PMAxx-RT-qPCR to Determine Human Norovirus Inactivation Following High-Pressure Processing of Oysters.

Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.

Oyster Heat Shock Protein 70 Plays a Role in Binding of Human Noroviruses.

Human noroviruses (HuNoVs) are important foodborne pathogens causing acute gastroenteritis. Oysters are an important vehicle for the transmission of HuNoVs. Histo-blood group antigen (HBGA)-like substances are considered the primary ligands for bioaccumulation of HuNoVs in oyster tissues. In this study, proteinaceous ligands for specific binding of HuNoVs were mined from oyster tissues using a bacterial cell surface display system. The macromolecular target was captured and identified in proteomic analysis. The distribution of viral particles, oyster heat shock protein 70 (oHSP 70), and type A HBGA (positive control) in oyster tissue was investigated by multiplex immunofluorescence assays after artificial contamination with HuNoVs (GII.4). Our results demonstrated that oHSP 70 is a candidate vital ligand for specific binding of HuNoVs in oyster tissues. In addition, P proteins (GI.1 and GII.4) and viral particles (GI.1 and GII.4) were captured by recombinant oHSP 70 in an enzyme-linked immunosorbent assay with a sample signal/negative signal of 7.8, 6.3, 17.0, and 8.8, respectively. The findings suggested that oHSP 70 plays an important role in the binding of these foodborne viruses. IMPORTANCE Human noroviruses (HuNoVs) are the most important pathogen for nonbacterial epidemic gastroenteritis cases. Foodborne transmission plays an important role in HuNoVs infection. Oysters, filter-feeding epibenthic bivalves, can be contaminated by fecal discharge in harvest water. A new proteinaceous ligand for HuNoVs other than HBGA is identified in oyster tissues. The significance of our research is in identifying and verifying the ligands in oyster tissues for HuNoV binding. Our data will allow a better understanding of HuNoV attachment in and transmission by oysters, leading to the control of undesired foodborne disease.

Reduction in Virulence over Time in Ostreid herpesvirus 1 (OsHV-1) Microvariants between 2011 and 2015 in Australia.

Microvariant genotypes of Ostreid herpesvirus 1 (OsHV-1) are associated with mass mortality events of Pacific oysters in many countries. The OsHV-1 microvariant (µVar) emerged in France 2008 and caused significant economic losses as it became endemic and displaced the previously dominant OsHV-1 reference genotype. Recently, considerable genotypic variation has been described for OsHV-1 microvariants, however, less is known about variation in viral phenotype. This study used an in vivo laboratory infection model to assess differences in total cumulative mortality, peak viral load, transmissibility, and dose-response for three OsHV-1 isolates obtained between 2011 and 2015 from endemic waterways in Australia. This followed field observations of apparent reductions in the severity of mass mortalities over this time. Significantly higher hazard of death and cumulative mortality were observed for an isolate obtained in 2011 compared to isolates from 2014-2015. In keeping with other studies, the hazard of death was higher in oysters challenged by injection compared to challenge by cohabitation and the mortality was higher when the initial dose was 1 × 104 OsHV-1 DNA copies per oyster injection compared to 1 × 102 DNA copies. There was no difference in the quantity of OsHV-1 DNA at time of death that could be related to isolate or dose, suggesting similar pathogenetic processes in the individual oysters that succumbed to end-stage disease. While the isolates examined in this study were biased towards pathogenic types of OsHV-1, as they were collected during disease outbreaks, the variation in virulence that was observed, when combined with prior data on subclinical infections, suggests that surveillance for low virulence genotypes of OsHV-1 would be rewarding. This may lead to new approaches to disease management which utilize controlled exposure to attenuated strains of OsHV-1.

Predominance of Human Bocavirus Genotypes 1 and 2 in Oysters in Thailand.

Human bocavirus (HBoV) has been recognized as an important pathogen that causes respiratory infection and acute gastroenteritis in young children worldwide. HBoV is most likely transmitted by the respiratory route and by fecal-oral transmission. Recently, HBoV has been detected in several types of environmental water and in bivalve shellfish. However, study of the existence of HBoV in oysters is still undocumented in Thailand. In this study, 144 oyster samples collected from different markets in Chiang Mai, Thailand, in 2017 and 2018 were investigated for the presence of HBoV by nested PCR and sequencing. HBoV was detected in 11 out of 144 samples (7.6%). Nine HBoV-positive samples (81.8%) were identified as genotype 1 (HBoV1) and two (18.2%) as HBoV2. A monthly investigation of HBoV in oyster samples from July 2017 to June 2018 showed that HBoV was sporadically detected in particular months spanning the rainy and colder season, with a peak in January. This study demonstrates the presence and genotype diversity of HBoV in oyster samples in Thailand. The findings contribute to evaluating the risk of foodborne transmission of HBoV and to monitoring outbreaks of HBoV in Thailand and in other countries. IMPORTANCE Human bocavirus is recognized as an important cause of respiratory infection and of acute gastroenteritis in children worldwide. Human bocavirus has been widely detected in many clinical specimens, as well as in several types of environmental samples. Most previous studies describe the incidence of bocavirus infection in humans, whereas few data are available for the occurrence of human bocavirus in food materials, particularly that in bivalve shellfish. Our findings provide evidence for the existence and prevalence of human bocavirus in oysters, suggesting that further monitoring of the potential risk of food- and waterborne transmission of this virus to humans should be undertaken.

Detection of Rotavirus Vaccine Strains in Oysters and Sewage and Their Relationship with the Gastroenteritis Epidemic.

Rotavirus is one of the major causes of infectious gastroenteritis among infants and children, and live attenuated vaccines for rotavirus A (RVA), namely, Rotarix and RotaTeq, have recently become available in Japan. Rotavirus is known to be excreted from patients and accumulated in oysters similar to norovirus; however, the vaccine strains in aquatic environments or oysters have not yet been analyzed. In this study, we focused on wild-type RVA, which is highly important in considering the risk of infectious diseases. We quantified total RVA, Rotarix, and RotaTeq strains in oyster and sewage samples collected between September 2014 and July 2016 to assess the contamination levels of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of total RVA. The positive rates of wild-type RVA, Rotarix, and RotaTeq in oysters were 54, 14, and 31%, respectively. These rates were comparable to those of wild-type RVA (57%) and RotaTeq (35%) in sewage; however, Rotarix was not detected in any sewage samples. The comparison of viral concentrations in oysters and sewage suggested more efficient accumulation of the vaccine strains in oysters than the wild-type RVA. The concentration of wild-type RVA in oysters was significantly correlated with that in sewage with a lag time of -6 to 0 weeks which is required for viral transportation from wastewater treatment plants to oysters. On the other hand, no significant correlation was observed between wild-type RVA concentration in sewage and the number of rotavirus-associated gastroenteritis cases, implying the existence of asymptomatic RVA-infected individuals.IMPORTANCE We quantified rotavirus A (RVA), Rotarix, and RotaTeq strains in oyster and sewage samples during two gastroenteritis seasons and revealed the exact contamination of wild-type RVA by subtracting the quantitative value of rotavirus vaccine strains from that of RVA. The concentration of wild-type RVA was significantly correlated between oysters and sewage, although no significant correlation was seen between wild-type RVA concentration in sewage and the number of rotavirus-associated gastroenteritis cases. This finding suggested the existence of asymptomatic patients and that monitoring of rotavirus vaccine strain could be useful to understand the trend of wild-type RVA and rotavirus outbreak in detail. We believe that our study makes a significant contribution to the literature because it reports the detection of rotavirus vaccine strains in oysters.

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.

Investigation of F-RNA Bacteriophage as a Tool in Re-Opening Australian Oyster Growing Areas Following Sewage Spills.

Oysters contaminated with human enteric viruses from sewage are implicated in foodborne outbreaks globally. Bacteriophages have been identified as potential indicators for these viruses, but have not been used in shellfish management outside of the USA. This study aimed to determine the background levels of F-RNA phage in five Australian oyster growing areas with a history of sewage spills and closures, over an 18-month period. In addition, oysters from five growing areas impacted by adverse sewage events were investigated for F-RNA phage, Escherichia coli, norovirus (NoV) and hepatitis A virus (HAV). F-RNA phage ≤ 60 pfu/100 gm shellfish flesh were found to represent a conservative background level in the surveyed areas. Following two of the five sewage spills, elevated phage levels were observed in most sample sites less than 4 days post spill. By 7 days, most sites from all events had phage < 30 pfu/100 gm. NoV was detected in day 1 and day 6 samples from one event when all phage were ≤ 30 pfu/100 gm. NoV was also detected in a day 3 sample from another event with < 30 phage pfu/100 gm, however, multiple replicate samples had elevated phage levels. The results of this study add evidence on the potential use of F-RNA phage as a tool in early re-opening of oyster harvest areas post sewage spills. However, it also highlights the need to better understand situations where phage testing may be ineffectual, and the importance of sampling at multiple sites and over multiple time points, to effectively capture evidence of contamination.

Influence of fucosidase-producing bifidobacteria on the HBGA antigenicity of oyster digestive tissue and the associated norovirus binding.

Bivalve molluscan shellfish such as oysters are filter feeders and are able to accumulate human noroviruses (NoVs) largely due to the presence of human histo-blood group antigens (HBGAs)-like carbohydrates in their intestine. Since the fucose contents play a key role in the binding of NoVs to HBGAs, this study intended to investigate the influence of fucosidase-producing bifidobacteria on the HBGA antigenicity of oyster digestive tissue and the associated NoV binding. On the contrary to the expected, after a treatment of the oyster digestive tissue extracts with Bifidobacterium bifidum strain JCM 1254, the binding of human NoV GII.4 virus like particles (VLPs) to the oyster digestive tissue extracts enhanced significantly (OD450 from 1.15 ± 0.05 to 1.51 ± 0.02, P < 0.001) in an in vitro direct binding assay. The accumulation of human NoV GII·P16-GII.4 also enhanced significantly in the intestine of B. bifidum JCM 1254 treated oysters from 4.27 ± 0.25 log genomic copies/g oyster digestive tissue to 5.25 ± 0.29 log genomic copies/g oyster digestive tissue (P < 0.005) as observed in an in vivo test. Correspondingly, the type A antigenicity of the oyster digestive tissue extracts enhanced (OD450 from 0.77 ± 0.04 to 1.06 ± 0.05, P < 0.01) after the treatment with B. bifidum JCM 1254. These results could be explained by the substrate specificity of the B. bifidum JCM 1254 associated fucosidases. This study identified an indirect interaction possibly happening between the bacterial microbiota with human NoVs during their transmission in the food systems. We also supplied a potential strategy to mitigate the NoV contamination from shellfish, suppose bacterial strains with specified fucosidase production could be obtained in the future.

Strategies to reduce norovirus (NoV) contamination from oysters under depuration conditions.

Depuration of oysters can effectively reduce levels of E. coli, however, may not be effective in safeguarding against viral contamination (EFSA, 2012). These trials assess the removal of Norovirus Genogroups I and II (NoV GI and GII) and F + RNA bacteriophage genogroup II (FRNAP-II) from oysters under depuration using molecular and viability assay methods. Our results show consistently better removal of NoV GII compared with Nov GI. We found approximately 46% removal of NoV GII at 18 °C after 2 days and 60% after 5 days compared with a maximum of 16% NoV GI removal. Twice the rate of NoV GII removal was achieved at 18 °C compared with 8 °C after 5 days. Results suggest better NoV removal when depuration water salinity is close to that prevailing in the harvesting area. Trials investigating algal feeding, light/dark and disturbance from pump vibration did not show any significant effect. We found that FRNAP-II was more readily removed than NoV. No significant difference was found between the rate of removal (as measured by RT-qPCR) and inactivation (as measured by bioassay) of FRNAP-II. This indicates that reduction in FRNAP-II may be primarily due to physical removal (or destruction) rather than in situ inactivation of the virus.

Detection of Human polyomavirus 2 (HPyV2) in oyster samples in northern Brazil.

BACKGROUND: Human polyomavirus 2 (HPyV2 or JCPyV) is persistent in the environment due to its excretion in urine and feces; it is detected in samples of wastewater, surface water and drinking water. A lack of basic sanitation and sewage collection results in the presence of this virus in food, especially in oysters, since they are bioaccumulators and are consumed in their natural form, thus posing a risk to human health. METHODS: This study investigated the frequency of HPyV2 in samples of oysters marketed in northeastern Pará State, Brazil, and optimized a real-time PCR (qPCR) protocol for the detection of an endogenous oyster control. A total of 217 oysters in 22 pools from five municipalities in the state of Pará were analyzed. Samples underwent dissection and total maceration of oyster tissue using a viral concentration technique, followed by DNA extraction with phenol-chloroform and amplification of the VP1 region for molecular detection via qPCR. RESULTS: HPyV2 was detected in 18.2% (4/22) of the pooled samples, with frequencies of 25, 20, 20 and 16% in the municipalities of Salinópolis, Augusto Corrêa, São Caetano de Odivelas and Curuçá, respectively. Notably, the sample pool from the municipality of Bragança did not have detectable HPyV2 and this was the only sampled location with a water treatment station. In this study, Crassostrea genus-specific primers (AFL52 ribosomal RNA gene) of oyster were developed for use as an endogenous control in the qPCR analysis, which will be useful for future studies. CONCLUSIONS: The detection of HPyV2 in oyster samples commercialized in the state of Pará shows the circulation of this virus in the studied municipalities. Thus, it is necessary to implement measures for improving sewage collection and basic sanitation to avoid contamination of water and food with HPyV2.

Marine virus predation by non-host organisms.

Viruses are the most abundant biological entities in marine environments, however, despite its potential ecological implications, little is known about virus removal by ambient non-host organisms. Here, we examined the effects of a variety of non-host organisms on the removal of viruses. The marine algal virus PgV-07T (infective to Phaeocystis globosa) can be discriminated from bacteriophages using flow cytometry, facilitating its use as a representative model system. Of all the non-host organisms tested, anemones, polychaete larvae, sea squirts, crabs, cockles, oysters and sponges significantly reduced viral abundance. The latter four species reduced viral abundance the most, by 90, 43, 12 and 98% over 24 h, respectively. Breadcrumb sponges instantly removed viruses at high rates (176 mL h-1 g tissue dry wt-1) which continued over an extended period of time. The variety of non-host organisms capable of reducing viral abundance highlights that viral loss by ambient organisms is an overlooked avenue of viral ecology. Moreover, our finding that temperate sponges have the huge potential for constant and effective removal of viruses from the water column demonstrates that natural viral loss has, thus far, been underestimated.

Metagenomic to evaluate norovirus genomic diversity in oysters: Impact on hexamer selection and targeted capture-based enrichment.

Human virus transmission through food consumption has been identified since many years and the international trade increases the risk of dissemination of viral pathogens. The development of metagenomic approach holds many promises for the surveillance of viruses in food and water. This work aimed to analyze norovirus diversity and to evaluate strain-dependent accumulation patterns in three oyster types by using a metagenomic approach. Different hexamer sets to prime cDNA were evaluated before capture-based approach to enhance virus reads recovery during deep sequencing. The study includes the use of technical replicates of artificially contaminated oysters and the analysis of multiple negatives controls. Results showed a clear impact of the hexamer set used for cDNA synthesis. A set of In-house designed (I-HD) hexamers, selected to lower mollusk amplification, gave promising results in terms of viral reads abundancy. However, the best correlation between CT values, thus concentrations, and number of reads was observed using random hexamers. Random hexamers also provided the highest numbers of reads and allowed the identification of sequence of different human enteric viruses. Regarding human norovirus, different genogroups and genotypes were identified among contigs longer than 500 bp. Two full genomes and six sequences longer than 3600 bases were obtained allowing a precise strain identification. The use of technical triplicates was found valuable to increase the chances to sequence viral strains present at low concentrations. Analyzing viral contamination in shellfish samples is quite challenging, however this work demonstrates that the recovery of full genome or long contigs, allowing clear identification of viral strains is possible.

Large concomitant outbreaks of acute gastroenteritis emergency visits in adults and food-borne events suspected to be linked to raw shellfish, France, December 2019 to January 2020.

On 27 December 2019, the French Public Health Agency identified a large increase in the number of acute gastroenteritis and vomiting visits, both in emergency departments and in emergency general practitioners' associations providing house-calls. In parallel, on 26 and 27 December, an unusual number of food-borne events suspected to be linked to the consumption of raw shellfish were reported through the mandatory reporting surveillance system. This paper describes these concomitant outbreaks and the investigations' results.

Optimisation and evaluation of an automated system for extraction of viral RNA from oysters.

The NucliSENS MiniMAG (Minimag) system from bioMérieux is widely used for extraction of viral RNA from oysters and is included as informative material in the ISO method for quantification of hepatitis A virus (HAV) and norovirus genogroups I and II (GI and GII) in food (ISO 15216-1:2017). However, the system is no longer on sale within the EU and alternative methods are therefore needed. We optimised and evaluated an automated benchtop system for extraction of viral RNA from oysters artificially contaminated with HAV, norovirus GI, norovirus GII and mengovirus, using the same reagents and a similar protocol as with the Minimag method. Using the automated system instead of Minimag increased measured viral concentration by on average 1.3 times, suggesting that the automated system extracts viral RNA more efficiently than Minimag. A drawback with the automated system was that it displayed higher variability in measured concentration for mengovirus. The median viral recovery was 17%, 37%, 44% and 41% for samples extracted with the automated system and 15%, 27%, 34% and 23% for samples extracted with Minimag for HAV, norovirus GI, norovirus GII and mengovirus, respectively. All samples displayed <75% inhibition in RT-qPCR when extracted with the automated system or Minimag. Together, these results suggest that the automated system can be a suitable alternative to Minimag in analysis of HAV, norovirus GI and norovirus GII in oysters. However, verification using naturally contaminated oysters is needed before it can be used for food safety control purposes.

Evaluation of Inactivation of Murine Norovirus in Inoculated Shell Oysters by High Hydrostatic Pressure Treatment.

One of the major foods causing norovirus gastroenteritis is bivalve shellfish, such as oysters. Depuration and relaying methods have been used to control norovirus. However, these methods may be inadequate to control norovirus gastroenteritis. The present study aimed to investigate the effectiveness of high hydrostatic pressure (HHP) treatment in controlling norovirus in shelled oysters, by evaluating the inactivating effect of HHP on murine norovirus strain 1 (MNV-1) inoculated into a buffer, oyster homogenate, and shelled oysters. First, MNV-1 was inoculated (infectivity of 4.5 log PFU/mL) into the buffer and oyster homogenate, with a pH of 6.3 and salinity (NaCl) of 1.5%, mimicking the habitats of the Pacific oyster (Crassostrea gigas). HHP treatment at 100, 200, 275, and 300 MPa for 2 and 5 min was conducted at an initial temperature of 0 or 5°C. The infectivity of MNV-1 in both the buffer and the oyster homogenate was lower when the initial temperature was 0°C. In the buffer, the infectivity of MNV-1 decreased to 1.8 log PFU/mL after HHP treatment (200 MPa for 5 min at 0°C), and the inactivating effect was higher in the buffer than in the oyster homogenate. MNV-1 was inoculated into shelled oysters (4.8 log PFU per oyster), and HHP treatment was done at 275, 300, and 350 MPa for 5 min at the initial temperature of 0°C. The infectivity of MNV-1 decreased to 2.8 log PFU per oyster after HHP treatment at 275 MPa for 5 min. The results indicate that the inactivating effect of HHP treatment varies, depending on the medium surrounding the viral particles. Inactivation was best in buffer, followed by oyster homogenate and shelled oysters. The data could inform the development of methods to control norovirus in oysters.

GII.17 norovirus infections in outbreaks of acute nonbacterial gastroenteritis in Osaka City, Japan during two decades.

Norovirus (NoV) is a major cause of viral gastroenteritis, and GII.4 has been the predominant genotype worldwide since the mid-1990s. During the 2014 to 2015 winter, a rare genotype, NoV GII.17, emerged and became prevalent mainly in East Asia. Over the past two decades, NoV molecular surveillance in Osaka City, Japan, has revealed that NoV GII.17 was detected for the first time in February 2001 and that NoV GII.17-associated outbreaks remarkably increased during the 2014 to 2015 season, with higher incidence recorded in January to March 2015. Genetic analysis indicated that 28 GII.17 outbreak strains were closely related to the novel GII.P17-GII.17 variants represented by the Kawasaki308/2015/JP strain, similar to that in other regions. Statistical analysis showed that NoV GII.17 infections were more common in adults than GII.3 and GII.4 infections, suggesting that the affected adults most likely did not have antibodies against NoV GII.17 and the novel GII.17 variant had recently appeared. Regarding transmission, food was one of the most important factors involved in the spread of NoV GII.17 among adults; 61% of GII.17 outbreaks were foodborne, with oysters being the most common vehicle. Interplay between pathogens, hosts, and environmental factors was considered to be important in the 2014 to 2015 NoV GII.17 epidemic.

Occurrence of Human Enteric Adenoviruses in Fresh Tropical Seafood from Retail Markets and Landing Centers.

Human adenoviruses (HAdVs) are the foodborne enteric pathogens transmitted by the consumption of contaminated shellfish. In this study, the occurrence of enteric adenoviruses in finfish and shellfish was investigated by virus concentration and polymerase chain reaction (PCR). Total plate count, total coliform, and fecal coliform levels were determined and correlated with the presence of adenovirus. Samples of fish, bivalve mollusks, crustaceans, and cephalopods were collected from supermarkets, landing centers, and retail fish markets of Mumbai, India for the study. Overall, the adenovirus DNA was detected in 21.27% of all the samples analyzed. The highest incidence was detected in clams (14.89%), followed by oysters, shrimps, and finfish (2.13% each). High prevalence of enteric adenovirus in filter-feeding bivalves, such as clams and oysters, as well as in fish suggests persistent fecal contamination of coastal waters in the region of study. The occurrence of adenoviruses in samples showed a positive correlation with the bacteriological indicators of fecal contamination, suggesting that fecal indicator bacteria may be used to monitor the presence of adenoviruses in seafood. PRACTICAL APPLICATION: This research demonstrates the occurrence of human adenoviruse (HAdV) in fresh seafood and the utility of fecal coliforms as indicators of HAdV presence in seafood. The study emphasizes the need to identify HAdV in seafood as a human health hazard and implement measures to prevent sewage pollution of fish and shellfish harvesting areas in India.

Norovirus Monitoring in Oysters Using Two Different Extraction Methods.

Detection of noroviruses in bivalve shellfish is difficult because of the low concentration of norovirus and the presence of reverse transcription (RT)-PCR inhibitors. This study aimed to assess the presence of noroviruses in oysters extracted using a proteinase K extraction (ISO 15216 method) and an adsorption-elution method. Seventy oyster samples were extracted using the two extraction methods and evaluated using RT-nested PCR. The results showed norovirus detection rates at an equal frequency of 28.6%, of which a total of 48 (68.6%) samples had corresponding positive or negative results, while there were 22 (31.4%) samples with discrepant results. Norovirus genogroup (G)I, GII, and mixed GI and GII were detected in 20%, 4.3%, and 4.3% of samples, respectively, by the proteinase K extraction method, which comprised of GI.2, GI.5b, GI.6b, GII.4, and GII.17 genotypes. With the adsorption-elution method noroviruses were detected in 17.1%, 8.6%, and 2.9% of samples, respectively, which comprised of GI.2, GII.2, GII.4, and GII.17 genotypes. All norovirus-positive oyster samples were further estimated for genome copy number using RT-quantitative PCR. The oyster samples processed using the adsorption-elution method contained norovirus GI of 3.36 × 101-1.06 × 105 RNA copies/g of digestive tissues and GII of 1.29 × 103-1.62 × 104 RNA copies/g. Only GII (2.20 × 101 and 7.83 × 101 RNA copies/g) could be quantified in samples prepared using the proteinase K extraction method. The results demonstrate the different performance of the two sample-processing methods, and suggest the use of either extraction method in combination with RT-nested PCR for molecular surveillance of norovirus genotypes in oysters.

Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.