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Noroviruses are a leading cause of endemic and epidemic acute gastroenteritis in all age groups. However, in Latin America, there are limited and updated data regarding circulating genotypes. The aim of this study was to assess the prevalence and genetic diversity of norovirus outbreaks in Argentina from 2013 to 2018. Stool samples from 29 acute gastroenteritis (AGE) outbreaks were available for viral testing. Norovirus was detected in samples from 18 (62.1%) outbreaks (2 GI and 16 GII). Both GI outbreaks were typed as GI.6[P11] whereas 10 different GII genotypes were detected, in which GII.4 viruses were the most frequently detected (29.4%, associated with GII.P31 and GII.P16) followed by GII.1[P33] and GII.6[P7] (17.6% each). Like GII.4 viruses, GII.2 viruses were also detected in association with different polymerases (GII.P2 and GII.P16). Our findings underscore the importance of dual RNA-dependent RNA polymerase-VP1 typing since recombinant strains with new polymerase sequences emerge frequently suggesting a possible role in improved fitness of these viruses. This study represents the most recent multi-year assessment of the molecular epidemiology of norovirus strains associated with AGE outbreaks in Argentina. Molecular surveillance of norovirus has to be considered to monitor possible changes in dominant genotypes which may assist to inform the formulation of future vaccines.

Noroviruses are recognized as the leading cause of acute gastroenteritis globally. With improved molecular diagnostics developed over the last two decades, archived clinical specimens are increasingly used to investigate the historic prevalence and molecular epidemiology of human norovirus. Yet the impact of long-term storage on viral integrity in clinical specimens has not been evaluated. In this study, we retested 994 stool specimens collected between 1996 and 2017 that originally tested norovirus-positive to quantify the loss of norovirus RT-PCR positivity with increasing sample storage time at 4 °C. In all, 79% of samples tested positive after retesting, but there was an approximate 3% decline in the positivity ratio and 4% decline in the percentage of samples that could be genotyped with each additional year of sample storage. For samples that were originally quantified by real-time RT-PCR (collected between 2003 and 2017), there was an estimated 1-log loss of viral titer occurring every 7 years of sample storage. Few samples contained PCR inhibitors, assessed using a MS2 extraction control, indicating that loss of RT-PCR signal was due primarily to loss of viral RNA integrity after long-term storage of stool samples at 4 °C. Our results indicate that norovirus positive stool samples can be stored with minimal loss in RT-PCR positivity when stored less than a decade. Longer periods of storage may impair norovirus detection, potentially impacting historic estimates of norovirus prevalence and molecular epidemiology if derived by testing archival clinical specimens.

Background Diarrhea remains the leading cause of childhood illness in China. Better understanding of burden and etiology of diarrheal diseases is important for development of effective prevention measures. Methods Population-based diarrhea surveillance was conducted in Sanjiang (southern China) year-round and Zhengding (northern China) in autumn/winter. Stool specimens were collected from children, Key Points Currently bacterial and viral agents in the gastrointestinal tract are under-detected and poorly defined in developing countries. A new sensitive TAC assay provided comprehensive identification of microorganisms, including neglected pathogens, in Chinese children with moderate and severe diarrhea. Supplementary Information The online version contains supplementary material available at 10.1186/s13099-021-00428-2.

Noroviruses are a leading cause of acute gastroenteritis (AGE) among adults and children worldwide. NoroSurv is a global network for norovirus strain surveillance among children

Background Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. Objective To evaluate RIDA®GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. Study design Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014–2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5′end of the norovirus ORF2 gene. Results A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5–93.5) and 99.1% (98.0–99.6) and for GII was 94.8% (90.8–97.2) and 98.6% (96.9–99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. Conclusions The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE.

Noroviruses are the most frequent cause of epidemic acute gastroenteritis in the United States. Between September 2013 and August 2016, 2,715 genotyped norovirus outbreaks were submitted to CaliciNet. GII.4 Sydney viruses caused 58% of the outbreaks during these years. A GII.4 Sydney virus with a novel GII.P16 polymerase emerged in November 2015, causing 60% of all GII.4 outbreaks in the 2015-2016 season. Several genotypes detected were associated with more than one polymerase type, including GI.3, GII.2, GII.3, GII.4 Sydney, GII.13, and GII.17, four of which harbored GII.P16 polymerases. GII.P16 polymerase sequences associated with GII.2 and GII.4 Sydney viruses were nearly identical, suggesting common ancestry. Other common genotypes, each causing 5 to 17% of outbreaks in a season, included GI.3, GI.5, GII.2, GII.3, GII.6, GII.13, and GII.17 Kawasaki 308. Acquisition of alternative RNA polymerases by recombination is an important mechanism for norovirus evolution and a phenomenon that was shown to occur more frequently than previously recognized in the United States. Continued molecular surveillance of noroviruses, including typing of both polymerase and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.

Norovirus is the leading cause of endemic and epidemic acute gastroenteritis in the United States (1). New variant strains of norovirus GII.4 emerge every 2-4 years (2-4) and are often associated with increased disease and health care visits (5-7). Since 2009, CDC has obtained epidemiologic data on norovirus outbreaks from state health departments through the National Outbreak Reporting System (NORS) (8) and laboratory data through CaliciNet (9). NORS is a web-based platform for reporting waterborne, foodborne, and enteric disease outbreaks of all etiologies, including norovirus, to CDC. CaliciNet, a nationwide electronic surveillance system of local and state public health and regulatory agency laboratories, collects genetic sequences of norovirus strains associated with gastroenteritis outbreaks. Because these two independent reporting systems contain complementary data, integration of NORS and CaliciNet records could provide valuable public health information about norovirus outbreaks. However, reporting lags and inconsistent identification codes in NORS and CaliciNet records have been an obstacle to developing an integrated surveillance system.

Noroviruses are the most frequent cause of epidemic acute gastroenteritis in the United States. Between September 2013 and August 2016, 2,715 genotyped norovirus outbreaks were submitted to CaliciNet. GII.4 Sydney viruses caused 58% of the outbreaks during these years. A GII.4 Sydney virus with a novel GII.P16 polymerase emerged in November 2015, causing 60% of all GII.4 outbreaks in the 2015-2016 season. Several genotypes detected were associated with more than one polymerase type, including GI.3, GII.2, GII.3, GII.4 Sydney, GII.13, and GII.17, four of which harbored GII.P16 polymerases. GII.P16 polymerase sequences associated with GII.2 and GII.4 Sydney viruses were nearly identical, suggesting common ancestry. Other common genotypes, each causing 5 to 17% of outbreaks in a season, included GI.3, GI.5, GII.2, GII.3, GII.6, GII.13, and GII.17 Kawasaki 308. Acquisition of alternative RNA polymerases by recombination is an important mechanism for norovirus evolution and a phenomenon that was shown to occur more frequently than previously recognized in the United States. Continued molecular surveillance of noroviruses, including typing of both polymerase and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.

Background The family Caliciviridae consists of a genetically diverse group of RNA viruses that infect a wide range of host species including noroviruses and sapoviruses which cause acute gastroenteritis in humans. Typing of these viruses relies on sequence-based approaches, and therefore there is a need for rapid and accurate web-based typing tools. Objective To develop and evaluate a web-based tool for rapid and accurate genotyping of noroviruses and sapoviruses. Methods The Human Calicivirus Typing (HuCaT) tool uses a set of curated reference sequences that are compared to query sequences using a k-mer (DNA substring) based algorithm. Outputs include alignments and phylogenetic trees of the 12 top matching reference sequences for each query. Results The HuCaT tool was validated with a set of 1310 norovirus and 239 sapovirus sequences covering all known human norovirus and sapovirus genotypes. HuCaT tool assigned genotypes to all queries with 100 % accuracy and was much faster (17 s) than BLAST (150 s) or phylogenetic analyses approaches. Conclusions The web-based HuCaT tool supports rapid and accurate genotyping of human noroviruses and sapoviruses.

Highlights • We validated genogroup-specific one-step conventional RT-PCR assays (PC assays) for sequence-based dual typing of GI and GII norovirus strains. • The PC assays use a combination of oligonucleotide primers that target a genomic region spanning the 3’-end of ORF1 and 5’end of ORF2 of GI and GII noroviruses. • The PC assays are sensitive (5 to 50 copies/rx) and detect all currently identified norovirus P-types and capsid genotypes from different geographic regions. • The PC assays have been successfully implemented by CaliciNet USA and CaliciNet China., Background Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. Objective Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. Results The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010–2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. Conclusion The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact.

Foodborne transmission accounts for 10% of outbreaks caused by GII.4, 27% by all other single genotypes, and 37% by mixtures of GII.4 and others, Worldwide, noroviruses are a leading cause of gastroenteritis. They can be transmitted from person to person directly or indirectly through contaminated food, water, or environments. To estimate the proportion of foodborne infections caused by noroviruses on a global scale, we used norovirus transmission and genotyping information from multiple international outbreak surveillance systems (Noronet, CaliciNet, EpiSurv) and from a systematic review of peer-reviewed literature. The proportion of outbreaks caused by food was determined by genotype and/or genogroup. Analysis resulted in the following final global profiles: foodborne transmission is attributed to 10% (range 9%%�?"11%) of all genotype GII.4 outbreaks, 27% (25%�?"30%) of outbreaks caused by all other single genotypes, and 37% (24%%�?"52%) of outbreaks caused by mixtures of GII.4 and other noroviruses. When these profiles are applied to global outbreak surveillance data, results indicate that �%^14% of all norovirus outbreaks are attributed to food.

Background Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) is the state-of-the-art diagnostic for norovirus. Cycle threshold (Ct), an indicator of viral load, may be associated with symptomatic disease as well as demographic and outbreak characteristics. Methods Data on (1) outbreak and sporadic cases and (2) asymptomatic controls in the United States and Latin America were analyzed. With multivariate regression models, we assessed relationships between various factors and Ct values, and we calculated odds ratios (ORs) for the presence of symptoms and attributable fractions of norovirus. Receiver-operating characteristic analysis was performed to define an optimal Ct cutoff to identify disease-causing infections. Results Cycle threshold values were lower (ie, higher viral loads) among symptomatic cases (model-adjusted mean ± standard error: 25.3 ± 1.2) compared with asymptomatic controls (28.5 ± 1.4). Cycle threshold values were significantly different across age groups, norovirus genogroups, timing of specimen collection, outbreak settings, and transmission modes. Genogroup II (GII) Ct values were associated with presence of symptoms (OR = 1.1), allowing us to estimate that 16% of diarrheal disease was attributable to norovirus. The optimized Ct cutoff led to poor sensitivity and specificity for genogroup I and GII. Conclusions Cycle threshold values were associated with host, pathogen, and outbreak factors. Cycle threshold values may not effectively distinguish disease-causing infection for individual patients, but they are useful for epidemiological studies aiming to attribute disease.

Noroviruses are the leading cause of acute gastroenteritis outbreaks worldwide. The majority of norovirus outbreaks are caused by genogroup II.4 (GII.4). Novel GII.4 strains emerge every 2 to 4 years and replace older variants as the dominant norovirus. Novel variants emerge through a combination of recombination, genetic drift, and selection driven by population immunity, but the exact mechanism of how or where is not known. We detected two previously unknown novel GII.4 variants, termed GII.4 UNK1 and GII.4 UNK2, and a diverse norovirus population in fecal specimens from immunocompromised individuals with diarrhea after they had undergone bone marrow transplantation. We hypothesized that immunocompromised individuals can serve as reservoirs for novel norovirus variants. To test our hypothesis, metagenomic analysis of viral RNA populations was combined with a full-genome bioinformatic analysis of publicly available GII.4 norovirus sequences from 1974 to 2014 to identify converging sites. Variable sites were proportionally more likely to be within two amino acids ( P < 0.05) of positively selected sites. Further analysis using a hypergeometric distribution indicated that polymorphic site distribution was random and its proximity to positively selected sites was dependent on the size of the norovirus genome and the number of positively selected sites.In conclusion, random mutations may have a positive impact on driving norovirus evolution, and immunocompromised individuals could serve as potential reservoirs for novel GII.4 strains. IMPORTANCE Norovirus is the most common cause of viral gastroenteritis in the United States. Every 2 to 3 years novel norovirus variants emerge and replace dominant strains. The continual emergence of novel noroviruses is believed to be caused by a combination of genetic drift, population immunity, and recombination, but exactly how this emergence occurs remains unknown. In this study, we identified two novel GII.4 variants in immunocompromised bone marrow transplant patients. Using metagenomic and bioinformatic analysis, we showed that most genetic polymorphisms in the novel variants occur near 0 to 2 amino acids of positively selected sites, but the distribution of mutations was random; clustering of polymorphisms with positively selected sites was a result of genome size and number of mutations and positively selected sites. This study shows that immunocompromised patients can harbor infectious novel norovirus variants, and although mutations in viruses are random, they can have a positive effect on viral evolution.

Noroviruses evolve by antigenic drift and recombination, which occurs most frequently at the junction between the non-structural and structural protein coding genomic regions. In 2015, a novel GII.P16-GII.4 Sydney recombinant strain emerged, replacing the predominance of GII.Pe-GII.4 Sydney among US outbreaks. Distinct from GII.P16 polymerases detected since 2010, this novel GII.P16 was subsequently detected among GII.1, GII.2, GII.3, GII.10 and GII.12 viruses, prompting an investigation on the unique characteristics of these viruses. Norovirus positive samples (n = 1807) were dual-typed, of which a subset (n = 124) was sequenced to yield near-complete genomes. CaliciNet and National Outbreak Reporting System (NORS) records were matched to link outbreak characteristics and case outcomes to molecular data and GenBank was mined for contextualization. Recombination with the novel GII.P16 polymerase extended GII.4 Sydney predominance and increased the number of GII.2 outbreaks in the US. Introduction of the novel GII.P16 noroviruses occurred without unique amino acid changes in VP1, more severe case outcomes, or differences in affected population. However, unique changes were found among NS1/2, NS4 and VP2 proteins, which have immune antagonistic functions, and the RdRp. Multiple polymerase-capsid combinations were detected among GII viruses including 11 involving GII.P16. Molecular surveillance of protein sequences from norovirus genomes can inform the functional importance of amino acid changes in emerging recombinant viruses and aid in vaccine and antiviral formulation.

Noroviruses are the leading cause of epidemic acute gastroenteritis in the United States. From September 2009 through August 2013, 3,960 norovirus outbreaks were reported to CaliciNet. Of the 2,895 outbreaks with a known transmission route, person-to-person and food-borne transmissions were reported for 2,425 (83.7%) and 465 (16.1%) of the outbreaks, respectively. A total of 2,475 outbreaks (62.5%) occurred in long-term care facilities (LTCF), 389 (9.8%) in restaurants, and 227 (5.7%) in schools. A total of 435 outbreaks (11%) were typed as genogroup I (GI) and 3,525 (89%) as GII noroviruses. GII.4 viruses caused 2,853 (72%) of all outbreaks, of which 94% typed as either GII.4 New Orleans or GII.4 Sydney. In addition, three non-GII.4 viruses, i.e., GII.12, GII.1, and GI.6, caused 528 (13%) of all outbreaks. Several non-GII.4 genotypes (GI.3, GI.6, GI.7, GII.3, GII.6, and GII.12) were significantly more associated with food-borne transmission (odds ratio, 1.9 to 7.1; P < 0.05). Patients in LTCF and people ≥65 years of age were at higher risk for GII.4 infections than those in other settings and with other genotypes ( P < 0.05). Phylogeographic analysis identified three major dispersions from two geographic locations that were responsible for the GI.6 outbreaks from 2011 to 2013. In conclusion, our data demonstrate the cyclic emergence of new (non-GII.4) norovirus strains, and several genotypes are more often associated with food-borne outbreaks. These surveillance data can be used to improve viral food-borne surveillance and to help guide studies to develop and evaluate targeted prevention methods such as norovirus vaccines, antivirals, and environmental decontamination methods.

We report an increase in the proportion of genotype GI.6 norovirus outbreaks in the United States from 1.4% in 2010 to 7.7% in 2012 (p

Background Norovirus (NoV) is the leading cause of epidemic gastroenteritis worldwide. The lack of a cell culture has significantly hampered the development of effective therapies against human NoV. Clinically approved nucleoside and non-nucleoside analogues have been used successfully against RNA viruses. Methods In this study, we evaluated the efficacy of four nucleoside analogues (2′-C-MeC, 2′-F-2′-C-MeC, β-D-N(4)-hydroxycytidine [NHC] and lamivudine) on Norwalk virus (NV) RNA levels and protein expression in NV replicon-harbouring cells (HG23 cells), and their efficacy in blocking murine norovirus (MNV) replication in RAW 264.7 cells. Results 2′-C-MeC and 2′-F-2′-C-MeC reduced MNV RNA levels and infectivity in RAW 264.7 cells in a concentration- and time-dependent manner. The median effective concentrations (EC50) of 2′-C-MeC and 2′-F-2′-C-MeC were 6.9 μM and 12.7 μM, respectively. 2′-C-MeC, 2′-F-2′-C-MeC and NHC reduced NV RNA levels and protein expression in HG23 cells. For the NV replicon, the EC50 of 2′-C-MeC (1.3 μM) was comparable to the antiviral activity of NHC (1.5 μM) and twofold more potent than 2′-F-2′-C-MeC (3.2 μM). The combination of 2′-C-MeC/ ribavirin resulted in modest synergistic activity, whereas NHC/ribavirin was antagonistic for NV replication in HG23 cells. Conclusions The antiviral activity of 2′-C-MeC against strains of two different NoV genogroups and the low EC50 suggest that this nucleoside analogue may be effective against the more prevalent GII NoVs. In the absence of a vaccine, antiviral agents could be an effective intervention to control the spread of human NoV in populations at a high risk for NoV disease.

To identify the prevalence and genetic variability of noroviruses in dogs, we tested fecal samples by using reverse transcription–PCR. We found canine norovirus in 40% and 9% of dogs with and without diarrhea, respectively. The virus was genetically unrelated to other noroviruses and constitutes a tentative new genogroup.

Alcohol-based hand sanitizers are widely used to disinfect hands to prevent the spread of pathogens including noroviruses. Alcohols inactivate norovirus by destruction of the viral capsid, resulting in the leakage of viral RNA (virolysis). Since conflicting results have been reported on the susceptibility of human noroviruses against alcohols, we exposed a panel of 30 human norovirus strains (14 GI and 16 GII strains) to different concentrations (50%, 70%, 90%) of ethanol and isopropanol and tested the viral RNA titer by RT-qPCR. Viral RNA titers of 10 (71.4%), 14 (100%), 3 (21.4%) and 7 (50%) of the 14 GI strains were reduced by > 1 log10 RNA copies/ml after exposure to 70% and 90% ethanol, and 70% and 90% isopropanol, respectively. RNA titers of 6 of the 7 non-GII 4 strains remained unaffected after alcohol exposure. Compared to GII strains, GI strains were more susceptible to ethanol than to isopropanol. At 90%, both alcohols reduced RNA titers of 8 of the 9 GII.4 strains by ≥ 1 log10 RNA copies/ml. After exposure to 70% ethanol, RNA titers of GII.4 Den Haag and Sydney strains decreased by ≥ 1.9 log10, whereas RNA reductions for GII.4 New Orleans strains were < 0.5 log10. To explain these differences, we sequenced the complete capsid gene of the 9 GII.4 strains and identified 17 amino acid substitutions in the P2 region among the 3 GII.4 variant viruses. When comparing with an additional set of 200 GII.4 VP1 sequences, only S310 and P396 were present in all GII.4 New Orleans viruses but not in the ethanol-sensitive GII.4 Sydney and GII.4 Den Haag viruses Our data demonstrate that alcohol susceptibility patterns between different norovirus genotypes vary widely and that virolysis data for a single strain or genotype are not representative for all noroviruses.

TOC Summary: The launch of CaliciNet in March 2009 was a milestone., CaliciNet, the outbreak surveillance network for noroviruses in the United States, was launched in March 2009. As of January 2011, twenty state and local health laboratories had been certified to submit norovirus sequences and epidemiologic outbreak data to CaliciNet. During the network’s first year, 552 outbreaks were submitted to CaliciNet, of which 78 (14%) were associated with foodborne transmission. A total of 395 (72%) outbreaks were typed as GII.4, of which 298 (75%) belonged to a new variant, GII.4 New Orleans, which first emerged in October 2009. Analysis of the complete capsid and P2 region sequences confirmed that GII.4 New Orleans is distinct from previous GII.4 variants, including GII.4 Minerva (2006b).