GI) And II Research Articles

Piloting wastewater-based surveillance of norovirus in England

Wastewater-based epidemiology (WBE) gained widespread use as a tool for supporting clinical disease surveillance during the COVID-19 pandemic. There is now significant interest in the continued development of WBE for other pathogens of clinical significance. In this study, approximately 3,200 samples of wastewater from across England, previously collected for quantification of SARS-CoV-2, were re-analysed for the quantification of norovirus genogroup I (GI) and II (GII). Overall, GI and GII were detected in 93% and 98% of samples respectively, and at least one of the genogroups was detected in 99% of samples. GI was found at significantly lower concentrations than GII, but the proportion of each genogroup varied over time, with GI becoming more prevalent than GII in some areas towards the end of the study period (May 2021 – March 2022). Using relative strength indices (RSI), it was possible to study the trends of each genogroup, and total norovirus over time. Increases in norovirus levels appeared to coincide with the removal of COVID-19 related lockdown restrictions within England. Local Moran's I analyses indicated several localised outbreaks of both GI and GII across England, notably the possible GI outbreak in the north of England in early 2022. Comparisons of national average norovirus concentrations in wastewater against concomitant norovirus reported case numbers showed a significant linear relationship. This highlights the potential for wastewater-based monitoring of norovirus as a valuable approach to support surveillance of norovirus in communities.

Point-of-Care Testing for Norovirus Typing Using CRISPR/Cas12a Combined with Reverse Transcription Recombinase Polymerase Amplification.

Noroviruses (NoVs) are one of the leading causes of acute gastroenteritis in humans. This study combined reverse transcription recombinase polymerase amplification (RT-RPA) with a clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) nucleic acid detection system to develop a point-of-care testing (POCT) technology for typing NoVs. The detection can be completed within 35 min at 37 °C, covering each genotype of genogroup I (GI) and II (GII) NoVs. The sensitivity of this method is 10 copies/μL for GI and 1 copy/μL for GII NoV plasmids. For the detection of clinical samples, the detection results of this method for NoV infected samples are consistent with the RT-qPCR detection method in the laboratory, and this detection method has no cross-reactivity with rotavirus and adenovirus. Therefore, the detection method established in this study enables the diagnosis and screening of suspected patients and close contacts by POCT, which is important for the timely identification and control of NoV outbreaks. In addition, the typing detection of GI and GII NoVs can achieve a precise diagnosis and treatment of patients infected with NoVs.

Norovirus Genetic Diversity in Children under Five Years Old with Acute Diarrhea in Mozambique (2014-2015).

Norovirus (NoV) is the second most important cause of viral diarrheal disease in children worldwide after rotavirus and is estimated to be responsible for 17% of acute diarrhea in low-income countries. This study aimed to identify and report NoV genotypes in Mozambican children under the age of five years with acute diarrhea. Between May 2014 and December 2015, stool specimens were collected within the Mozambique Diarrhea National Surveillance (ViNaDia) and tested for NoV genogroups I (GI) and II (GII) using conventional reverse transcriptase-polymerase chain reaction (RT-PCR). Partial capsid and RNA-dependent RNA polymerase (RdRp) nucleotide sequences were aligned using the Muscle tool, and phylogenetic analyses were performed using MEGA X. A total of 204 stool specimens were tested for NoV. The detection rate of NoV was 14.2% (29/204). The presence of NoV was confirmed, by real-time RT-PCR (RT-qPCR), in 24/29 (82.8%) specimens, and NoV GII predominated (70.8%; 17/24). NoV GII.4 Sydney 2012[P31] was the predominant genotype/P-type combination detected (30.4%; 7/23). This is the first study which highlights the high genetic diversity of NoV in Mozambican children and the need to establish a continuous NoV surveillance system.

Occurrence of enteric viruses and faecal indicators in submarine groundwater discharges in the coastal environment of the Mexican Caribbean

AbstractThe occurrence of faecal microorganisms such as total coliforms (TCs), Escherichia coli (E. coli), coliphages (somatic and F+ specific) and the genomes of faecal viruses: human adenovirus (HAdV), norovirus (NoV) genogroup I (GI) and II (GII), enterovirus (EV), aichi virus (AiV) and Pepper mild mottle virus (PMMoV), was explored in submarine groundwater discharges (SGDs) in the Mexican Caribbean. Ultra‐filtrated water samples were subjected to nucleic acid extraction and real‐time quantitative polymerase chain reaction (PCR). Results showed the presence of TC bacteria at all sites, whereas the detection of E. coli occurred only during the dry season. Coliphages occurred during the rainy and dry seasons, with concentrations ranging from 10 to 2560 plaque forming units (PFU/100 ml). HAdV and AiV were detected in 50% and 41% of the samples, at concentrations ranging from 102 to 104 genome copies per litre (GC/L) and from 102 to 106 GC/L, respectively. NoV GI and II were detected in 25% and 16% of the samples, at concentrations of 101 GC/L and 102–103 GC/L, respectively. PMMoV was detected in 50% of the samples at concentrations ranging from 101 to 103 GC/L. The EV genomes were not detected. These findings demonstrate that faecal microorganisms can be transported through SGDs in the Mexican Caribbean, potentially contributing to human health risks for recreation.

Occurrence of Norovirus, Rotavirus, Hepatitis a Virus, and Enterovirus in Berries in Argentina

Berries have been implicated as the probable vehicle of infection in multiple outbreaks of norovirus and hepatitis A virus (HAV). These foods often receive minimal or no processing and may be exposed to virus contamination at each stage of production. In an increasingly globalized world, berries have a wide distribution and can give rise to the spread of diseases in distant parts of the world. With the aim of describing the virological quality of the berries cultivated in Argentina, a total of 184 soft fruits of different varieties (strawberries, blueberries, raspberries, blackberries, currants, pomegranate arils, cassis, and elder) were collected during the periods 2016-2018 and 2020. Viral particles were eluted and concentrated by polyethylene glycol precipitation according to ISO 15216-2:2019 guidelines. Genome detection of norovirus (NoV) genogroups I (GI) and II (GII), HAV, rotavirus, and enterovirus was performed by real-time RT-PCR with TaqMan probes. Positive samples were amplified by conventional RT-PCR and the amplicons were purified and sequenced in both directions. Phylogenetic analysis was performed using the Neighbor-Joining method based on the evolutionary model Kimura-2-parameters. NoV GII.6 was detected in 1/184 (0.5%) of the soft fruits, corresponding to a raspberry sample obtained during the fall of 2017. No presence of other human enteric viruses was found in the other berries analyzed. The collected data are the first in Argentina in relation to the prevalence of enteric viruses in berries and is useful as reference data for a risk assessment of soft fruits as vehicles of foodborne pathogenic viruses.

Molecular Epidemiology and Characterization of Picobirnavirus in Wild Deer and Cattle from Australia: Evidence of Genogroup I and II in the Upper Respiratory Tract.

Picobirnaviruses (PBVs) have been detected in several species of animals worldwide; however, data pertaining to their presence in Australian wild and domestic animals are limited. Although PBVs are mostly found in faecal samples, their detection in blood and respiratory tract samples raises questions concerning their tropism and pathogenicity. We report here PBV detection in wild deer and cattle from southeastern Australia. Through metagenomics, the presence of PBV genogroups I (GI) and II (GII) were detected in deer serum and plasma. Molecular epidemiology studies targeting the partial RNA-dependent RNA polymerase gene were performed in a wide range of specimens (serum, faeces, spleen, lung, nasal swabs, and trachea) collected from wild deer and cattle, with PCR amplification obtained in all specimen types except lung and spleen. Our results reveal the predominance of GI and concomitant detection of both genogroups in wild deer and cattle. In concordance with other studies, the detected GI sequences displayed high genetic diversity, however in contrast, GII sequences clustered into three distinct clades. Detection of both genogroups in the upper respiratory tract (trachea and nasal swab) of deer in the present study gives more evidence about the respiratory tract tropism of PBV. Although much remains unknown about the epidemiology and tropism of PBVs, our study suggests a wide distribution of these viruses in southeastern Australia.

Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints.

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.

Attribution of Pediatric Acute Gastroenteritis Episodes and Emergency Department Visits to Norovirus Genogroups I and II

Norovirus is a leading cause of acute gastroenteritis. With vaccines in development, population-based estimates of norovirus burden are needed to identify target populations, quantify potential benefits, and understand disease dynamics. We estimated the attributable fraction (AF) for norovirus infections in children, defined as the proportion of children testing positive for norovirus whose gastroenteritis was attributable to norovirus. We calculated the standardized incidence and emergency department (ED) visit rates attributable to norovirus using provincial gastroenteritis visit administrative data. From 3731 gastroenteritis case patients and 2135 controls we determined that the AFs were 67.0% (95% confidence interval [CI], 31.5%-100%) and 91.6% (88.8%-94.4%) for norovirus genogroups I (GI) and II (GII), respectively. Norovirus GII AF varied by season but not age. We attributed 116 episodes (95% CI, 103-129) and 59 (51-67) ED visits per 10 000 child-years to norovirus GII across all ages, accounting for 20% and 18% of all medically attended gastroenteritis episodes and ED visits, respectively. In children, a large proportion of norovirus GII detections reflect causation, demonstrating significant potential for norovirus GII vaccines. Seasonal variation in the norovirus GII AF may have implications for understanding the role asymptomatic carriage plays in disease dynamics.

Leafy vegetables as potential transmitters of Norovirus in Cordoba city, Argentina.

The Norovirus (NoV) constitute a genus within the viral family Caliciviridae, being the main cause of outbreaks of food origin among humans. Fresh vegetables are susceptible to being contaminated with these pathogens during their cultivation, harvest, transport, processing and handling. So it was intended to determine the frequency of detection of NoV in plant samples of leaves of the City of Córdoba, and adapt a method of viral concentration with polyethylene glycol for the recovery of viral particles from the surface of vegetables and characterize the genogroups of NoV detected. 19 samples of leafy vegetables were taken between June and December 2012. A viral concentration technique previously validated in the laboratory was applied (elution and precipitation with polyethylene glycol). The viral RNA was extracted to the concentrates of the samples using Trizol and precipitation with isopropyl alcohol. The nucleic acid was amplified by Rt-PCR with specific primers to identify genogroups I (GI) and II (GII). The products of the amplification were revealed by polyacrylamide gel electrophoresis and silver staining. We found 57.89% positive samples. Ten of the detected strains belonged to genogroup I (GI) and one to genogroup II (GII). They were identified throughout the study period, particularly during the months of August, September and November. These pathogens were detected with a prevalence of 57.89%. The strains belonged mainly to the GI, representing a potential risk for the population.

Quantitative PCR Detection of Enteric Viruses in Wastewater and Environmental Water Sources by the Lisbon Municipality: A Case Study

Current regulations and legislation require critical revision to determine safety for alternative water sources and water reuse as part of the solution to global water crisis. In order to fulfill those demands, Lisbon municipality decided to start water reuse as part of a sustainable hydric resources management, and there was a need to confirm safety and safeguard for public health for its use in this context. For this purpose, a study was designed that included a total of 88 samples collected from drinking, superficial, underground water, and wastewater at three different treatment stages. Quantitative Polimerase Chain Reaction (PCR) detection (qPCR) of enteric viruses Norovirus (NoV) genogroups I (GI) and II (GII) and Hepatitis A (HepA) was performed, and also FIB (E. coli, enterococci and fecal coliforms) concentrations were assessed. HepA virus was only detected in one untreated influent sample, whereas NoV GI/ NoV GI were detected in untreated wastewater (100/100%), secondary treated effluent (47/73%), and tertiary treated effluent (33/20%). Our study proposes that NoV GI and GII should be further studied to provide the support that they may be suitable indicators for water quality monitoring targeting wastewater treatment efficiency, regardless of the level of treatment.

Application of a multiplex salivary immunoassay to detect sporadic incident norovirus infections

Norovirus is one of the most common causes of gastroenteritis. Following infection, anti-norovirus salivary immunoglobulin G (IgG) rises steeply within 2 weeks and remains elevated for several months; this immunoconversion can serve as an indicator of infection. We used a multiplex salivary immunoassay to study norovirus infections among 483 visitors to a Lake Michigan beach in 2015. Saliva was collected on the day of the beach visit (S1); after 10–14 days (S2); and after 30–40 days (S3). Luminex microspheres were coupled to recombinant antigens of genogroup I (GI) and II (GII) noroviruses and incubated with saliva. Immunoconversion was defined as at least 4-fold increase in anti-norovirus IgG antibody response from S1 to S2 and a 3-fold increase from S1 to S3. Ten (2.1%) immunoconverted to either GI (2) or GII (8) norovirus. Among those who immunoconverted, 40% reported at least one gastrointestinal symptom and 33% reported diarrhea, compared to 15% (p = 0.06) and 8% (p = 0.04) among those who did not immunoconvert, respectively. The two participants who immunoconverted to GI norovirus both swallowed water during swimming (p = 0.08). This study demonstrated the utility of a non-invasive salivary immunoassay to detect norovirus infections and an efficient approach to study infectious agents in large cohorts.

645. Singulex Clarity Norovirus Assay (In Development) Provides Ultrasensitive Detection of Norovirus Genogroups I and II

BackgroundCommercially available enzyme immunoassays (EIAs) for detection of norovirus antigen have poor sensitivity and are limited to use in investigations of a gastroenteritis outbreak. Hence, there remains a need for a standalone high-sensitivity assay that enables rapid and accurate detection of norovirus antigen.MethodsThe Singulex Clarity norovirus assay is currently in development for use on the Singulex Clarity® system (Singulex Inc., Alameda, CA, USA), a fully-automated platform powered by Single Molecule Counting technology (registered with the FDA and CE marked). The assay uses paramagnetic microparticles bound to capture antibody and a fluorescently labeled reporter antibody to detect virion capsid protein of norovirus genogroups I (GI) and II (GII) in the stool. For the development of Clarity Norovirus assay, diagnostic performance of 4 antibody pairs (as Capture and Detection reagent) were evaluated by testing 137 stool samples from patients with suspected norovirus infection. Samples were sourced from three providers: (1) 90 genotyped samples of which 75 were positive (19 different genotypes) and 15 were negative by the CDC assay, (2) 3 samples positive and 5 samples negative by the BioFire® FilmArray® Gastrointestinal Panel, and (3) 39 samples negative by a lab-developed test using Cepheid reagents (SmartCycler®).ResultsFrom all the antibody pairs tested, one of the pairs had best performance with the area under the receiver operating characteristic (AuROC) curve demonstrating a C-Statistic of 0.959 (95% CI 0.921–0.997), compared with AuROC C-statistic of 0.943 (95% CI 0.896–0.990), 0.871 (95% CI 0.807–0.936), and 0.914 (95% CI 0.863–0.964) for the three other pairs. The Clarity assay detected all 19 different genotypes tested (figures).ConclusionThe ultrasensitive and rapid Clarity norovirus assay (in development) for detection of GI and GII demonstrated excellent performance with one of the antibody pairs tested and detected all 19 tested genotypes. The Clarity assay may offer a standalone solution for norovirus diagnostics. Disclosures All authors: No reported disclosures.

Detection and differentiation of norovirus genogroups I and II from clinical stool specimens using real-time PCR

A real-time RT-PCR assay was designed to detect and differentiate norovirus genogroups I (GI) and II (GII), with primers and probes targeting the nonstructural polyprotein gene. Stool samples (n = 100) submitted for routine testing by the BioFire FilmArray® GI panel were also tested by the norovirus GI/GII real-time PCR assays. When compared to the FilmArray GI panel, the norovirus real-time PCR assay demonstrated a sensitivity of 77.5% (62/80) and specificity of 95% (19/20). Specimens yielding discordant results (n = 19) were tested at two outside laboratories for adjudication. Following discordant resolution, the adjusted sensitivity and specificity of the norovirus real-time PCR assays were 96.9% (63/65) and 100% (35/35), respectively. These results suggest that the real-time PCR assays are able to accurately detect and differentiate norovirus GI/GII from clinical stool specimens. Furthermore, our report highlights a potential issue with the specificity of the BioFire FilmArray® norovirus assay, which warrants additional investigation.

Risk factors for norovirus contamination of shellfish water catchments in England and Wales

This study examines the relationships between concentrations of human noroviruses (NoV) genogroups I (GI) and II (GII) and Escherichia coli monitored in oysters from 31 commercial harvesting areas on the coast of England and Wales from May 2009 to April 2011 and demographic, hydrometric, climatic and pollution source characteristics of upstream river catchments using multiple regression techniques. The predictive environmental factors for E. coli contamination in the oysters were rainfall (cumulative 7days before sampling) while the predictive factors for NoV (GI+GII) were water temperature, catchment area and the combined volume of continuous sewage discharges in the catchment. Oysters from cold waters (<5°C) had significantly higher NoV content than those from warmer waters (>10°C). The association with water temperature may be consequential on the seasonal prevalence of the virus in the community or linked with oyster metabolic function. In a group of 10 study sites, mean concentrations of NoV increased as the number of stormwater spills at those sites also increased. The results of this study could be used to evaluate the likely impact of sewerage infrastructure improvements in catchments at risk of NoV contamination and to help identify sites suitable for shellfish farming.

Distribution of Human Norovirus in the Coastal Waters of South Korea.

The presence of human norovirus in the aquatic environment can cause outbreaks related to recreational activities and the consumption of norovirus-contaminated clams. In this study, we investigated the prevalence of norovirus genogroups I (GI) and II (GII) in the coastal aquatic environment in South Korea (March 2014 to February 2015). A total of 504 water samples were collected periodically from four coastal areas (total sites = 63), of which 44 sites were in estuaries (clam fisheries) and 19 were in inflow streams. RT-PCR analysis targeting ORF2 region C revealed that 20.6% of the water samples were contaminated by GI (13.3%) or GII (16.6%). The prevalence of human norovirus was higher in winter/spring than in summer/fall, and higher in inflow streams (50.0%) than in estuaries (7.9%). A total of 229 human norovirus sequences were identified from the water samples, and phylogenetic analysis showed that the sequences clustered into eight GI genotypes (GI.1, 2, 3, 4, 5, 6, 7, and 9) and nine GII genotypes (GII.2, 3, 4, 5, 6, 11, 13, 17, and 21). This study highlighted three issues: 1) a strong correlation between norovirus contamination via inflow streams and coastal areas used in clam fisheries; 2) increased prevalence of certain non-GII.4 genotypes, exceeding that of the GII.4 pandemic variants; 3) seasonal shifts in the dominant genotypes of both GI and GII.

Occurrence of enteric viruses in reclaimed and surface irrigation water: relationship with microbiological and physicochemical indicators.

To assess the prevalence of enteric viruses in different irrigation water sources and in the irrigated produce, and the possible links with microbiological and physicochemical water characteristics. The prevalence and levels of Escherichia coli, Norovirus (NoV) genogroup I (GI) and II (GII), as well as Hepatitis A virus were assessed in three types of water: surface water (surface-W), reclaimed water subjected to secondary treatment (secondary-W) and reclaimed water subjected to tertiary treatment (tertiary-W), as well as in zucchini irrigated with these irrigation water sources. Chemical oxygen demand (COD), turbidity, total suspended solids, alkalinity and maximum filterable volume (MFV) were also measured in the water. Higher prevalence of NoV in secondary-W (GI 100%, GII 55·6%) and tertiary-W (GI 91·7%, GII 66·7%) compared with surface-W (GI 58·4%, GII 22·2%) was observed. Nov GI showed positive correlation with E.coli (Spearman's correlation coefficient=0·68, P<0·01), and with some physicochemical parameters such as COD (0·52, P<0·01), turbidity (0·52, P<0·01) and MFV (0·54, P<0·01). Escherichia coli and enteric viruses were not detected in zucchini. There is a potential risk of contamination of crops with NoV when reclaimed water is used for irrigation. Increase the knowledge on the prevalence of enteric viruses in different irrigation water sources, and its consequences for fresh produce safety.

Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water

Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoculated lettuce. Pretreatment with 50μM PMAxx and 0.5% Triton X-100 (Triton) for 10min reduced the signal of thermally inactivated NoV by ca. 1.8 logs for both genogroups in lettuce concentrates. Additionally, this pretreatment reduced the signal of thermally inactivated NoV GI between 1.4 and 1.9 logs in spinach and romaine and lamb's lettuces and by >2 logs for NoV GII in romaine and lamb's lettuce samples. Moreover this pretreatment was satisfactorily applied to naturally-contaminated water samples with NoV GI and GII. Based on the obtained results this pretreatment has the potential to be integrated in routine diagnoses to improve the interpretation of positive NoV results obtained by RT-qPCR.

Clinical and epidemiological characteristics of norovirus gastroenteritis among hospitalized children in Lebanon.

AIMTo assess the burden of norovirus (NoV) and to determine the diversity of circulating strains among hospitalized children in Lebanon.METHODSStool samples were collected from children presenting with acute gastroenteritis to six major hospitals in Lebanon. A total of 739 eligible stool samples, testing negative for diarrhea caused by rotavirus as a possible viral pathogen, were collected between January 2011 and June 2013. A standardized questionnaire including demographic, epidemiological and clinical observations was used at the time of hospitalization of children presenting with diarrhea. Viral RNA was extracted from stool samples followed by reverse transcription polymerase chain reaction and nucleotide sequencing of a fragment of the viral protein 1 capsid gene. Multiple sequence alignments were carried out and phylogenetic trees were constructed using the MEGA 6 software.RESULTSOverall, 11.2% of stool samples collected from children aged < 5 years tested positive for NoV genogroups I (GI) and II (GII). GII accounted for 10.6% of the gastroenteritis cases with only five samples being positive for GI (0.7%). The majority of hospitalized children showed symptoms of diarrhea, dehydration, vomiting and fever. Upon sequencing of positive samples and based on their clustering in the phylogenetic tree, 4/5 of GI gastroenteritis cases were designated GI.3 and one case as GI.4. GII.4 was predominantly detected in stool of our study participants (68%). We report a JB-15/KOR/2008 GII.4 Apeldoorn 2008-like variant strain circulating in 2011; this strain was replaced between 2012 and 2013 by a variant sharing homology with the Sydney/NSW0514/2012/AUS GII.4 Sydney 2012 and Sydney 2012/FRA GII.4 strains. We also report the co-circulation of non-GII.4 genotypes among hospitalized children. Our data show that NoV gastroenteritis can occur throughout the year with the highest number of cases detected during the hot months.CONCLUSIONThe majority of NoV-associated viral gastroenteritis cases among our participants are attributable to GII.4, which is compatible with results reported worldwide.

Prevalence and genetic diversity of noroviruses in adults with acute gastroenteritis in Huzhou, China, 2013–2014

Norovirus (NoV) infection is the most common cause of nonbacterial acute gastroenteritis, which affects both adults and children. However, the molecular epidemiology of NoV in adults with acute gastroenteritis in China has not been investigated extensively. In this study, we investigated the occurrence of NoV infections and analyzed the genetic diversity of NoV in adults with acute gastroenteritis in Huzhou, China. A total of 796 fecal samples were collected from outpatients (≥16 years of age) between March 2013 and February 2014. Real-time RT-PCR was performed to detect NoV genogroups I (GI) and II (GII). For genotyping, the capsid and RNA-dependent RNA polymerase (RdRp) genes were partially amplified and sequenced for phylogenetic analysis. NoVs were detected in 26.51 % (211/796) of the specimens, with GII being predominant, representing 96.20 % of the NoV infections. At least nine genotypes were identified among GI and GII specimens, including GI.P2/GI.2, GI.P3/GI.3, GI.P4/GI.4, GII.Pe/GII.4 Sydney_2012, GII.P12/GII.3, GII.P7/GII.6, GII.P16/GII.13, GII.Pe, and GII.Pg (RdRp only). This is the first report of a GII.P16/GII.13 recombinant virus in adults in China. GII.Pe/GII.4 Sydney_2012 was the most prevalent genotype and the only GII.4 variant identified during the study period. Our findings suggested that NoV was a common causative agent of acute gastroenteritis in adults in Huzhou, China. During the study period, the NoVs circulating in adults in Huzhou were predominantly GII.4 Sydney_2012 variants and GII NoV recombinants.

Detection and quantification of hepatitis A virus and norovirus in Spanish authorized shellfish harvesting areas

An 18-month survey was conducted in ten class “B” harvesting areas from two Galician Rias (NW of Spain), the most important bivalve production area in Europe, to determine the prevalence of hepatitis A virus (HAV) and human norovirus (NoV), including genogroups I (GI) and II (GII). Quantification was performed by reverse transcription real-time PCR (RT-qPCR), according to the recently developed standard method ISO/TS 15216-1:2013. Four bivalve species were studied, including wild and cultured mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and Venerupis decussata) and cockles (Cerastoderma edule). Overall, 55.4% of samples were contaminated by at least one of the studied viruses, being detected the simultaneous presence of two or three viruses in 11.3% of the cases. NoV GI was the most prevalent virus (32.1%), followed by NoV GII (25.6%) and HAV (10.1%). Cultured mussels showed the highest percentage of positive samples (61.4%), followed by cockles (59.4%), wild mussels (54.3%) and clams (38.7%). Viral contamination levels for most of the positive samples ranged from 102 to 103 RNA copies/g of digestive tissue (RNAc/g DT). The presence of viral contamination was statistically higher (P<0.0001) in warm months (April to September) than in cold months (October to March). The data presented here may contribute to the development of more representative sampling strategies, in monitoring and management of shellfish growing areas as well as being useful in a future scenario in which viral critical values are adopted in legislation.