Prevalence, distribution and environmental effects on faecal indicator bacteria and pathogens of concern in commercial shellfish production areas in a subtropical region of a developing country (Santa Catarina, Brazil).

This paper reviews recent literature on the abundance and distribution of faecal indicator bacteria and pathogens in shellfish production areas in the state of Santa Catarina, on the subtropical coast of Brazil. This state supplies > 95% of the national production of shellfish. Microbiological monitoring data were mapped using GIS and the results compared with those from other countries. Coastal human population is the main predictive parameter for faecal bacteria in the production areas. Temporal variations of the bacteria can also be predicted by solar radiation and rainfall. The prevalence of pathogens such as hepatitis A virus, human norovirus, Salmonella spp. and Vibrio spp. does not differ substantially from that in developed countries. The information reported here can be used to inform development of microbiological risk profiles for shellfish production areas.

Reservoirs of Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) in Squid and Shrimp Species of Northern Alboran Sea.

The production of the aquaculture industry has increased to be equal to that of the world fisheries in recent years. However, aquaculture production faces threats such as infectious diseases. Betanodaviruses induce a neurological disease that affects fish species worldwide and is caused by nervous necrosis virus (NNV). NNV has a nude capsid protecting a bipartite RNA genome that consists of molecules RNA1 and RNA2. Four NNV strains distributed worldwide are discriminated according to sequence homology of the capsid protein encoded by RNA2. Since its first description over 30 years ago, the virus has expanded and reassortant strains have appeared. Preventive treatments prioritize the RGNNV (red-spotted grouper nervous necrosis virus) strain that has the highest optimum temperature for replication and the broadest range of susceptible species. There is strong concern about the spreading of NNV in the mariculture industry through contaminated diet. To surveil natural reservoirs of NNV in the western Mediterranean Sea, we collected invertebrate species in 2015 in the Alboran Sea. We report the detection of the RGNNV strain in two species of cephalopod mollusks (Alloteuthis media and Abralia veranyi), and in one decapod crustacean (Plesionika heterocarpus). According to RNA2 sequences obtained from invertebrate species and reported to date in the Mediterranean Sea, the strain RGNNV is predominant in this semienclosed sea. Neither an ecosystem- nor host-driven distribution of RGNNV were observed in the Mediterranean basin.

Applicability Evaluation of Male-Specific Coliphage-Based Detection Methods for Microbial Contamination Tracking.

Outbreaks of food poisoning due to the consumption of norovirus-contaminated shellfish continue to occur. Male-specific (F+) coliphage has been suggested as an indicator of viral species due to the association with animal and human wastes. Here, we compared two methods, the double agar overlay and the quantitative real-time PCR (RT-PCR)-based method, for evaluating the applicability of F+ coliphage-based detection technique in microbial contamination tracking of shellfish samples. The RT-PCR-based method showed 1.6-39 times higher coliphage PFU values from spiked shellfish samples, in relation to the double agar overlay method. These differences indicated that the RT-PCR-based technique can detect both intact viruses and non-particle-protected viral DNA/RNA, suggesting that the RT-PCR based method could be a more efficient tool for tracking microbial contamination in shellfish. However, the virome information on F+ coliphage-contaminated oyster samples revealed that the high specificity of the RT-PCR- based method has a limitation in microbial contamination tracking due to the genomic diversity of F+ coliphages. Further research on the development of appropriate primer sets for microbial contamination tracking is therefore necessary. This study provides preliminary insight that should be examined in the search for suitable microbial contamination tracking methods to control the sanitation of shellfish and related seawater.

Aichi virus inactivation by heat in 2-ml glass vials.

Aichi virus (AiV) that results in gastroenteritis worldwide, is spread through contaminated shellfish and water. The resistance/tolerance of AiV to common inactivation processes along with the absence of commercially available vaccines makes it necessary to study its thermal inactivation kinetics. This research evaluated the heat inactivation of AiV in cell-culture media using 2-ml sterile glass vials by the linear and Weibull models. Heat treatments of AiV titers of 7 log plaque forming units (PFU)/ml were conducted thrice in a water-bath at 50, 54, and 58 °C for up to 90 min. Plaque assays for each dilution in duplicate were used to determine infectious virus titers. Linear model D-values for AiV at 50 ± 1 °C (± = standard error) (come-up time = 68 s), 54 ± 0.7 °C (130 s), and 58 ± 0.6°C (251 s) were 43.3 ± 4.23 (R2 = 0.40, RMSE = 0.56), 5.69 ± 0.28 (R2 = 0.80, RMSE = 0.43), and 1.20 ± 0.63 min (R2 = 0.69, RMSE = 0.39), respectively, and the linear model z-value was 5.14 ± 0.39°C (R2 = 0.99, RMSE = 0.08). For the same temperatures, the Weibull model td = 1 values were 20.98 ± 8.8 (R2 = 0.62, RMSE = 0.46, α (scale parameter) = 2.30, ß (shape parameter) = 0.38), 3.84 ± 0.69 (R2 = 0.85, RMSE = 0.38, α = 1.08, ß = 0.66), and 0.87 ± 0.10 min (R2 = 0.80, RMSE = 0.32, α = 0.22, ß = 0.61), respectively and the z-value (using Td = 1 ) was 5.79 ± 0.22 °C (R2 = 1.0, RMSE = 0.03). A better fit was obtained with the Weibull model for log reductions versus time with higher R2 and lower RMSE values. Application of AiV inactivation parameters can help reduce the risk of AiV outbreaks.

Hepatitis A virus strains identified in jogaejeot associated with outbreaks in Seoul, South Korea.

Jogaejeot, seasoned Venerupis philippinarum, is a traditional Korean fermented food, and hepatitis A virus (HAV) can be transmitted through contaminated food, especially bivalve shellfish, causing acute gastroenteritis worldwide. Here, we carried out a phylogenetic analysis to identify and characterize HAV strains in jogaejeot samples associated with hepatitis A (HA) outbreaks in Seoul, South Korea, in 2019. The HAV strains were identified using blast and molecular analysis of the amplified HAV VP1-P2B genome region. The HAV strains identified in the five jogaejeot samples shared at least 99% sequence identity, were all classified as genotype IA and were most closely related to strains that are widespread in East Asia. These results support a link between the consumption of jogaejeot and the HA outbreaks observed in 2019 in Seoul. In addition, they indicate a need for more stringent enforcement of food safety regulations for the shellfish industry, especially against HAV, and the value of widespread vaccination.

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.

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.

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.

Impact of chlorine dioxide and electron-beam irradiation for the reduction of murine norovirus in low-salted "jogaejeotgal", a traditional Korean salted and fermented clam.

Clam jeotgal, called "jogaejeotgal," is a Korean fermented seafood product with, generally, a high amount of added salt to inhibit the growth of pathogenic microorganisms. This study aimed to evaluate the efficacy of chlorine dioxide (ClO2) and sodium hypochlorite (NaOCl) against murine norovirus 1 (MNV-1), a surrogate for human norovirus, in salt-fermented clam, jogaejeotgal. The sequential effect of ClO2 and electron-beam (e-beam) irradiation on the inactivation of MNV-1 was also investigated. Treatments of up to 300 ppm ClO2 and 1000 ppm NaOCl were used to determine the disinfectant concentrations at which more than 1 log (90%) MNV-1 inactivation occurred. The sequential treatment of ClO2 (50-300 ppm) and e-beam (1-5.5 kGy) was performed after storage at 4 °C for 7 days. There was a 1.9-log reduction of the virus in seasoned clams irradiated at 5.5 kGy after ClO2 treatment at 300 ppm. No significant change (p > 0.05) in physicochemical quality was observed after the combined treatment, suggesting the potential for the use of a combined treatment using ClO2 (300 ppm) and e-beam (5.5 kGy) in the jeotgal manufacturing industry for the reduction of norovirus.

Incidence of hepatitis A and hepatitis E viruses and norovirus and rotavirus in fish and shrimp samples caught from the Persian Gulf / [Incidência de vírus da hepatite A e hepatite E e norovírus e rotavírus em amostras de peixes e camarões capturados no Golfo Pérsico]

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.(AU)

Vírus transmitidos por alimentos, incluindo hepatite A (HAV), norovírus (NoV), rotavírus (RoV) e hepatite E (HEV) são facilmente transmitidos através de frutos do mar contaminados. Esta pesquisa foi realizada para avaliar a incidência de RoV, NoV GI e GII, hAV e hEV em amostras de peixes e camarões capturadas no Golfo Pérsico, Irã. Foram coletadas 300 amostras de peixes e camarões. A presença de vírus transmitidos por alimentos foi avaliada por PCR em tempo real. Quarenta e nove das 320 amostras de peixes e camarões (15,31%) foram positivas para vírus transmitidos por alimentos. A distribuição de hAV, NoV GI e NoV GII entre as amostras estudadas foi 0,93%, 5,93% e 8,43%, respectivamente. Os vírus hEV e RoV não foram encontrados nas amostras estudadas. Os peixes Parastromateus niger e Scomberomorus commerson e o camarão Penaeus monodon foram as amostras mais frequentemente contaminadas. A incidência simultânea de hAV e NoV GI, e hAV e NoV GII foi de 0,31% e 0,93%, respectivamente. A distribuição dos vírus transmitidos por alimentos nas amostras coletadas na primavera, verão, outono e inverno foi de 14,28%, 9,33%, 11,76% e 24,44%, respectivamente. Os resultados demonstram que a incidência de vírus transmitidos por alimentos foi significativamente associada às espécies de frutos do mar e também à época da amostragem.(AU)

Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels.

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log10 between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

Occurrence of Human Enteric Viruses in Water Sources and Shellfish: A Focus on Africa.

Enteric viruses are a diverse group of human pathogens which are primarily transmitted by the faecal-oral route and are a major cause of non-bacterial diarrhoeal disease in both developed and developing countries. Because they are shed in high numbers by infected individuals and can persist for a long time in the environment, they pose a serious threat to human health globally. Enteric viruses end up in the environment mainly through discharge or leakage of raw or inadequately treated sewage into water sources such as springs, rivers, dams, or marine estuaries. Human exposure then follows when contaminated water is used for drinking, cooking, or recreation and, importantly, when filter-feeding bivalve shellfish are consumed. The human health hazard posed by enteric viruses is particularly serious in Africa where rapid urbanisation in a relatively short period of time has led to the expansion of informal settlements with poor sanitation and failing or non-existent wastewater treatment infrastructure, and where rural communities with limited or no access to municipal water are dependent on nearby open water sources for their subsistence. The role of sewage-contaminated water and bivalve shellfish as vehicles for transmission of enteric viruses is well documented but, to our knowledge, has not been comprehensively reviewed in the African context. Here we provide an overview of enteric viruses and then review the growing body of research where these viruses have been detected in association with sewage-contaminated water or food in several African countries. These studies highlight the need for more research into the prevalence, molecular epidemiology and circulation of these viruses in Africa, as well as for development and application of innovative wastewater treatment approaches to reduce environmental pollution and its impact on human health on the continent.

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.

Hepatitis A Virus Strains Circulating in the Campania Region (2015-2018) Assessed through Bivalve Biomonitoring and Environmental Surveillance.

The genetic diversity of Hepatitis A Virus (HAV) circulating in the Campania Region in years 2015-2018 was investigated through the monitoring of sentinel bivalve shellfish and water matrices. Overall, 463 water samples (71 sewage samples, 353 coastal discharge waters, and 39 seawaters samples), and 746 bivalve shellfish samples were analyzed. Positivity for HAV was detected in 20/71 sewage samples, 14/353 coastal discharge waters, 5/39 seawaters, and 102/746 bivalve shellfish. Sixty-one of the positive samples were successfully sequenced and were characterized as genotype IA (n = 50) and IB (n = 11). The prevalent strain circulating in 2015 in both bivalves and waters was the IA strain responsible for the outbreak occurring around the same time in the Naples area. This variant was no longer identified in subsequent years (2017-2018) when, instead, appeared two of the IA variants of the multistate outbreak affecting men who have sex with men (MSM), VRD_521_2016, and RIVM-HAV16-090, with the former prevailing in both shellfish and water environments. HAV IB isolates were detected over the years in shellfish and in water matrices, but not in clinical samples, suggesting that this genotype had been circulating silently. An integrated surveillance system (environment/food/clinical cases) can be a useful tool to monitor changes in viral variants in the population, as well as an early warning system.

Comparative Transcriptome Analysis of Litopenaeus vannamei Reveals That Triosephosphate Isomerase-Like Genes Play an Important Role During Decapod Iridescent Virus 1 Infection.

Decapod iridescent virus 1 (DIV1) results in severe economic losses in shrimp aquaculture. However, little is known about the physiological effect of DIV1 infection on the host. In this study, we found that the lethal dose 50 of DIV1-infected Litopenaeus vannamei after 48, 72, 96, and 156 h were 4.86 × 106, 5.07 × 105, 2.13 × 105, and 2.38 × 104 copies/µg DNA, respectively. In order to investigate the mechanisms of DIV1 infection, a comparative transcriptome analysis of hemocytes from L. vannamei, infected or not with DIV1, was conducted. The BUSCO analysis showed that the transcriptome was with high completeness (complete single-copy BUSCOs: 57.3%, complete duplicated BUSCOs: 41.1%, fragmentation: 0.8%, missing: 0.8%). A total of 168,854 unigenes were assembled, with an average length of 601 bp. Based on homology searches, Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and cluster of orthologous groups of proteins (KOG) analysis, 62,270 (36.88%) unigenes were annotated. Among them, 1,112 differentially expressed genes (DEGs) were identified, of which 889 genes were up-regulated and 223 genes were down-regulated after DIV1 infection. These genes were mainly annotated to the major metabolic processes such as fructose and mannose metabolism, carbon metabolism, and inositol phosphate metabolism. Among these metabolic pathways, the triosephosphate isomerase (TPI) family was the most eye-catching DEG as it participates in several metabolic processes. Three types of TPI, LvTPI-like, LvTPI-Blike, and LvTPI-Blike1, were obtained for gene silencing by RNA interference. The results showed that LvTPI-like and LvTPI-Blike1 silencing caused a high mortality rate among L. vannamei. However, LvTPI-like and LvTPI-Blike silencing reduced DIV1 replication in DIV1-infected L. vannamei. All the results indicated that TPI-like genes play an important role during DIV1 infection, which provides valuable insight into the infection mechanism of DIV1 in shrimp and may aid in preventing viral diseases in shrimp culture.

A Targeted Metagenomics Approach to Study the Diversity of Norovirus GII in Shellfish Implicated in Outbreaks.

Human noroviruses (NoV) cause epidemics of acute gastroenteritis (AGE) worldwide and can be transmitted through consumption of contaminated foods. Fresh products such as shellfish can be contaminated by human sewage during production, which results in the presence of multiple virus strains, at very low concentrations. Here, we tested a targeted metagenomics approach by deep-sequencing PCR amplicons of the capsid (VP1) and polymerase (RdRp) viral genes, on a set of artificial samples and on shellfish samples associated to AGE outbreaks, to evaluate its advantages and limitations in the identification of strains from the NoV genogroup (G) II. Using artificial samples, the method allowed the sequencing of most strains, but not all, and displayed variability between replicates especially with lower viral concentrations. Using shellfish samples, targeted metagenomics was compared to Sanger-sequencing of cloned amplicons and was able to identify a higher diversity of NoV GII and GIV strains. It allowed phylogenetic analyses of VP1 sequences and the identification, in most samples, of GII.17[P17] strains, also identified in related clinical samples. Despite several limitations, combining RdRp- and VP1-targeted metagenomics is a sensitive approach allowing the study NoV diversity in low-contaminated foods and the identification of NoV strains implicated in outbreaks.

Estimating the distribution of norovirus in individual oysters.

Norovirus in oysters is a significant food safety risk. A recent ISO detection method allows for reliable and repeatable estimates of norovirus concentrations in pooled samples, but there is insufficient data to estimate a distribution of copies per animal from this. The spread of norovirus accumulated across individual oysters is useful for risk assessment models. Six sets of thirty individual Crassostrea gigas oysters were tested for norovirus concentration levels by reverse-transcription quantitative PCR (RT-qPCR): three from a commercial harvest site, and three post-depuration. Five sets had norovirus GII means above the limit of quantification (LOQ), and one below the LOQ, but above the limit of detection. No norovirus GI was detected in pooled tests, and individual oysters were not tested for norovirus GI. Depuration was shown to reduce the mean concentration of GII copies, but not to affect the shape of the distribution around the mean. Deconvoluting the uncertainty of the method, the coefficient of variation was stationary (0.45 ±â€¯0.2). The best fit distribution was either a lognormal distribution or a gamma. Multiplying these distributions by the weight of oyster digestive tissues gave an estimate for the count mean. This was used as the parameter λ in three compound Poisson distributions: Poisson-lognormal, Poisson-gamma, and Poisson-K. No model was found to fit better than the others, with advantages for each. All three could be used in future risk assessments. Preliminary validation of sampling uncertainty using repeated testing data from a previous study suggests that these results have predictive power.

Contamination of Clams with Human Norovirus and a Novel Hepatitis A Virus in Cameroon.

Shellfish constitute an important protein source but may be contaminated by viruses from various origins. A study performed on clams collected in Cameroon showed a high prevalence of norovirus and hepatitis A virus. After sequencing, the hepatitis A virus showed similarities with the genotype V simian strains.

Hepatitis E virus genotype 3 in echinoderms: First report of sea urchin (Paracentrotus lividus) contamination.

Hepatitis E virus (HEV) deriving from manure application runoffs and faecal waste spill over of swine and human origin bypass wastewater treatment plants and contaminate coastal waters. Shellfish bioaccumulate enteric viruses such as HEV from fecally contaminated coastal waters and under current European Regulations, shellfish sanitary status surveillance is mandatory but only by means of bacterial faecal indicators. The sea urchins are under the same regulations and their vulnerability to fecal contamination has been pointed out. Since they are consumed raw and with no steps to control/reduce hazards, sea urchin contamination with enteric viruses can represent a food safety risk. Hence, the aim of the present study was to screen sea urchin gonads destined for human consumption for the presence of HEV. HEV was detected and quantified in gonads of sea urchins collected in north Portugal by a reverse transcription-quantitative PCR (RT-qPCR) assay targeting the ORF3 region, followed by genotyping by a nested RT-PCR targeting the ORF2 region. Sequencing and phylogenetic analysis clustered the HEV sequence within genotype 3, subgenotype e. This the first study reporting HEV contamination of sea urchins. We hypothesize that like shellfish, sea urchins can also be a food vehicle for HEV transmission to humans.

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.