, we developed a highly sensitive RT-qPCR in combination with the bentonite-coated activated carbon (BCAC) assay for detection of norovirus from fruits and vegetables, which could be completed within 7 h and was about 10–100 fold more sensitive than the standard procedures (ISO 15216-1:2017). The extraction efficiencies of three surrogate viruses (MS2, MNV-1, and TV) from five fresh produce (lettuce, cherry tomato, blueberry, strawberry, and spinach) were higher with BCAC treatment than those of control groups, ranging from 17.82% to 98.60%. The average detection limit of these viruses using the BCAC-RT-qPCR method was stable at an average of 102 PFU/g or GC/g. Finally, this BCAC-RT-qPCR method was applied for detection of human norovirus GII.4 spiked onto lettuce and cherry tomato. The viral extraction efficiencies were up to 53.43% and 95.56%, respectively, which is almost four and seven times better than those without BCAC. Therefore, the BCAC-RT-qPCR method can be used to detect low levels of foodborne viruses from produce.

Functional and structural characterization of Norovirus GII.6 in recognizing histo-blood group antigens

Noroviruses (NoVs) are the primary cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) are receptors or attachment factors that affect the prevalence and host susceptibility of NoVs. GII.6 NoV is one of the predominant genotypes in humans, which recognizes the type ABO secretor of HBGAs. However, the structural basis of GII.6 NoV's interaction with HBGAs receptors remains elusive. In this study, we investigated the binding features of the GII.6 strain to HBGAs using saliva- and glycan-ELISA assays and characterized the molecular basis of the GII.6 virus that recognizes H disaccharide. We showed that the GII.6 ​P domain recognized some A and O secretor's saliva samples, most B secretor's saliva samples, and H disaccharide antigen, but did not bind non-secretors’ saliva. Further, we determined the crystal structures of GII.6 and its complex with H disaccharides at 1.7 ​Å, revealing that the P domain of GII.6 shares the conventional binding interface and mode of GII HBGAs. Single residue mutations at the GII.6-H binding sites could inhibit the binding of GII.6 to HBGAs, demonstrating that the interaction residues were crucial in maintaining NoV-glycan integrity. Finally, structural and sequence analyses showed that the major residues of the GII.6-H interaction were conserved among NoVs in the GII genogroup. Taken together, our study characterized the functional and structural features of GII.6 that allow it to interact with HBGAs, and shed light on NoV evolution, epidemiology, and anti-viral drug development.

Impact of COVID-19 Nonpharmaceutical Interventions on the Extent of Norovirus Contamination in Oyster Production Areas in Ireland during Winter 2020 to 2021

A significant decrease in norovirus prevalence and concentration was observed in oyster production areas in Ireland during winter 2020 to 2021. Oyster production areas impacted by human wastewater discharges that had been undergoing norovirus surveillance since 2018 were investigated. Samples collected in the winter seasons of 2018 to 2019 and 2019 to 2020, prior to when the COVID-19 pandemic interventions were applied, showed a prevalence of 94.3 and 96.6%, respectively, and geometric mean concentrations of 533 and 323 genome copies per g, respectively. These values decreased significantly during the winter of 2020 to 2021 (prevalence of 63.2% and geometric concentration of below the limit of quantification), coinciding with the control measures to mitigate the transmission of severe acute respiratory syndrome coronavirus 2 of the genus Betacoronavirus. Divergence between norovirus GI and GII prevalence and concentrations was observed over the 3-year monitoring period. Norovirus GII was the dominant genogroup detected in winter 2020 to 2021, with over half of samples positive, although concentrations detected were significantly lower than prepandemic winters, with a geometric mean concentration of below the limit of quantification.

High pressure treatment and green tea extract synergistically control enteric virus contamination in beverages

Consumers are driving food production toward the use of natural preservatives and minimal processing technologies. Green tea extract (GTE) at low concentration could be combined with high pressure processing (HPP) for reduced treatment times and quality impact on foods in a hurdle concept for synergistic effects on foodborne viral pathogens, specifically human norovirus and hepatitis A virus (HAV). Viral inactivation by HPP (at 300, 400, and 500 MPa for 5 min) combined with 3.3 mg/mL aged-GTE was initially evaluated in buffer (PBS) against murine norovirus (MNV), a culturable human norovirus surrogate, and HAV. Furthermore, human norovirus inactivation was evaluated by the novel human intestinal enteroid system (HIE) and a capsid integrity binding assay (ISC-RT-qPCR). HPP treatment completely inhibits human norovirus GII.4 infectivity when applied at 500 MPa alone and at 400 MPa combined with aged-GTE. Additional experiments investigated the reduction of MNV and HAV infectivity in apple and horchata juices exposed to combined aged-GTE and HPP treatments. Results demonstrated that the addition of aged-GTE to the juices exposed to HPP significantly inactivated MNV and HAV at reduced holding pressure time. This synergistic effect of aged-GTE combined with HPP treatments represents a hurdle technology that could be exploited as a control measure to improve the food safety of beverages.

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay

BackgroundIntegrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens.MethodsRT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored.ResultsThe results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/μL within approximately 30–40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement.ConclusionsTogether, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

Application of a high-throughput quantitative PCR system for simultaneous monitoring of SARS-CoV-2 variants and other pathogenic viruses in wastewater

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuously emerging, highlighting the importance of regular surveillance of SARS-CoV-2 and other epidemiologically significant pathogenic viruses in the current context. Reverse transcription-quantitative PCR (RT-qPCR) is expensive, time-consuming, labor-intensive, requires a large reagent volume, and only tests a few targets in a single run. High-throughput qPCR (HT-qPCR) utilizing the Biomark HD system (Fluidigm) can be used as an alternative. This study applied an HT-qPCR to simultaneously detect SARS-CoV-2, SARS-CoV-2 nucleotide substituted RNA, and other pathogenic viruses in wastewater. Wastewater samples were collected from the coronavirus disease 2019 (COVID-19) quarantine facility between October 2020 and February 2021 (n = 4) and from the combined and separated sewer lines of a wastewater treatment plant (WWTP) in Yokkaichi, Mie Prefecture, Japan, between March and August 2021 (n = 23 each). The samples were analyzed by HT-qPCR using five SARS-CoV-2, nine SARS-CoV-2 spike gene nucleotide substitution-specific, five pathogenic viruses, and three process control assays. All samples from the quarantine facility tested positive for SARS-CoV-2 and the nucleotide substitutions N501Y and S69-70 del (Alpha variant) were detected in the December 2020 sample, coinciding with the first clinical case in Japan. Only three WWTP samples were positive when tested with a single SARS-CoV-2 assay, whereas more than eight samples were positive when tested with all assays, indicating that using multiple assays increases the likelihood of detection. The nucleotide substitution L452R (Delta variant) was detected in the WWTP samples of Mie Prefecture in April 2021, but the detection of Delta variant from patients had not been reported until May 2021. Aichi virus 1 and norovirus GII were prevalent in WWTP samples. This study demonstrated that HT-qPCR may be the most time- and cost-efficient method for tracking COVID-19 and broadly monitoring community health.

Development and Characterization of Synthetic Norovirus RNA for Use in Molecular Detection Methods.

Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials. Norovirus GI and GII RNA sequences including the ORF1-ORF2 junction region were designed based on 1,495 reported norovirus sequences and synthesized via plasmid preparation and in vitro transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial norovirus detection kits used in Korea and subjected to homogeneity and stability analyses. A multicenter study involving five laboratories and using four commercial real-time PCR norovirus detection assays was conducted for synthetic norovirus RNA characterization and uncertainty measurements. The synthetic norovirus GI and GII RNAs were positively detected using the six commercial norovirus detection kits and were homogeneous and stable for one year when stored at -20°C or -70°C. All data from the five laboratories were within a range of 1.0 log copies/μL difference for each RNA, and the overall mean concentrations for norovirus GI and GII RNAs were 7.90 log copies/μL and 6.96 log copies/μL, respectively. The synthetic norovirus GI and GII RNAs are adequate for quality control based on commercial molecular detection reagents for noroviruses with high sequence variability. The synthetic RNAs can be used as reference materials in norovirus molecular detection methods.

Human rotavirus VP4 and VP7 genetic diversity and detection of GII norovirus in Ibadan as Nigeria introduces rotavirus vaccine.

ObjectiveThis cross-sectional study investigated the circulating strains of rotavirus and screened for noravirus in Ibadan, Nigeria as the country introduces the rotavirus vaccine into its national immunization program.MethodsSixty-five stool samples were collected from children younger than 5 years with clinically diagnosed diarrhea and screened for the presence of rotavirus and norovirus using RT-PCR. Rotavirus-positive samples were further analyzed to determine the G and P genotypes using semi-nested multiplex PCR.ResultsThe rates of rotavirus and norovirus positivity were 30.8% and 10.8%, respectively, whereas the rate of rotavirus and norovirus mixed infection was 4.6%. G1 was the predominant VP7 genotype, followed by G2, G9, and G1G2G9, whereas the predominant VP4 genotype was P[4], followed by P[6], P[8], and P[9]. The mixed P types P[4]P[8] and P[4]P[6] were also detected. G1P[4] was the most common VP4 and VP7 combination, followed by G2P[4], G1[P6], G1P[8], G2P[6], G2P[9], G9P[6], G2G9P[4], G2P[4]P[6], G1P[4]P[8], G2G9P[8], G1G2G9P[8], and G1[non-typable] P[non-typable], which were detected in at least 5% of the samples. Four samples had a combination of non-typable G and P types.ConclusionsIt is essential to monitor the circulation of virus strains prior to and during the implementation of the immunization program.

Impact of COVID-19 Social Distancing Mandates on Gastrointestinal Pathogen Positivity: Secondary Data Analysis.

BackgroundAcute gastrointestinal (GI) illnesses are of the most common problems evaluated by physicians and some of the most preventable. There is evidence of GI pathogen transmission when people are in close contact. The COVID-19 pandemic led to the sudden implementation of widespread social distancing measures in the United States. There is strong evidence that social distancing measures impact the spread of SARS-CoV-2, and a growing body of research indicates that these measures also decrease the transmission of other respiratory pathogens.ObjectiveThis study aims to investigate the impact of COVID-19 social distancing mandates on the GI pathogen positivity rates.MethodsDeidentified GI Panel polymerase chain reaction test results from a routinely collected diagnostic database from January 1, 2019, through August 31, 2020, were analyzed for the GI pathogen positivity percentage. An interrupted time series analysis was performed, using social distancing mandate issue dates as the intervention date. The following 3 target organisms were chosen for the final analysis to represent different primary transmission routes: adenovirus F40 and 41, norovirus GI and GII, and Escherichia coli O157.ResultsIn total, 84,223 test results from 9 states were included in the final data set. With the exception of E coli O157 in Kansas, Michigan, and Nebraska, we observed an immediate decrease in positivity percentage during the week of social distancing mandates for all other targets and states. Norovirus GI and GII showed the most notable drop in positivity, whereas E coli O157 appeared to be least impacted by social distancing mandates. Although we acknowledge the analysis has a multiple testing problem, the majority of our significant results showed significance even below the .01 level.ConclusionsThis study aimed to investigate the impact of social distancing mandates for COVID-19 on GI pathogen positivity, and we discovered that social distancing measures in fact decreased GI pathogen positivity initially. The use of similar measures may prove useful in GI pathogen outbreaks. The use of a unique diagnostic database in this study exhibits the potential for its use as a public health surveillance tool.

Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberries

Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15–145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.

Molecular epidemiology and characterization of norovirus and sapovirus in pediatric patients with acute diarrhea in Thailand, 2019–2020

BackgroundHuman enteric pathogens in the family Caliciviridae including norovirus (NoV) and sapovirus (SaV) are associated with acute diarrheal disease globally and are considered as one of the viruses with high genetic diversity. MethodsIn order to investigate the epidemiology of NoV and SaV in pediatric patients with acute diarrhea in Chiang Mai, Thailand from January 2019 to December 2020, a total of 675 stool specimens were collected and examined for the presence of NoV and SaV by RT-multiplex PCR. Results126 (18.7 %) and 6 (0.9 %) stool samples were positive for NoV and SaV, respectively. Mixed infection of NoV and SaV was detected in one patient (0.2 %). Among 10 different NoV strains detected in this study, NoV genogroup II genotype 4 (GII.4) Sydney 2012 was the most predominant genotype (51.2 %) followed by GII.3, GII.2, GII.6, GII.12, GII.7, GII.17, GI.4, GII.14, and GI.3. Interestingly, monthly distribution of NoV genotypes revealed that NoV GII.3 increased dramatically in August 2019, suggesting an outbreak of NoV GII.3 might occur in the community. In addition, 3 genotypes of SaV were detected in this study with SaV GI.1 being the most common genotype (71.4 %) followed by GI.2 and GII.5 (each at 14.3 %). ConclusionsThis study demonstrates the prevalence and genetic diversity of NoV and SaV circulating in pediatric patients with acute gastroenteritis in Chiang Mai, Thailand during 2019–2020 and shows an emergence of NoV GII.3 infection in 2019.

Fucoidan from Fucus vesiculosus can inhibit human norovirus replication by enhancing the host innate immune response

• IFN signaling-related genes and ISGs upregulated in hNoV infected zebrafish larvae. • Fucoidan inhibited hNoV replication in zebrafish larvae. • Fucoidan enhanced the innate immune response of zebrafish larvae. Fucoidan extracted from Fucus vesiculosus was able to inhibit the replication of human noroviruses (hNoV) GII.4[P16] in zebrafish ( Danio rerio ) larvae. In the hNoV infected zebrafish larvae, a transcriptomic analysis showed significant upregulation (p < 0.01) of the interferons (IFNs) signaling related genes as well as a series of IFN-stimulated genes (ISGs) encoding antiviral effectors compared to the mock-infected controls. Accordingly, injection of fucoidan together with hNoV resulted in significant upregulation of 834 genes (p < 0.01) and the innate immune system was calculated as the top cluster with 94 gene counts involved and a log 10 (p) value of −25.41. Tulane virus (TV), a very commonly used surrogate for hNoV, served as a complementary tool showing that the fucoidan was not able to block the virions irreversibly. Enhancement of the host innate immune response could be one of the important mechanisms behind the anti-hNoV effect of fucoidan.

Norovirus GI and GII and hepatitis A virus in berries and pomegranate arils in Canada

Between 2016 and 2021, the Canadian Food Inspection Agency (CFIA) collected 4218 samples of fresh and frozen berries (blackberries, blueberries, raspberries, strawberries and mixed berries) and pomegranate arils at retail across 11 major cities in Canada and tested these samples for the presence of norovirus GI, norovirus GII and hepatitis A virus RNA. The purpose of this testing was to provide information on the prevalence of these viruses in berries and pomegranate arils on the Canadian marketplace. Of the 926 fresh fruit samples tested, norovirus GI RNA was detected in one raspberry sample and norovirus GII RNA was detected in one strawberry sample. Of the 3292 frozen fruit samples tested, norovirus GI RNA was detected in one blackberry sample, one raspberry sample and one strawberry sample, and norovirus GII RNA was detected in one blueberry sample, three raspberry samples, four strawberry samples, one pomegranate arils sample and one mixed berry sample. None of the fresh or frozen fruit samples tested positive for hepatitis A virus RNA. No statistically significant associations were observed between the prevalence of viral RNA in samples of fresh and frozen fruit, between the prevalence of viral RNA in samples of domestic and imported fruit or between the prevalence of viral RNA in samples of specific fruit types. Overall, the prevalence of norovirus GI and GII RNA together in fresh and frozen fruit samples in Canada was 0.36 %. The results of this study may be used to refine surveillance programs for norovirus and hepatitis A virus in fresh and frozen berries and pomegranate arils, e.g. by adapting the commodities tested and/or the numbers of planned samples to better target these hazards. This information may also be used to inform other Government of Canada approaches to better understand the controls associated norovirus and hepatitis A virus in fresh and frozen berries and pomegranate arils.