Oyster Contamination with Human Noroviruses Impacted by Urban Drainage and Seasonal Flooding in Vietnam

This study investigated the level of norovirus contamination in oysters collected at a lagoon receiving urban drainage from Hue City for 17 months (August 2015–December 2016). We also investigated the genetic diversity of norovirus GI and GII in oyster and wastewater samples by using pyrosequencing to evaluate the effect of urban drainage on norovirus contamination of oysters. A total of 34 oyster samples were collected at two sampling sites (stations A and B) in a lagoon. Norovirus GI was more frequently detected than GII (positive rate 79 vs. 41%). Maximum concentrations of GI and GII were 2.4 × 10⁵ and 2.3 × 10⁴ copies/g, respectively. Co-contamination with GI and GII was observed in 35% of samples. Norovirus GII concentration was higher at station A in the flood season than in the dry season (P = 0.04, Wilcoxon signed-rank test). Six genotypes (GI.2, GI.3, GI.5, GII.2, GII.3, and GII.4) were identified in both wastewater and oyster samples, and genetically similar or identical sequences were obtained from the two types of samples. These observations suggest that urban drainage and seasonal flooding contribute to norovirus contamination of oysters in the study area.     

A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia

Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP’s outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75 % and AsV in 67 %. HAV were not detected in wastewater. Overall, 92 % of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92 % of samples and at concentrations up to 7.7 × 10³ genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66 % of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach.     

Prevalence and Molecular Genotyping of Noroviruses in Market Oysters,Mussels, and Cockles in Bangkok,Thailand

Noroviruses are the most common cause of acute gastroenteritis associated with bivalve shellfish consumption. This study aimed to detect and characterize noroviruses in three bivalve shellfish species: oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The virus concentration procedure (adsorption-twice elution-extraction) and a molecular method were employed to identify noroviruses in shellfish. RT-nested PCR was able to detect known norovirus GII.4 of 8.8 × 10^?2 genome copies/g of digestive tissues from oyster and cockle concentrates, whereas in mussel concentrates, the positive result was seen at 8.8 × 10² copies/g of digestive tissues. From August 2011 to July 2012, a total of 300 shellfish samples, including each of 100 samples from oysters, cockles, and mussels were collected and tested for noroviruses. Norovirus RNA was detected in 12.3 % of shellfish samples. Of the noroviruses, 7.7 % were of the genogroup (G) I, 2.6 % GII, and 2.0 % were mixed GI and GII. The detection rate of norovirus GI was 2.1 times higher than GII. With regards to the different shellfish species, 17 % of the oyster samples were positive, while 14.0 and 6.0 % were positive for noroviruses found in mussels and cockles, respectively. Norovirus contamination in the shellfish occurred throughout the year with the highest peak in September. Seventeen norovirus-positive PCR products were characterized upon a partial sequence analysis of the capsid gene. Based on phylogenetic analysis, five different genotypes of norovirus GI (GI.2, GI.3, GI.4, GI.5, and GI.9) and four different genotypes of GII (GII.1, GII.2, GII.3, and GII.4) were identified. These findings indicate the prevalence and distribution of noroviruses in three shellfish species. The high prevalence of noroviruses in oysters contributes to the optimization of monitoring plans to improve the preventive strategies of acute gastroenteritis.     

Environmental Surveillance for Noroviruses in Selected South African Wastewaters 2015–2016: Emergence of the Novel GII.17

Norovirus (NoV) GII.4 is the predominant genotype associated with gastroenteritis pandemics and new strains emerge every 2–3 years. Between 2008 and 2011, environmental studies in South Africa (SA) reported NoVs in 63% of the sewage-polluted river water samples. The aim of this study was to assess whether wastewater samples could be used for routine surveillance of NoVs, including GII.4 variants. From April 2015 to March 2016, raw sewage and effluent water samples were collected monthly from five wastewater treatment plants in SA. A total of 108 samples were screened for NoV GI and GII using real-time RT-qPCR. Overall 72.2% (78/108) of samples tested positive for NoVs with 4.6% (5/108) GI, 31.5% (34/108) GII and 36.1% (39/108) GI + GII strains being detected. Norovirus concentrations ranged from 1.02 × 10² to 3.41 × 10⁶ genome copies/litre for GI and 5.00 × 10³ to 1.31 × 10⁶ genome copies/litre for GII. Sixteen NoV genotypes (GI.2, GI.3, GI.4, GI.5, GI.6, GII.2, GII.3, GII.4, GII.7, GII.9, GII.10, GII.14, GII.16, GII.17, GII.20, and GII.21) were identified. Norovirus GII.2 and GII.17 co-dominated and the majority of GII.17 strains clustered with the novel Kawasaki 2014 variant. Sewage surveillance facilitated detection of Kawasaki 2014 in SA, which to date has not been detected with surveillance in children with gastroenteritis <5 years of age. Combined surveillance in the clinical setting and environment appears to be a valuable strategy to monitor emergence of NoV strains in countries that lack NoV outbreak surveillance.     

Distribution of Naturally Occurring Norovirus Genogroups I,II, and IV in Oyster Tissues

This study evaluated different tissues of naturally contaminated oysters (Crassostrea belcheri) for the presence of noroviruses. RNA from digestive tissues, gills, and mantle of the oysters was extracted and tested for norovirus genogroup (G) I, GII, and GIV using RT-nested PCR. In spiking experiments with a known norovirus, GII.4, the detection limits were 2.97 × 10² RNA copies/g of digestive tissues, 2.62 × 10² RNA copies/g of gills, and 1.61 × 10³ RNA copies/g of mantle. A total of 85 oyster samples were collected from a fresh market in Bangkok, Thailand. Noroviruses were found in the oyster samples (40/85, 47%): GI (29/85, 34.1%), GII (9/85, 10.5%), mixed GI and GII (1/85, 1.2%), and GIV (1/85, 1.2%). All three genogroups were found in the digestive tissues of oysters. Norovirus GI was present in all three tissues with the highest frequency in the mantle, and was additionally detected in multiple tissues in some oysters. GII was also detected in all three tissues, but was not detected in multiple tissues in the same oyster. For genogroup I, only GI.2 could be identified and it was found in all tissues. For genogroup II, three different genotypes were identified, namely GII.4 which was detected in the gills and the mantle, GII.17 which was detected in the digestive tissues, and GII.21 which was detected in the mantle. GIV.1 was identified in the digestive tissues of one oyster. This is the first report on the presence of human GIV.1 in oyster in Thailand, and the results indicate oyster as a possible vehicle for transmission of all norovirus genogroups in Thailand.     

One-year Surveillance of Human Enteric Viruses in Raw and Treated Wastewaters,Downstream River Waters,and Drinking Waters

Human enteric viruses are a major cause of waterborne diseases, and can be transmitted by contaminated water of all kinds, including drinking and recreational water. The objectives of the present study were to assess the occurrence of enteric viruses (enterovirus, norovirus, adenovirus, hepatitis A and E virus) in raw and treated wastewaters, in rivers receiving wastewater discharges, and in drinking waters. Wastewater treatment plants’ (WWTP) pathogen removal efficiencies by adenovirus quantitative real-time PCR and the presence of infectious enterovirus, by cell culture assays, in treated wastewaters and in surface waters were also evaluated. A total of 90 water samples were collected: raw and treated wastewaters (treated effluents and ultrafiltered water reused for industrial purposes), water from two rivers receiving treated discharges, and drinking water. Nested PCR assays were used for the identification of viral DNA/RNA, followed by direct amplicon sequencing. All raw sewage samples (21/21), 61.9 % of treated wastewater samples (13/21), and 25 % of ultrafiltered water samples (3/12) were contaminated with at least one viral family. Multiple virus families and genera were frequently detected. Mean positive PCRs per sample decreased significantly from raw to treated sewage and to ultrafiltered waters. Moreover, quantitative adenovirus data showed a reduction in excess of 99 % in viral genome copies following wastewater treatment. In surface waters, 78.6 % (22/28) of samples tested positive for one or more viruses by molecular methods, but enterovirus-specific infectivity assays did not reveal infectious particles in these samples. All drinking water samples tested negative for all viruses, demonstrating the effectiveness of treatment in removing viral pathogens from drinking water. Integrated strategies to manage water from all sources are crucial to ensure water quality.     

Simple and Rapid Detection of Human Norovirus from Produce Using SYBR Green I-based Real-time RT-PCR

Several foodborne norovirus gastroenteritis outbreaks have been linked to fresh produce. Rapid and sensitive detection can help prevent the release of contaminated produce items in the market. The objectives of this study were to apply a relatively inexpensive SYBR Green I-based real-time RT-PCR assay for the rapid detection of human norovirus (NoV) GI and GII on the surfaces of lettuce, cherry tomatoes, and green onions. Each washed produce commodity (25 g) was spiked with serial dilutions of NoV GI and GII stool samples. RNA was eluted from the produce surface and extracted using the TRIzol^? method. This was followed by detection using SYBR Green I real-time RT-PCR with primers specific for NoV GI (COG1F-COG1R) and GII (COG2F-COG2R) along with an internal RNA amplification control. End-point detection limits from lettuce and tomatoes were found to be 10 RT-PCR units/25 g for GI and GII and 1 RT-PCR unit/25 g for GI and 10 RT-PCR units/25 g for GII from green onions. These results were confirmed by Tm analysis (showing peaks at 81.5 and 84°C for GI and GII, respectively; and 83°C for the IAC) as well as agarose gel electrophoresis that confirmed products of ~95 bp for GI and GII and ~155 bp for the RNA IAC. Results could be obtained within one working day, showing potential for routine use in diagnostics and monitoring of NoV contamination by the produce industry.     

Virus Type-Specific Removal in a Full-Scale Membrane Bioreactor Treatment Process

We investigated removal of noroviruses, sapoviruses, and rotaviruses in a full-scale membrane bioreactor (MBR) plant by monitoring virus concentrations in wastewater samples during two gastroenteritis seasons and evaluating the adsorption of viruses to mixed liquor suspended solids (MLSS). Sapoviruses and rotaviruses were detected in 25% of MBR effluent samples with log reduction values of 3- and 2-logs in geometric mean concentrations, respectively, while noroviruses were detected in only 6% of the samples. We found that norovirus and sapovirus concentrations in the solid phase of mixed liquor samples were significantly higher than in the liquid phase (P < 0.01, t test), while the concentration of rotaviruses was similar in both phases. The efficiency of adsorption of the rotavirus G1P[8] strain to MLSS was significantly less than norovirus GI.1 and GII.4 and sapovirus GI.2 strains (P < 0.01, t test). Differences in the adsorption of viruses to MLSS may cause virus type-specific removal during the MBR treatment process as shown by this study.     

Persistent Norovirus Contamination of Groundwater Supplies in Two Waterborne Outbreaks

Microbiological contamination of groundwater supplies causes waterborne outbreaks worldwide. In this study, two waterborne outbreaks related to microbiological contamination of groundwater supplies are described. Analyses of pathogenic human enteric viruses (noroviruses and adenoviruses), fecal bacteria (Campylobacter spp. and Salmonella spp.), and indicator microbes (E. coli, coliform bacteria, intestinal enterococci, Clostridium perfringens, heterotrophic plate count, somatic and F-specific coliphages) were conducted in order to reveal the cause of the outbreaks and to examine the effectiveness of the implemented management measures. Moreover, the long-term persistence of noro- and adenovirus genomes was investigated. Noroviruses were detected in water samples from both outbreaks after the intrusion of wastewater into the drinking water sources. In the outbreak I, the removal efficiency of norovirus genome (3.0 log₁₀ removal) in the sand filter of onsite wastewater treatment system (OWTS) and during the transport through the soil into the groundwater well was lower than the removal efficiencies of E. coli, coliform bacteria, intestinal enterococci, and spores of C. perfringens (6.2, 6.0, > 5.9, and > 4.8 log₁₀ removals, respectively). In the outbreak II, cleaning of massively contaminated groundwater well and drinking water distribution network proved challenging, and noro- and adenovirus genomes were detected up to 3 months (108 days). The long-term persistence study showed that noro- and adenovirus genomes can remain detectable in the contaminated water samples up to 1277 and 1343 days, respectively. This study highlights the transport and survival properties of enteric viruses in the environment explaining their potency to cause waterborne outbreaks.     

Spatial and Temporal Distribution of Norovirus and <Emphasis Type="Italic">E. coli</Emphasis> in Sydney Rock Oysters Following a Sewage Overflow into an Estuary

This paper reports a study of norovirus (NoV) GII distribution and persistence in Sydney rock oysters (SRO) (Saccostrea glomerata) located in an estuary after a pump station sewage overflow. SRO were strategically placed at six sites spanning the length of the estuary from the pump station to the sea. The spatial and temporal distribution of NoV, hepatitis A virus (HAV) and Escherichia coli (E. coli) in oysters was mapped after the contamination event. NoV GI and GII, HAV and E. coli were quantified for up to 48 days in oysters placed at six sites ranging from 0.05 to 8.20 km from the sewage overflow. NoV GII was detected up to 5.29 km downstream and persisted in oysters for 42 days at the site closest to the overflow. NoV GII concentrations decreased significantly over time; a reduction rate of 8.5% per day was observed in oysters (p < 0.001). NoV GII concentrations decreased significantly as a function of distance at a rate of 5.8% per km (p < 0.001) and the decline in E. coli concentration with distance was 20.1% per km (p < 0.001). HAV and NoV GI were not detected. A comparison of NoV GII reduction rates from oysters over time, as observed in this study and other published research, collectively suggest that GII reduction rates from oysters may be broadly similar, regardless of environmental conditions, oyster species and genotype.     

Viruses Surveillance Under Different Season Scenarios of the Negro River Basin,Amazonia, Brazil

The Negro River is located in the Amazon basin, the largest hydrological catchment in the world. Its water is used for drinking, domestic activities, recreation and transportation and water quality is significantly affected by anthropogenic impacts. The goals of this study were to determine the presence and concentrations of the main viral etiological agents of acute gastroenteritis, such as group A rotavirus (RVA) and genogroup II norovirus (NoV GII), and to assess the use of human adenovirus (HAdV) and JC polyomavirus (JCPyV) as viral indicators of human faecal contamination in the aquatic environment of Manaus under different hydrological scenarios. Water samples were collected along Negro River and in small streams known as igarapés. Viruses were concentrated by an organic flocculation method and detected by quantitative PCR. From 272 samples analysed, HAdV was detected in 91.9 %, followed by JCPyV (69.5 %), RVA (23.9 %) and NoV GII (7.4 %). Viral concentrations ranged from 10² to 10⁶ GC L^?1 and viruses were more likely to be detected during the flood season, with the exception of NoV GII, which was detected only during the dry season. Statistically significant differences on virus concentrations between dry and flood seasons were observed only for RVA. The HAdV data provides a useful complement to faecal indicator bacteria in the monitoring of aquatic environments. Overall results demonstrated that the hydrological cycle of the Negro River in the Amazon Basin affects the dynamics of viruses in aquatic environments and, consequently, the exposure of citizens to these waterborne pathogens.     

Persistence of Viruses by qPCR Downstream of Three Effluent-Dominated Rivers in the Western United States

This study was designed to determine the quantitative polymerase chain reaction (qPCR) signal persistence of viruses in three effluent-dominated streams. Samples were collected from the effluent outfall of three wastewater treatment plants in the Western United States and downstream at different locations. All samples were tested for the presence of pepper mild mottle virus (PMMoV), adenoviruses, norovirus GI and GII, Aichi virus, and enteroviruses using qPCR. PMMoV was detected most frequently in 54/57 (94.7%) samples, followed by adenoviruses which was detected in 21/57 (36.8%) samples. PMMoV was detected at all locations downstream and up to 32 km from the discharge point. This study demonstrated that the detection signal of PMMoV was able to persist in wastewater discharges to a greater degree than human enteric viruses in effluent-dominated rivers.     

Occurrence of Norovirus and Hepatitis A Virus in Wild Mussels Collected from the Baltic Sea

The aim of the study was to define the occurrence of human noroviruses of genogroup I and II (NoV GI and NoV GII) and hepatitis A virus (HAV) in the Baltic Sea mussels. The shellfish samples were taken at the sampling sites located on the Polish coast. In total, 120 shellfish were tested as pooled samples using RT-PCR and hybridisation with virus specific probes. NoV GI was detected in 22 (18.3 %), NoV GII in 28 (23.3 %), and HAV in 9 (7.5 %) of the shellfish. The nucleotide sequence analysis of the detected NoV GII strains showed a 97.3–99.3 % similarity to GII.4 virus strain. This is the first report describing the NoV and HAV occurrence in wild Baltic mussels and their possible role as bioindicators of seawater contamination with human enteric viruses.     

An Outbreak of Norovirus Infection from Shellfish Soup Due to Unforeseen Insufficient Heating During Preparation

Norovirus causes large outbreaks involving all age groups and are considered the most common cause of infectious foodborne diseases worldwide. The aim of this study was to describe a norovirus outbreak connected to insufficient heat treatment during preparation of a shellfish soup in serving portions, during a company Christmas celebration in Norway, December 2013. A questionnaire sent to the employees, showed that 67 % (n = 43) of the celebration participants, reported gastrointestinal symptoms including stomach pain, vomiting, diarrhoea and light fever in the period between 24 and 48 h post celebration. Several dishes were served, including shellfish soup made with carpet shell clams (Tapes rhomboides) in porcelain cups. Consuming this soup, was the only significant risk factor for infection. Norovirus GI and GII were detected in the remaining raw shellfish. To mimic the time and temperature obtained during bivalve soup preparation, raw chopped shellfish tissue and raw cepa onion were added in porcelain cups tempered to 20 °C. To each of these cups, boiling soup base was added. The temperature in the shellfish tissue was continuously recorded, and showed a maximum of 49 °C in the period between 3 and 7 min after adding the boiling soup base. After 1 h the temperature was 30 °C. This time and temperature combination was obviously not sufficient for inactivation of norovirus present in the shellfish tissue. In conclusion, the heat-absorbing capacity of cold ingredients, utensils and table wear porcelain should not be underestimated during food production. Consumers who want to avoid eating raw shellfish, should not assume that the shellfish tissue in preparation as described in our study is adequately heat treated.