Norovirus GI Research Articles

Norovirus passive surveillance as an alternative strategy for genetic diversity assessment in developing countries

In developing countries, the acute gastroenteritis outbreaks submitted for viral testing are limited due to deficient surveillance programs. The aim of this study was to analyze a passive surveillance strategy for monitoring the molecular epidemiology of norovirus (NV) and counterbalance the genetic diversity data gap.Laboratory-confirmed rotavirus negative sporadic stool samples (N = 523) collected between 2010 and 2017 from children were selected from our archival collection and were tested for NV and sequencing was performed on the positive samples. Passive surveillance information was compared with the genetic diversity data that was available from local norovirus-confirmed gastroenteritis outbreaks.Each year, norovirus detection in the sporadic samples ranged from 12 to 29%. GI and GII norovirus were detected in 7 (1.3%) and 101 (19.3%) of the specimens, respectively. Four GI and six GII capsid genotypes were identified. Six out of 9 strains detected in the NV outbreaks panel were also identified in the set of sporadic samples either coincidently in the same year, the previous or the later year. Also, this set of samples depicted even better the circulating epidemic strain. Thus, implementing norovirus testing and genotyping in stool samples collected with other purposes represent a suitable strategy for providing genetic diversity information.

Microbial water quality improvement associated with transitioning from intermittent to continuous water supply in Nagpur, India

Nearly half a billion people living in Indian cities receive their drinking water from an intermittent water supply (IWS), which can be associated with degraded water quality and risk of waterborne disease. The municipal water supply in Nagpur, India is transitioning from intermittent to continuous supply in phases. We conducted cross-sectional sampling to compare microbial water quality under IWS and continuous water supply (CWS) in Nagpur. In 2015 and 2017, we collected 146 grab samples and 90 large-volume dead-end ultrafiltration (DEUF) samples (total volume: 6,925 liters). In addition to measuring traditional water quality parameters, we also assayed DEUF samples by droplet digital PCR (ddPCR) for waterborne pathogen gene targets. At household taps served by IWS, we detected targets from enterotoxigenic E. coli, Shigella spp./enteroinvasive E. coli, norovirus GI and GII, adenovirus A-F, Cryptosporidium spp., and Giardia duodenalis. We observed a significant increase in the proportion of grab samples positive for culturable E. coli (p = 0.0007) and DEUF concentrates positive for waterborne pathogen gene targets (p = 0.0098) at household taps served by IWS compared to those served by CWS. IWS continues to be associated with fecal contamination, and, in this study, with increased prevalence of molecular evidence of waterborne pathogens. These findings add mounting evidence that, despite the presence of piped on premise infrastructure, IWS is less likely to meet the requirements for safely-managed drinking water as defined by the Sustainable Development Goals. Importantly, these findings demonstrate the transition from IWS to CWS in Nagpur is yielding meaningful improvements in microbial water quality.

Salivary Antibodies against Multiple Environmental Pathogens Found in Individuals Recreating at an Iowa Beach.

Detecting environmental exposures and mitigating their impacts are growing global public health challenges. Antibody tests show great promise and have emerged as fundamental tools for large-scale exposure studies. Here, we apply, demonstrate and validate the utility of a salivary antibody multiplex immunoassay in measuring antibody prevalence and immunoconversions to six pathogens commonly found in the environment. The study aimed to assess waterborne infections in consenting beachgoers recreating at an Iowa riverine beach by measuring immunoglobulin G (IgG) antibodies against select pathogens in serially collected saliva samples. Results showed that nearly 80% of beachgoers had prior exposures to at least one of the targeted pathogens at the beginning of the study. Most of these exposures were to norovirus GI.1 (59.41%), norovirus GII.4 (58.79%) and Toxoplasma gondii (22.80%) and over half (56.28%) of beachgoers had evidence of previous exposure to multiple pathogens. Of individuals who returned samples for each collection period, 6.11% immunoconverted to one or more pathogens, largely to noroviruses (GI.1: 3.82% and GII.4: 2.29%) and T. gondii (1.53%). Outcomes of this effort illustrate that the multiplex immunoassay presented here serves as an effective tool for evaluating health risks by providing valuable information on the occurrence of known and emerging pathogens in population surveillance studies.

Development of a Tetraplex qPCR for the Molecular Identification and Quantification of Human Enteric Viruses, NoV and HAV, in Fish Samples.

Human enteric viruses such as norovirus (NoV) and hepatitis A virus (HAV) are some of the most important causes of foodborne infections worldwide. Usually, infection via fish consumption is not a concern regarding these viruses, since fish are mainly consumed cooked. However, in the last years, raw fish consumption has become increasingly common, especially involving the use of seabass and gilthead seabream in dishes like sushi, sashimi, poke, and carpaccio. Therefore, the risk for viral infection via the consumption of raw fish has also increased. In this study, a virologic screening was performed in 323 fish specimens captured along the Portuguese coast using a tetraplex qPCR optimised for two templates (plasmid and in vitro transcribed RNA) to detect and quantify NoV GI, NoV GII and HAV genomes. A difference of approximately 1-log was found between the use of plasmid or in vitro transcribed RNA for molecular-based quantifications, showing an underestimation of genome copy-number equivalents using plasmid standard-based curves. Additionally, the presence of NoV genomic RNA in a pool of seabass brains was identified, which was shown to cluster with a major group of human norovirus sequences from genogroup I (GI.1) by phylogenetic analysis. None of the analysed fish revealed the presence of NoV GII or HAV. This result corroborates the hypothesis that enteric viruses circulate in seawater or that fish were contaminated during their transportation/handling, representing a potential risk to humans through raw or undercooked fish consumption.

Bacteriophages Are Good Estimators of Human Viruses Present in Water.

The detection of fecal viral pathogens in water is hampered by their great variety and complex analysis. As traditional bacterial indicators are poor viral indicators, there is a need for alternative methods, such as the use of somatic coliphages, which have been included in water safety regulations in recent years. Some researchers have also recommended the use of reference viral pathogens such as noroviruses or other enteric viruses to improve the prediction of fecal viral pollution of human origin. In this work, phages previously tested in microbial source tracking studies were compared with norovirus and adenovirus for their suitability as indicators of human fecal viruses. The phages, namely those infecting human-associated Bacteroides thetaiotaomicron strain GA17 (GA17PH) and porcine-associated Bacteroides strain PG76 (PGPH), and the human-associated crAssphage marker (crAssPH), were evaluated in sewage samples and fecal mixtures obtained from different animals in five European countries, along with norovirus GI + GII (NoV) and human adenovirus (HAdV). GA17PH had an overall sensitivity of ≥83% and the highest specificity (>88%) for human pollution source detection. crAssPH showed the highest sensitivity (100%) and specificity (100%) in northern European countries but a much lower specificity in Spain and Portugal (10 and 30%, respectively), being detected in animal wastewater samples with a high concentration of fecal indicators. The correlations between GA17PH, crAssPH, or the sum of both (BACPH) and HAdV or NoV were higher than between the two human viruses, indicating that bacteriophages are feasible indicators of human viral pathogens of fecal origin and constitute a promising, easy to use and affordable alternative to human viruses for routine water safety monitoring.

Monitoring of human enteric virus and coliphages throughout water reuse system of wastewater treatment plants to irrigation endpoint of leafy greens

One solution to current water scarcity is the reuse of treated wastewater. Water reuse systems have to be examined as a whole, including the efficacy of water-reclamation treatments and the operation steps from the wastewater inlet into the WWTP to the irrigation endpoint, including the irrigated crop. In this study, the monitoring of human enteric viruses and coliphages were assessed in two water reused systems. The presence of hepatitis A virus (HAV) and human noroviruses genogroups I and II (GI and GII) were analyzed by real-time RT-PCR (RT-qPCR) in water (n = 475) and leafy green samples (n = 95). Total coliphages were analyzed by the double-layer agar plaque technique. The prevalence of HAV in water samples was very low (c.a. 2%), mostly linked to raw sewage, while for leafy green samples, none was positive for HAV. In leafy greens, prevalence of norovirus was low (less than 5–6%). The highest reductions for norovirus were observed in samples taken from the water reservoirs used by the growers near the growing field. The virus die-off during water storage due to solar radiation could be considered as an additional improvement. Reclamation treatments significantly reduced the prevalence and the counts of noroviruses GI and GII and coliphages in reclaimed water. However, the coliphage reductions (c.a. 5 log) do not comply with the specifications included in the new European regulation on reclaimed water (≥6.0 log). Correlations between noroviruses GI and GII and coliphages were found only in positive samples with high concentrations (>4.5 log PFU/100 mL). A high percentage of samples (20–25%) negative for total coliphages showed moderate norovirus counts (1–3 logs), indicating that coliphages are not the most suitable indicator for the possible presence of human enteric viruses.

Assessment of enteric viruses during a hepatitis outbreak in Detroit MI using wastewater surveillance and metagenomic analysis.

This study investigates enteric viruses in wastewater during an outbreak of acute hepatitis caused by hepatitis A virus (HAV) in a large metropolitan area. Emphasis is given to caliciviruses and HAV. Metagenomic analysis was performed to characterize enteric viruses excreted by the population of Detroit MI, during a hepatitis A outbreak that occurred in 2017 and 2018. Additionally, HAV, norovirus GII, and sapovirus were quantified, using qPCR, in 54 untreated wastewater samples collected over the course of 4months. Correlation analysis was performed to identify associations between the number of disease cases and HAV concentrations in wastewater. HAV obtained the highest relative abundance among other enteric viruses detected in wastewater metagenomes. Metagenomic analysis also detected several other enteric viruses including astrovirus, enterovirus and hepatitis E virus. Average sapovirus concentrations of 1·36×106 gcl-1 were significantly greater than norovirus GII concentrations (2·94×104 gcl-1 ). Additionally, norovirus GI and GII along with sapovirus GI.1 were detected using metagenomics. HAV loads in wastewater were significantly correlated with the number of disease cases reported 1week after wastewater sampling. Surveying untreated wastewater is a promising method for detecting early signs of hepatitis A outbreaks and for routine environmental monitoring of enteric viruses circulating in the environment. Authors demonstrate the usefulness of metagenomics for genogrouping and enteric viral surveillance.

Evaluation of Norovirus Reduction in Environmentally Contaminated Pacific Oysters During Laboratory Controlled and Commercial Depuration

Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT-PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17–21 °C and after 4 days at 11–15 °C, compared to 44% reduction at 7–9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram (gcg−1; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant difference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg−1. These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry.

Enteric virus in reclaimed water from treatment plants with different multi-barrier strategies: Trade-off assessment in treatment extent and risks

Reclaimed water is an alternative water resource to mitigate water scarcity. To promote safe reuse, this paper aims to monitor the enteric virus concentration in the reclaimed water generated by two sewage treatment plants (STP) with different multibarrier technologies, and to assess if stringent treatment extent is required in a low-resource setting to achieve minimal viral risks arising from non-potable reuse. Through a 9-month surveillance, it was observed that a higher diversity and abundance of enteric DNA and RNA viruses were detected in treated wastewater generated from conventional activated sludge (i.e., site B) compared to that from membrane bioreactor-based STP (i.e., site A). To exemplify, enteric RNA viruses were detected in up to 1.13, 4.1, 4.9, 4.5, and 4.5 log10 copies/L for Aichi virus (AiV), rotavirus (RV), enterovirus (EV), norovirus GI and GII (NoV GII, GII) respectively, at site B. For enteric DNA virus, up to 4.3 and 5.35 log10 copies/L of adenovirus (AdV) and polyoma BK virus (BKV) were also found in site B. This is in contrast to the absence of AiV, RV and NoV detected in samples from site A. However, when translated to risks outcome from NoV GII, it was noted that recreational users at both sites A and B are exposed to acceptable disease burden (<10−4 DALYpppy). Occupational workers at site B faced burden risk of 2.01 × 10−4 to 3.85 × 10−4 DALYpppy, which is slightly higher than the acceptable 10−4 DALYpppy but such level of risks can be reduced by minimizing exposure frequency and/or adoption of best management practices. Findings from this study suggest that additional implementation of treatment barriers that incur higher capital investment or energy costs in low resource countries may not be mandatory to mitigate risks arising from enteric viruses for non-potable reuse purposes studied here.

Quantitative levels of norovirus and hepatitis A virus in bivalve molluscs collected along the food chain in the Netherlands, 2013–2017

Contamination of bivalve molluscs with viruses is well recognized as a food safety risk. A microbiological criterion for norovirus (NoV) and hepatitis A virus (HAV) in shellfish, however, does not exist in the European Union currently. The aim of this study was to evaluate the contamination levels of these viruses for fluctuation over a long period (2013–2017) in oyster (n = 266) and mussel samples (n = 490) using a method based on ISO/TS 15216-1: 2013. Samples were taken at different points in the food chain, either directly post-harvest, at Dutch dispatch centers or in retail stores, from September until March of each year. Altogether, 53.1% of the mussel and 31.6% of the oyster samples tested positive for NoV RNA. Simultaneous presence of NoV GI and GII RNA was observed in 31.6% of mussel and 10.2% of oyster samples. Contamination levels in NoV positive mussel samples collected post-harvest from B-areas were significantly higher than in those collected post-harvest from A-areas, or at dispatch centers or retail stores. Levels in oysters from dispatch were significantly lower than those collected in retail stores. Ready for sale mussels and oysters contained 2.04 and 1.76 mean log10 transformed NoV genome copies/gram (gc/g), respectively. GII levels were at a constant level in ready for sale mussels throughout all sampling periods in the study. This seemed to be true for oysters as well. HAV RNA was detected in only one of the tested mussel samples (n = 392) (typed HAV 1A) and in none of the tested oyster samples (n = 228). Critical evaluation of NoV and HAV levels in shellfish can be of help for risk assessment and risk management actions.

Interlaboratory Evaluation of a Method for Quantification of Norovirus RNA as an Alternative Use for ISO 15216-1:2017 to Conduct Japan Baseline Survey of Oysters.

In this study, we aimed to investigate the standard method used for quantification of norovirus in oysters in Japan for the provisional adaptation of the method as an alternative to ISO 15216-1:2017, to conduct a Japan baseline survey of norovirus in oysters. For this purpose, the method provided by the Japan Committee for Standardization of Virus Detection in Food was subjected to an interlaboratory study to determine the performance characteristics of the standard method used in Japan. As a result, the theoretical limit of quantification for norovirus GI and GII in oysters by the standard method used in Japan was expected to be 1.92 and 1.85 log10 copies/g, respectively. The repeatability standard deviations (Sr) were 0.26 and 0.30 log10 copies/g for GI and GII, respectively, and the reproducibility standard deviations (SR) were 0.47 and 0.44 log10 copies/g for GI and GII, respectively. Through the interlaboratory study, we specified several critical points to obtain scientifically reliable results by using the standard method used in Japan. Especially, necessity for application of using process control virus was the most crucial point that needed to be improved. In addition, there are many participating laboratories that could not handle dilution of standard and quantify or detect the viruses in the test samples. To ensure scientifically reliable test result, capacity building of laboratories and implementation of proficiency testing should be considered for future tasks in combination with an application of process control materials in the method. On the assumption that the problems revealed in this study will be solved, the standard method used in Japan would be suitable for use in Japan baseline survey of norovirus in oysters, which will contribute to the international action against norovirus in oysters, led by the EU.

Incidence of hepatitis A and hepatitis E viruses and norovirus and rotavirus in fish and shrimp samples caught from the Persian Gulf

ABSTRACT Foodborne viruses including hepatitis A virus (HAV), norovirus (NoV), rotavirus (RoV) and hepatitis E virus (HEV) are easily transmitted through contaminated seafoods. The current research was done to assess the incidence of RoV, NoV GI and GII,hAV and hEV in fish and shrimp samples caught from the Persian Gulf, Iran. Three-hundred and twenty fish and shrimp samples were collected. The presence of foodborne viruses were assessed by the real-time PCR. Forty-nine out of 320 (15.31%) fish and shrimp samples were positive for foodborne viruses. Distribution of hAV, NoV GI and NoV GII amongst all studied samples were 0.93%, 5.93% and 8.43%, respectively. hEV and RoV viruses were not found in studied samples. Parastromateus niger and Scomberomorus commerson fish and Penaeus monodon shrimp were the most frequently contaminated samples. Simultaneous incidence of hAV and NoV GI and hAV and NoV GII were 0.31% and 0.93%, respectively. Distribution of foodborne viruses in samples collected through spring, summer, autumn and winter seasons were 14.28%, 9.33%, 11.76% and 24.44%, respectively. Findings revealed that the incidence of foodborne viruses was significantly associated with seafood species and also season of sampling.

Virus Etiology, Diversity and Clinical Characteristics in South African Children Hospitalised with Gastroenteritis.

Background: Viral gastroenteritis remains a major cause of hospitalisation in young children. This study aimed to determine the distribution and diversity of enteric viruses in children ≤5 years, hospitalised with gastroenteritis at Kalafong Provincial Tertiary Hospital, Pretoria, South Africa, between July 2016 and December 2017. Methods: Stool specimens (n = 205) were screened for norovirus GI and GII, rotavirus, sapovirus, astrovirus and adenovirus by multiplex RT-PCR. HIV exposure and FUT2 secretor status were evaluated. Secretor status was determined by FUT2 genotyping. Results: At least one gastroenteritis virus was detected in 47% (96/205) of children. Rotavirus predominated (46/205), followed by norovirus (32/205), adenovirus (15/205), sapovirus (9/205) and astrovirus (3/205). Norovirus genotypes GI.3, GII.2, GII.3, GII.4, GII.7, GII.12, GII.21, and rotavirus strains G1P[8], G2P[4], G2P[6], G3P[4], G3P[8], G8P[4], G8P[6], G9P[6], G9P[8] and sapovirus genotypes GI.1, GI.2, GII.1, GII.4, GII.8 were detected; norovirus GII.4[P31] and rotavirus G3P[4] predominated. Asymptomatic norovirus infection (GI.3, GI.7, GII.4, GII.6, GII.13) was detected in 22% of 46 six-week follow up stools. HIV exposure (30%) was not associated with more frequent or severe viral gastroenteritis hospitalisations compared to unexposed children. Rotavirus preferentially infected secretor children (p = 0.143) and norovirus infected 78% secretors and 22% non-secretors. Conclusion: Rotavirus was still the leading cause of gastroenteritis hospitalisations, but norovirus caused more severe symptoms.

Detection and phylogenetic analysis of norovirus from individual septic tanks in the drainage basin of the coastal area located in the Jaran Bay of Korea

Norovirus (NoV) prevalence was investigated in 100 sewage samples collected from 35 individual septic tanks around the drainage basin of Jaran Bay, Korea in January, May, and July of 2017. Genotypes and diversity of NoV strains detected in sewage samples were also assessed using the conventional RT-PCR and phylogenetic analysis. NoV GI or GII were detected in 22 (22.0%) and 24 (24.0%) samples, respectively. Thirteen genotypes were identified with three dominant genotypes (GI.9, GII.5 and GII.17) and GII.17 showed relatively higher prevalence during the survey period. GII.17 strains were clustered into recombinant type variant or NoV GII.17 Kawasaki variant. NoV GII.17 strains were considered emergent epidemic variants with widespread circulation. NoV surveillance strategy should include both environmental (sewage) and clinical data to reveal minor NoV genotypes likely cause of asymptomatic or underreported infections in the local population.

Occurrence of respiratory viruses on school desks

Schools represent high occupancy environments and well-documented high-risk locations for the transmission of respiratory viruses. The goal of this study was to report on the area density, occurrence, and type of respiratory viruses on desks in primary school classrooms. Quantitative reverse transcription polymerase chain reaction (qPCR) techniques were employed to measure nucleic acid area densities from a broad range of human adenoviruses and rhinoviruses, as well as coronavirus OC43, influenza A, and norovirus GI. Every two weeks, virus monitoring was conducted on the desks of four primary school classrooms in Colorado, USA, during the 2019 respiratory virus season. DNA and RNA from respiratory viruses and norovirus were recovered from more than 20% of the desks sampled; occurrence patterns that indicate a greater than 60% probability of encountering any virus, if more than five desks were occupied in a day. Rhinoviruses and adenoviruses were the most commonly detected viruses as judged by the composite of occurrence and number of gene copies recovered. Desktop adenosine triphosphate monitoring did not predict the recovery of viral genomic materials on desks. School desks can be commonly contaminated with respiratory viruses. Genomic surveys of the identity, distribution and abundance of human viruses on "high-touch" surfaces, can help inform risk assessments, design cleaning interventions, and may be useful for infection surveillance.

Characterization of Antigenic Relatedness Among GI Norovirus Genotypes Using Serum Samples From Norovirus-Infected Patients and Mouse Sera.

Characterizing diversity and the antigenic relatedness of norovirus remains a primary focus in understanding its biological properties and vaccine designs. The precise antigenic and serological features of GI genotypes have not been studied. The study represented an investigation on a gastroenteritis outbreak related to GI.3 norovirus and the three most detected GI genotypes, GI.2 (belonging to immunotype B), GI.3 and GI.9 (belonging to immunotype C), were selected to characterize their phylogenetic relationship, HBGA binding profiles and antigenic relatedness within (intra-immunotype), and between (inter-immunotypes) genotypes using mouse sera and patient’s serum samples from the GI.3 related outbreak. Wide HBGA binding profiles and evolution of binding affinity were observed in the three GI genotypes studied. A low specific blockade antibody to GI.3 in the population generated the pool of susceptible individuals and supported virus spread in the outbreak. We found strong blockade immune response in homologous strains, moderate intra-immunotype blockade but weak inter-immunotypes blockade in humans following GI.3 norovirus infections. These findings further support the immunotypes grouping and will be valuable for optimizing the design of norovirus vaccine.

Disulfide stabilization of human norovirus GI.1 virus-like particles focuses immune response toward blockade epitopes

Human noroviruses are non-enveloped, single-strand RNA viruses that cause pandemic outbreaks of acute gastroenteritis. A bivalent vaccine containing GI.1 and GII.4 virus-like particles (VLPs) has been shown to be safe and highly immunogenic, but its efficacy and durability have been limited. Here, we show that norovirus GI.1 VLPs are unstable and contain a substantial fraction of dissociated VLP components. Broadly reactive, non-neutralizing antibodies isolated from vaccinated donors bound to the dissociated components, but not to the intact VLPs. Engineering of interprotomer disulfide bonds within the shell domain prevented disassembly of the VLPs, while preserving antibody accessibility to blockade epitopes. Without adjuvant, mice immunized with stabilized GI.1 VLPs developed faster blockade antibody titers compared to immunization with wild-type GI.1 VLPs. In addition, immunization with stabilized particles focused immune responses toward surface-exposed epitopes and away from occluded epitopes. Overall, disulfide-stabilized norovirus GI.1 VLPs elicited improved responses over the non-disulfide-stabilized version, suggesting their promise as candidate vaccines.

Circulation of GII.4 norovirus strains in children

Background: GII.4 norovirus strains have caused the majority of norovirus outbreaks and sporadic cases globally since the mid-1990s. New norovirus GII.4 strains emerge every 2–4 years, circulate widely, and replace older variants. Novel variants emerge through a combination of recombination, genetic drift, and selection possibly driven by population immunity. Variants of GII.4 viruses have caused all the 6 pandemics of acute gastroenteritis caused by noroviruses in the last 2 decades. Methods and materials: In order to study the diversity of circulating norovirus GII.4 strains in Vellore (south India) over a 13 years period, stool samples collected from children <5 years hospitalized for acute watery diarrhea at Christian Medical College, Vellore, from December 2005 to December 2018, were tested for norovirus GI and GII by multiplex one-step real-time PCR (qRT-PCR). Norovirus positive samples were genotyped by performing conventional PCR that amplified the ORF1-ORF2 junction region, followed by Sanger sequencing, and sequence data analysis of the ORF2 (major capsid protein VP gene). Results: A total of 3303 stool samples were tested, of which 13.7% (451/3303) were positive for norovirus. Of the 451 samples positive for norovirus by qRT-PCR, 69.8% (315/451) were genotyped (19 GI and 296 GII genotypes). 63.9% (189/296) of the GII positive samples were GII.4 genotype. During 2005–2006, GII.4 Yerseke (60%) was the most common GII.4 variant, followed by GII.4 Hunter (33.3%). During 2007, GII4 Den Haag (55%) emerged as the predominant strain, followed by GII.4 Osaka (35%). During 2008, GII.4 Osaka was the most common (81.8%), which was replaced by GII.4 New Orleans (50%) in 2009. GII.4 New Orleans continued to be the most common variant in 2011. In 2012, GII.4 Sydney (42.9%) emerged as the most common GII.4 variant, along with GII.4 New Orleans (42.9%). During the successive years from 2013 to 2018, GII.4 Sydney remained the predominant GII.4 variant in Vellore. Conclusion: The circulation of norovirus GII.4 variants in our setting is similar to that reported globally.

Detection of Norovirus in Saliva Samples from Acute Gastroenteritis Cases and Asymptomatic Subjects: Association with Age and Higher Shedding in Stool.

Norovirus infections are a leading cause of acute gastroenteritis outbreaks worldwide and across all age groups, with two main genogroups (GI and GII) infecting humans. The aim of our study was to investigate the occurrence of norovirus in saliva samples from individuals involved in outbreaks of acute gastroenteritis in closed and semiclosed institutions, and its relationship with the virus strain, virus shedding in stool, the occurrence of symptoms, age, and the secretor status of the individual. Epidemiological and clinical information was gathered from norovirus outbreaks occurring in Catalonia, Spain during 2017–2018, and stool and saliva samples were collected from affected and exposed resident individuals and workers. A total of 347 saliva specimens from 25 outbreaks were analyzed. Further, 84% of individuals also provided a paired stool sample. For GII infections, norovirus was detected in 17.9% of saliva samples from symptomatic cases and 5.2% of asymptomatic individuals. Positivity in saliva occurred in both secretors and nonsecretors. None of the individuals infected by norovirus GI was positive for the virus in saliva. Saliva positivity did not correlate with any of the studied symptoms but did correlate with age ≥ 65 years old. Individuals who were positive in saliva showed higher levels of virus shedding in stool. Mean viral load in positive saliva was 3.16 ± 1.08 log10 genome copies/mL, and the predominance of encapsidated genomes was confirmed by propidium monoazide (PMA)xx-viability RTqPCR assay. The detection of norovirus in saliva raises the possibility of oral-to-oral norovirus transmission during the symptomatic phase and, although to a lesser extent, even in cases of asymptomatic infections.

Variations among Viruses in Influent Water and Effluent Water at a Wastewater Plant over One Year as Assessed by Quantitative PCR and Metagenomics.

Influent wastewater and effluent wastewater at the Rya treatment plant in Gothenburg, Sweden, were continuously monitored for enteric viruses by quantitative PCR (qPCR) during 1 year. Viruses in effluent wastewater were also identified by next-generation sequencing (NGS) in samples collected during spring, early summer, and winter. Samples of incoming wastewater were collected every second week. Seasonal variations in viral concentrations in incoming wastewater were found for noroviruses GII, sapovirus, rotavirus, parechovirus, and astrovirus. Norovirus GI and GIV and Aichi virus were present in various amounts during most weeks throughout the year, while hepatitis A virus, enterovirus, and adenovirus were identified less frequently. Fluctuations in viral concentrations in incoming wastewater were related to the number of diagnosed patients. The viruses were also detected in treated wastewater, however, with a 3- to 6-log10 reduction in concentration. Seven different hepatitis E virus (HEV) strains were identified in the effluents. Five of these strains belonged to genotype 3 and have been isolated in Sweden from swine, wild boars, and humans and in drinking water. The other two strains were divergent and had not been identified previously. They were similar to strains infecting rats and humans. Surveillance of enteric viruses in wastewater is a tool for early detection and follow-up of gastroenteritis outbreaks in society and for the identification of new viruses that can cause infection in humans.IMPORTANCE Both influent wastewater and treated wastewater at a wastewater treatment plant (WWTP) contain a high variety of human viral pathogens with seasonal variability when followed for 1 year. The peak of the amount of 11 different viruses in the inlet wastewater preceded the peak of the number of diagnosed patients by 2 to 4 weeks. The treatment of wastewater reduced viral concentrations by 3 to 6 log10 Despite the treatment of wastewater, up to 5 log10 virus particles per liter were released from into the surrounding river. Hepatitis E virus (HEV) strains previously identified in drinking water and two new strains, similar to those infecting rats and humans, were identified in the treated wastewater released from the WWTP.