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Mass Mortality Events (MMEs) affecting the noble pen shell Pinna nobilis have been reported since 2016. In this work, we used an in vitro flow cytometric assay to evaluate phagocytosis, coupled with cytology and Electron Microscopy (TEM), to define animal immunocompetence following infection by P. nobilis Picornavirus (PnPV). The study was performed on 27 animals in July 2021 and May 2022 on two natural population from the Ebro Delta (Catalonia, Spain) and animals maintained in captivity at facilities in Valencia and Murcia Aquarium. Hemolymph was collected in the field and in captivity as a non-destructive sampling method. Based on dimension and internal complexity, flow cytometry identified three haemocyte types, distinguished in granulocytes, hyalinocytes and a third type, biggest in size and with high internal complexity and granularity. Those cells corresponded at ultrastructure to hemocytes with advanced phases of PnPV infection and related to cytopathic effect of the replicating virus displaying numerous Double Membrane Vesicles (DMVs) and cells corpse fusion. The results showed that pen shell in captivity had significantly lower Total Hemocyte Count (THC) compared with natural population of Alfacs Bay (mean number of 7-9 x 10 4 vs 2-5 x 10 5  cells/mL, respectively). FACS (Fluorescence-activated cell sorting) based phagocytosis analysis demonstrate that animals in captivity at IMEDMAR-UCV and Murcia Aquarium, had scarce or absent ability to phagocyte the two stimuli (Staphylococcus aureus and Zymosan A) (10,2 % ± 1,7 of positives) if compared with the natural population in Alfacs Bay (28,5 % ± 5,6 of positive). Ultrastructure images showed that PnPV itself can lead to an alteration of the hemocyte cytoskeleton, impairing the capabilities to perform an active phagocytosis and an efficient phagolysosome fusion., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Channel catfish (Ictalurus punctatus) are a food fish extensively reared in aquaculture facilities throughout the world and are also among the most abundant wild catfish species in North America, making them a popular target of anglers. Furthermore, channel catfish are important members of aquatic ecosystems; for example, they serve as a glochidial host for the endangered winged mapleleaf mussel (Quadrula fragosa), making them critical for conserving this species through hatchery-based restoration efforts. During a routine health inspection, a novel aquareovirus was isolated from channel catfish used in mussel propagation efforts at a fish hatchery in Wisconsin. This virus was isolated on brown bullhead cells (ATCC CCL-59) and identified through metagenomic sequencing as a novel member of the family Spinareoviridae, genus Aquareovirus. The virus genome consists of 11 segments, as is typical of the aquareoviruses, with phylogenetic relationships based on RNA-dependent RNA polymerase and major outer capsid protein amino acid sequences showing it to be most closely related to golden shiner virus (aquareovirus C) and aquareovirus C/American grass carp reovirus (aquareovirus G) respectively. The potential of the new virus, which we name genictpun virus 1 (GNIPV-1), to cause disease in channel catfish or other species remains unknown., (© 2024 John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Viruses are crucial for regulating deep-sea microbial communities and biogeochemical cycles. However, their roles are still less characterized in deep-sea holobionts. Bathymodioline mussels are endemic species inhabiting cold seeps and harboring endosymbionts in gill epithelial cells for nutrition. This study unveiled a diverse array of viruses in the gill tissues of Gigantidas platifrons mussels and analyzed the viral metagenome and transcriptome from the gill tissues of Gigantidas platifrons mussels collected from a cold seep in the South Sea. The mussel gills contained various viruses including Baculoviridae, Rountreeviridae, Myoviridae and Siphovirdae, but the active viromes were Myoviridae, Siphoviridae, and Podoviridae belonging to the order Caudovirales. The overall viral community structure showed significant variation among environments with different methane concentrations. Transcriptome analysis indicated high expression of viral structural genes, integrase, and restriction endonuclease genes in a high methane concentration environment, suggesting frequent virus infection and replication. Furthermore, two viruses (GP-phage-contig14 and GP-phage-contig72) interacted with Gigantidas platifrons methanotrophic gill symbionts (bathymodiolin mussels host intracellular methanotrophic Gammaproteobacteria in their gills), showing high expression levels, and have huge different expression in different methane concentrations. Additionally, single-stranded DNA viruses may play a potential auxiliary role in the virus-host interaction using indirect bioinformatics methods. Moreover, the Cro and DNA methylase genes had phylogenetic similarity between the virus and Gigantidas platifrons methanotrophic gill symbionts. This study also explored a variety of viruses in the gill tissues of Gigantidas platifrons and revealed that bacteria interacted with the viruses during the symbiosis with Gigantidas platifrons. This study provides fundamental insights into the interplay of microorganisms within Gigantidas platifrons mussels in deep sea., (© 2024. The Author(s).)

Viruses in the family Rhabdoviridae display remarkable genomic variation and ecological diversity. This plasticity occurs despite the fact that, as negative sense RNA viruses, rhabdoviruses rarely if ever recombine. Here, we describe nonrecombinatorial evolutionary processes leading to genomic diversification in the Rhabdoviridae inferred from two novel rhabdoviruses of freshwater mussels (Mollusca: Bivalvia: Unionida). Killamcar virus 1 (KILLV-1) from a plain pocketbook ( Lampsilis cardium ) is closely related phylogenetically and transcriptionally to finfish-infecting viruses in the subfamily Alpharhabdovirinae . KILLV-1 offers a novel example of glycoprotein gene duplication, differing from previous examples in that the paralogs overlap. Evolutionary analyses reveal a clear pattern of relaxed selection due to subfunctionalization in rhabdoviral glycoprotein paralogs, which has not previously been described in RNA viruses. Chemarfal virus 1 (CHMFV-1) from a western pearlshell ( Margaritifera falcata ) is closely related phylogenetically and transcriptionally to viruses in the genus Novirhabdovirus , the sole recognized genus in the subfamily Gammarhabdovirinae , representing the first known gammarhabdovirus of a host other than finfish. The CHMFV-1 G-L noncoding region contains a nontranscribed remnant gene of precisely the same length as the NV gene of most novirhabdoviruses, offering a compelling example of pseudogenization. The unique reproductive strategy of freshwater mussels involves an obligate parasitic stage in which larvae encyst in the tissues of finfish, offering a plausible ecological mechanism for viral host-switching. IMPORTANCE Viruses in the family Rhabdoviridae infect a variety of hosts, including vertebrates, invertebrates, plants and fungi, with important consequences for health and agriculture. This study describes two newly discovered viruses of freshwater mussels from the United States. One virus from a plain pocketbook ( Lampsilis cardium ) is closely related to fish-infecting viruses in the subfamily Alpharhabdovirinae . The other virus from a western pearlshell ( Margaritifera falcata ) is closely related to viruses in the subfamily Gammarhabdovirinae , which until now were only known to infect finfish. Genome features of both viruses provide new evidence of how rhabdoviruses evolved their extraordinary variability. Freshwater mussel larvae attach to fish and feed on tissues and blood, which may explain how rhabdoviruses originally jumped between mussels and fish. The significance of this research is that it improves our understanding of rhabdovirus ecology and evolution, shedding new light on these important viruses and the diseases they cause., Competing Interests: The authors declare no conflict of interest.

Hepatitis A virus (HAV) continues to be a public health concern and has caused large foodborne outbreaks and economic losses worldwide. Rapid detection of HAV in foods can help to confirm the source of outbreaks in a timely manner and prevent more people getting infected. In order to efficiently detect HAV at low levels of contamination in foods, rapid and easy-to-use techniques are required to separate and concentrate viral particles to a small volume. In the current study, HAV particles were eluted from green onion, strawberry, and mussel using glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and suspended viral particles were captured using protamine-coated magnetic nanoparticles (PMNPs). This process caused a selective concentration of the viral particles, which could be followed by quantitative real-time RT-PCR analysis. Results showed that pH, NaCl concentration, and PMNP amount used for the capturing had significant effects on the recovery efficiency of HAV (P < 0.05). The highest recovery rate was obtained at pH 9.0, 0.14 M NaCl, and 50 μL of PMNPs. The optimized PMNP capturing method enabled the rapid capture and concentration of HAV. A sensitive real-time RT-PCR test was developed with detection limits of 8.3 × 10 0  PFU/15 g, 8.3 × 10 1  PFU/50 g, and 8.3 × 10 0  PFU/5 g of HAV in green onion, strawberry, and mussel, respectively. In conclusion, the PMNP method is rapid and convenient in capturing HAV from complex solid food samples and can generate concentrated HAV sample solutions suitable for high-sensitivity real time RT-PCR detection of the virus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (© 2021 The Society for Applied Microbiology.)

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 log 10 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.

Aims: This study aims to investigate the presence and spatial-seasonal variability of human and fish viruses in coastal marine systems using Ravenna's harbour area (Adriatic Sea, Italy) as a model., Methods and Results: Human viruses (noroviruses and hepatitis A virus) and one of the most threatening finfish pathogens, the nervous necrosis virus (NNV), were investigated in mussels living inside and offshore Ravenna's harbour. Thirty-three and 36·7% of tested mussel samples resulted contaminated by human and fish viruses respectively. A different spatial-seasonal distribution was observed. Human viruses were detected mainly in inner port sites during colder months, while NNV was detected in both inside and offshore of Ravenna's harbour, mainly during warmer months., Conclusions: The presence of human viruses in the inner port close to the city centre could be attributed to wastewaters carrying pathogens in the port environment and this arises public health concerns, however, the presence of these viruses limited to the canal port during the winter can greatly reduce the risk to human health. Regarding NNV, the accumulation and release of viable virus by mussels, could represent a viral source for susceptible finfish. These findings reflect the different epidemiological features of these infections and indicate the importance to choose the correct indicator to monitor viral contaminations., Significance and Impact of the Study: The high frequency of viral contamination pointed out in the study stresses the imperative to monitor the viral presence in all coastal habitats where the high natural value meets several recreational and commercial activities such as the Ravenna's harbour area. Particularly, this study could represent a novel starting point for the development of a more structured bio-monitoring program, in order to ensure improved environmental management and safety of coastal areas., (© 2020 The Society for Applied Microbiology.)

Vibrio parahaemolyticus is a major foodborne pathogen and is also pathogenic to shrimp. Due to the emergence of multidrug-resistant V. parahaemolyticus strains, bacteriophages have shown promise as antimicrobial agents that could be used for controlling antibiotic-resistant strains. Here, a V. parahaemolyticus phage, vB&#95;VpaP&#95;MGD2, was isolated from a clam (Meretrix meretrix) and further characterized to evaluate its potential capability for biocontrol. Podophage vB&#95;VpaP&#95;MGD2 had a wide host range and was able to lyse 27 antibiotic-resistant V. parahaemolyticus strains. A one-step growth curve showed that vB&#95;VpaP&#95;MGD2 has a short latent period of 10 min and a large burst size of 244 phages per cell. Phage vB&#95;VpaP&#95;MGD2 was able to tolerate a wide range of temperature (30 °C-50 °C) and pH (pH 3-pH 10). Two multidrug-resistant strains (SH06 and SA411) were suppressed by treatment with phage vB&#95;VpaP&#95;MGD2 at a multiplicity of infection of 100 for 24 h without apparent regrowth of bacterial populations. The frequency of mutations causing bacteriophage resistance was relatively low (3.1 × 10 -6 ). Phage vB&#95;VpaP&#95;MGD2 has a double-stranded DNA with a genome size of 45,105 bp. Among the 48 open reading frames annotated in the genome, no lysogenic genes or virulence genes were detected. Sequence comparisons suggested that vB&#95;VpaP&#95;MGD2 is a member of a new species in the genus Zindervirus within the subfamily Autographivirinae. This is the first report of a member of the genus Zindervirus that can infect V. parahaemolyticus. These findings suggest that vB&#95;VpaP&#95;MGD2 may be a candidate biocontrol agent against early mortality syndrome/acute hepatopancreatic necrosis disease (EMS/AHPND) caused by multidrug-resistant V. parahaemolyticus in shrimp production.

The complement C1r/C1s, Uegf, and Bmp1 (CUB) domains, which are most exclusively found in extracellular and plasma membrane-related proteins, are involved in various biological processes. In this study, a CUB domain-containing protein (designed as HcCDCP) was cloned and characterized from freshwater pearl mussel (Hyriopsis cumingii). The 2280 bp complete cDNA of the HcCDCP contained a 1002 bp open reading frame, which encoded a protein with 333 amino acids. The predicted HcCDCP protein contained a typical CUB domain and a transmembrane region. The tissue distribution analysis indicated that the HcCDCP was detected in all tissues, and the highest expression was found in hepatopancreas followed by gills. After infection with bacteria (i.e., Staphylococcus aureus and Vibrio parahaemolyticus), virus (white spot syndrome virus) and virus analogs (poly[I:C]), the mRNA level of the HcCDCP was significantly upregulated, suggesting that the HcCDCP might be involved in host immune defense response. The RNA interference revealed that the silencing of the HcCDCP could evidently inhibit the expression levels of lysozyme and tumor necrosis factor. Moreover, the recombinant protein of the CUB domain (rCUB) possessed binding capacity to eight different kinds of bacteria. The polysaccharide binding assay showed that the rCUB specifically bound to lipopolysaccharide, peptidoglycan, and D-mannose. This study provided valuable information for exploring the biological roles of CDCPs in the host defense system of mollusks., (Copyright © 2020. Published by Elsevier Inc.)

Human noroviruses are recognized as the leading worldwide cause of sporadic and epidemic viral gastroenteritis, causing morbidity and mortality in impoverished developing countries and engendering enormous economic losses in developed countries. Transmitted faecal-orally, either via person-to-person contact, or by consumption of contaminated foods or water, norovirus outbreaks are often reported in institutional settings or in the context of communal dining. Bivalve molluscs, which accumulate noroviruses via filter feeding and are often eaten raw or insufficiently cooked, are a common food vehicle implicated in gastroenteritis outbreaks. The involvement of bivalve molluscs in norovirus outbreaks and epidemiology over the past two decades are reviewed. The authors describe how their physiology of filter feeding can render them concentrated vehicles of norovirus contamination in polluted environments and how high viral loads persist in molluscs even after application of depuration practices and typical food preparation steps. The global prevalence of noroviruses in bivalve molluscs as detected by different monitoring efforts is determined and the various methods currently utilized for norovirus extraction and detection from bivalve matrices described. An overview of gastroenteritis outbreaks affirmatively associated with norovirus-contaminated bivalve molluscs as reported in the past 18 years is also provided. Strategies for risk reduction in shellfish contamination and subsequent human infection are discussed., (© 2019 Blackwell Verlag GmbH.)

Human bocavirus (HBoV) has a global distribution and is associated with respiratory and enteric infections, particularly in the paediatric population. In this study, raw sewage and mussel samples were analysed for the presence of HBoV using nested PCR with primers targeting the VP1/VP2 junction. Amplification and sequencing of the 382 bp region followed by phylogenetic analysis indicated the presence of HBoV 2 in mussel samples and HBoV 3 in sewage samples. This is the first report describing the presence of enteric-associated HBoV in environmental samples from South Africa and in mussel samples from the African continent. The results signify the need for further studies examining the potential risk of foodborne transmission of HBoV and highlight the importance of continued screening to determine the prevalence and epidemiology of HBoV in South Africa.

Susceptibility of hard clams Meretrix lusoria to birnavirus (BV) infections caused by temperature variations, from a mechanistic perspective, has rarely been explored. We used a deterministic susceptible-infectious-mortality (SIM) model to derive temperature-dependent key epidemiologic parameters based on data sets of viral infections in hard clams subjected to acute temperature changes. To parameterize seasonal pattern dependence, we estimated monthly based cumulative mortality and basic reproduction numbers (R 0 ) between 1997 and 2017 by way of statistical analysis. Two alternative disease control models were also proposed to assess status of controlled temperature-mediated BV infection by using, respectively, control reproduction number (R C )-control line criterion and removal strategy-based control measure. We showed that based on R C -control strategy, when temperatures ranged from 15 to 26.8°C, proportion of susceptible hard clams removed should be at least 0.22%. Based on removal-control strategy, we found that by limiting pond water temperature to 25-30°C, together with increased removal rates and periods to remove hard clams, it is better to remove hard clams from June and August to reduce both mortality rate and spread of BV. Our results can be used to monitor BV transmission potential in hard clams that will contribute to government control strategy to eradicate future BV epidemics., (© 2019 John Wiley & Sons Ltd.)

Norovirus (NoV) is the leading cause of acute viral gastroenteritis outbreaks in the world. These outbreaks are frequently associated with bivalve shellfish consumption, particularly because these products are often eaten raw or only slightly cooked. In Morocco, regulations concerning the acceptable levels of enteric bacteria indicator organisms in these products have been put in place. However, these regulations do not take into account the risk of viral contamination, and many gastroenteritis outbreaks have been linked to the ingestion of bivalve shellfish from areas that comply with the current food safety criteria. The aim of this study was to investigate NoV presence in shellfish samples (n = 104) collected at four sites owcff Oualidia lagoon (Moroccan Atlantic coast) from November 2015 to February 2017. Samples were analysed using real-time RT-PCR in accordance with the ISO 15216-2 method. NoVs of the genogroup II were detected in 7% of samples that were all collected during the winter months. Moreover, 71% of NoV-positive samples were harvested at sites upstream of the lagoon. These results highlight the need of regularly monitoring viral contamination in bivalve shellfish to limit the risk of viral gastroenteritis outbreaks.

Calich Lagoon is a Mediterranean coastal lagoon located along the northwestern coast of Sardinia (Italy). The connection to marine and fresh water determines the high productivity of this coastal lagoon. Despite its great potential and the presence of natural beds of bivalve mollusks ( Mytilus galloprovincialis ), the lagoon has not yet been classified for shellfish production. In this study, through a multidisciplinary approach, the presence of several bacterial pathogens ( Escherichia coli, Salmonella spp., and Vibrio spp.) and viral pathogens (hepatitis A virus and norovirus genogroups I and II) was evaluated from March 2017 to February 2018. In addition, phytoplankton composition in lagoon waters and associated algal biotoxins (paralytic and diarrhetic shellfish poisoning) in mussels were also monitored. The aim of this study was to provide useful data to improve knowledge about their seasonal presence and to assess the potential risk for public health, as well as to provide input for future conservation and management strategies. In mussels, Salmonella spp. were found in spring, along with E. coli, but Salmonella spp. were not found in autumn or winter, even though E. coli was detected in these seasons. Vibrio parahaemolyticus was found in autumn and winter, but not in spring. Norovirus genogroups I and II were found in winter samples. None of the bacteria were found in summer. Algal biotoxins have never been detected in mussel samples. Among potentially harmful phytoplankton, only Pseudo-nitzschia spp. were present, mainly in summer. The results showed that a possible bacterial and viral contamination, together with the presence of potentially toxic microalgae, is a real problem. Therefore, the development of natural resource management strategies is necessary to ensure the good quality of waters and guarantee the protection of consumers.

This study was conducted to evaluate the microbiological quality of a mangrove estuary in the Vitória Bay region, Espírito Santo, Brazil. We analyzed the presence and concentration of enteric viruses and thermotolerant coliforms in water, mussels (Mytella charruana and Mytella guyanensis), and oysters (Crassostrea rhizophorae), collected over a 13-month period. Human adenovirus, rotavirus A (RVA), and norovirus genogroup II were analyzed by quantitative PCR. The highest viral load was found in RVA-positive samples with a concentration of 3.0 × 10 4  genome copies (GC) L -1 in water samples and 1.3 × 10 5  GC g -1 in bivalves. RVA was the most prevalent virus in all matrices. Thermotolerant coliforms were quantified as colony-forming units (CFU) by the membrane filtration method. The concentration of these bacteria in water was in accordance with the Brazilian standard for recreational waters (< 250 CFU 100 mL -1 ) during most of the monitoring period (12 out of 13 months). However, thermotolerant coliform concentrations of 3.0, 3.1, and 2.6 log CFU 100 g -1 were detected in M. charruana, M. guyanensis, and C. rhizophorae, respectively. The presence of human-specific viruses in water and bivalves reflects the strong anthropogenic impact on the mangrove and serves as an early warning of waterborne and foodborne disease outbreaks resulting from the consumption of shellfish and the practice of water recreational activities in the region.

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., (© 2019 Institute of Food Technologists®.)

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015-2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV; 8.9%), norovirus GI (NoVGI; 10.8%) and GII (NoVGII; 39.7%), rotavirus (RV; 9.0%), astrovirus (AsV; 20.8%), sapovirus (SaV; 18.8%), aichivirus-1 (AiV-1; 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.

The hepatitis E virus (HEV) affects almost 20 million individuals annually, causing approximately 3.3 million acute liver injuries, 56,600 deaths, and huge healthcare-associated economic losses. Shellfish produced close to urban and livestock areas can bioaccumulate this virus and transmit it to the human population. The aim of this study was to evaluate the presence of HEV in molluscan shellfish, in order to deepen the knowledge about HEV prevalence in Galicia (northwestern Spain), and to investigate this as a possible route of HEV transmission to humans. A total of 168 shellfish samples was obtained from two different Galician rías (Ría de Ares-Betanzos and Ría de Vigo). The samples were analyzed by reverse transcription-quantitative PCR (RT-qPCR). RT-nested PCR and sequencing were used for further genotyping and phylogenetic analysis of positive samples. HEV was detected in 41 (24.4%) samples, at quantification levels ranging from non-quantifiable (<10 2 copies of the RNA genome (RNAc)/g tissue) to 1.1 × 10 5 RNAc/g tissue. Phylogenetic analysis based on the open reading frame (ORF)2 region showed that all sequenced isolates belonged to genotype 3, and were closely related to strains of sub-genotype e, which is of swine origin. The obtained results demonstrate a significant prevalence of HEV in bivalve molluscs from Galician rías, reinforcing the hypothesis that shellfish may be a potential route for HEV transmission to humans.

The discharge of human-derived wastewater represents a major threat to water quality with the potential for waterborne disease outbreaks mainly associated with enteric viruses. To prevent illnesses, indicators associated with fecal contamination are monitored in polluted areas, however, their prevalence often does not correlate well with viral pathogens. In this study, we used crAssphage, a recently discovered human-specific gut-associated bacteriophage, for the surveillance of wastewater-derived viral contamination. Untreated and treated wastewater, surface water, sediment and mussel samples were collected monthly over 1 year from the Conwy River and estuary (UK) and were analyzed for crAssphage marker by quantitative PCR. This is the first long-term catchment-to-coast scale study of environmental crAssphage concentrations. CrAssphage was detected in all sample types and showed no distinct seasonal pattern. CrAssphage concentrations were 2 × 10 5 -10 9 genome copies (gc)/L in all untreated wastewater influent and 10 7 -10 8  gc/L in secondary treated effluent samples, 3 × 10 3  gc/L-3 × 10 7  gc/L in surface water samples (94% positive) and 2 × 10 2 -10 4  gc/g sediment (68% positive) and mussel digestive tissue (79% positive). CrAssphage concentrations were 1-5 log 10 higher than human enteric virus titers (norovirus, sapovirus, adenovirus, polyomavirus). Our results indicate that crAssphage is well suited to tracking human wastewater contamination and pollution risk assessment in aquatic environments.

A prospective matched case-control study was conducted to evaluate associations between dietary histories, including consumption of bivalves, diarrhea, and norovirus positive diarrhea in adult ambulatory patients at an outpatient clinic of a hospital in Tokyo, Japan. Ambulatory cases with diarrhea were matched with nondiarrheal control patients, who visited the same clinic. A standardized questionnaire was used to obtain patients' information, including histories of food consumption and clinical information. Norovirus infection was confirmed using real-time reverse transcription polymerase chain reaction. A total of 207 patients, including 69 diarrheal cases and 138 nondiarrheal cases were included in the analysis. Among them, 60 (29.0%) participants reported consuming bivalves. Norovirus was detected in 35% (24/69) of diarrheal cases. Of those, 10 (41.7%) reported consumption of bivalves and of those, 6 (60.0%) consumed raw bivalves. The proportion of those who consumed raw bivalves was significantly higher in norovirus-positive diarrheal cases than in norovirus-negative diarrheal cases (25.0% vs 6.7%; odds ratio [OR], 4.67; 95% confidence interval [CI], 1.1-20.7) and matched nondiarrheal controls (25.0% vs 6.3%, OR: 5.00; 95% CI, 1.1-22.2). The attributable fraction of consuming raw bivalves for norovirus-associated diarrhea to matched nondiarrheal controls was 20.0%. Consuming raw bivalves was substantially attributed to norovirus-associated diarrhea in adult ambulatory patients and preventive measures for reducing the risk associated with consumption of raw bivalves could decrease the incidence of norovirus-associated diarrhea., (© 2019 Wiley Periodicals, Inc.)

Anthropogenic contamination of beaches in the south of Brazil was assessed by detection of Escherichia coli, human mastadenovirus species C (HAdV-C) and F (HAdV-F) and hepatitis E virus (HEV). Sampling was carried out in October (2016), and in January, April and July (2017). Water, sediment, sea surface microlayer (SML), bivalves, and air sentinel samples were evaluated. Quantitative microbiological risk assessment (QMRA) was used to estimate the probability of swimmer infection. HAdV-C was present in 26% of the samples, for both qPCR and viral isolation. The highest rates of detection in genomic copies (GC) were in water (2.42E+10 GC/L), SML (2.08E+10 GC/L), sediment (3.82E+08 GC/g) and bivalves (3.91E+07 GC/g). QMRA estimated daily and annual risks with a maximum value (9.99E-01) in almost all of the samples. Viable HAdV-C was often detected in the SML, pointing that this is a source of infection for people bathing in these waters., (Copyright © 2018. Published by Elsevier Ltd.)

Viral enteropathogens are one of the leading causative agents of foodborne illnesses in both the United States and the European Union. While human noroviruses and hepatitis A virus cause the vast majority of outbreaks and illnesses, there are handful of human enteric viruses that contribute to sporadic outbreaks worldwide including astrovirus, sapovirus, rotavirus, enterovirus and Aichi virus. In addition, hepatitis E virus is increasingly being recognized as an emerging zoonotic threat within the food supply. This review aims to briefly describe the primary human enteric viruses of concern with respect to foodborne transmission. Next, we focus on the contamination and persistence of these viruses within three high-risk food commodities-leafy greens, soft red fruits and bivalve mollusks. As opposed to detailing the specific routes by which these foods can be contaminated with enteric viruses, we have chosen to focus on their persistence and specific interactions within the food itself. Therefore, the processes of attachment and internalization of the viruses in foods have been emphasized. Looking forward, the implications of these specific interactions of human enteric viruses with leafy greens, soft red fruits and bivalve mollusks are briefly considered within the context of future prevention and control strategies., (© 2018 The Society for Applied Microbiology.)

Recently, we reported an emerging pathology named Brown Muscle Disease (BMD) affecting Asari clams inhabiting the most productive area for this species in France, the Arcachon Bay. The main macroscopic feature of the pathology relies on the atrophy of the posterior adductor muscle, affecting the ability of clams to burry. The research of the etiological agent of BMD privileged a viral infection. Contrary to healthy clams, infected animals are always found at the surface of the sediment and exhibit 30 nm virus-like particles in muscle, granulocytic and rectal cells. In order to get more insights on the etiology and impacts of the BMD on clams, we took advantage in the present study of next generation sequencing technologies. An RNA-Seq approach was used (i) to test whether viral RNA sequences can be specifically found in the transcriptome of diseased animals and (ii) to identify the genes that are differentially regulated between diseased and healthy clams. Contrary to healthy buried animals, in diseased clams one sequence showing extensive homologies with retroviridae-related genes was detected. Among the biological processes that were affected in diseased clams, the synaptic transmission process was the most represented. To deepen this result, a new sampling was carried out and the transcription level of genes involved in synaptic transmission was determined in healthy and diseased clams but also in clams with no visible sign of pathology but located at the surface of the sediment. Our findings suggest that muscle atrophy is a latter sign of the pathology and that nervous system could be instead a primary target of the BMD agent., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Bivalve mollusks thrive in environments rich in microorganisms, such as estuarine and coastal waters, and they tend to accumulate various particles, including viruses. However, the current knowledge on mollusk viruses is mainly centered on few pathogenic viruses, whereas a general view of bivalve-associated viromes is lacking. This study was designed to explore the viral abundance and diversity in bivalve mollusks using transcriptomic datasets. From analyzing RNA-seq data of 58 bivalve species, we have reconstructed 26 nearly complete and over 413 partial RNA virus genomes. Although 96.4% of the predicted viral proteins refer to new viruses, some sequences belong to viruses associated with bivalve species or other marine invertebrates. We considered short non-coding RNAs (sncRNA) and post-transcriptional modifications occurring specifically on viral RNAs as tools for virus host-assignment. We could not identify virus-derived small RNAs in sncRNA reads obtained from the oyster sample richest in viral reads. Single Nucleotide Polymorphism (SNP) analysis revealed 938 A-to-G substitutions occurring on the 26 identified RNA viruses, preferentially impacting the AA di-nucleotide motif. Under-representation analysis revealed that the AA motif is under-represented in these bivalve-associated viruses. These findings improve our understanding of bivalve viromes, and set the stage for targeted investigations on the specificity and dynamics of identified viruses.

Aichi virus 1 (AiV-1) has a worldwide distribution and is associated with gastroenteritis in humans. In this study, raw sewage and mussel samples were analyzed for the presence of AiV-1 using reverse transcription-PCR (RT-PCR). Amplification and sequencing of the 3CD and VP1 genomic regions followed by phylogenetic analysis using selected genome sequences revealed the presence of AiV-1, genotype B. The results highlight the importance of further screening to evaluate the prevalence and epidemiology of this clinically important virus in South Africa.

Lack of wastewater treatment efficiency causes receiving seawaters and bivalve molluscan shellfish to become contaminated, which can lead to public health issues. Six wastewater samples, five seawater samples and three batches of giant clams from Tahiti (French Polynesia) were investigated for the presence of enteric viruses, but also if present, for the diversity, infectivity and integrity of human adenoviruses (HAdV). Enteroviruses (EV), sapoviruses (SaV) and human polyomaviruses (HPyV) were detected in all wastewater samples. In decreasing frequency, noroviruses (NoV) GII and HAdV, rotaviruses (RoV), astroviruses (AsV), NoV GI and finally hepatitis E viruses (HEV) were also observed. Nine types of infectious HAdV were identified. HPyV and EV were found in 80% of seawater samples, NoV GII in 60%, HAdV and SaV in 40% and AsV and RoV in 20%. NoV GI and HEV were not detected in seawater. Intact and infectious HAdV-41 were detected in one of the two seawater samples that gave a positive qPCR result. Hepatitis A viruses were never detected in any water types. Analysis of transcriptomic data from giant clams revealed homologues of fucosyltransferases (FUT genes) involved in ligand biosynthesis that strongly bind to certain NoV strains, supporting the giant clams ability to bioaccumulate NoV. This was confirmed by the presence of NoV GII in one of the three batches of giant clams placed in a contaminated marine area. Overall, all sample types were positive for at least one type of virus, some of which were infectious and therefore likely to cause public health concerns.

In a cluster of hepatitis A infections that occurred in Nagano Prefecture in 2017, hepatitis A virus (HAV) was detected in asari clams (reference food) and the patients' fecal samples. Initially, the asari clams were suspected to be the infection source. However, the exact infection route remained unknown because a patient who had not consumed an asari clam dish also developed the disease. Suspecting a secondary infection originating from the asari clams, we investigated the presence of HAV genomes in water used for washing and soaking the frozen asari clams and detected HAV in the soaking water. These results suggest that soaking water is a risk factor for secondary contamination because of the leakage of HAV accumulated in midgut gland of the asari clam. During the asari clam sand removal process, the water used to clean asari clams spread across a wide area in a concentric fashion, raising concerns that this process may aggravate contamination. In addition to HAV, diarrhea viruses, such as norovirus, have often been detected in bivalves, including asari clams. Thus, handling these foodstuffs requires adequate care.

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness, with common vehicles including bivalve molluscan shellfish, soft fruit and various vegetables. Outbreaks of viral illness due to contamination of the surfaces of foods, or food preparation surfaces by for example infected food handlers are also common. Virus analysis of food matrices can contribute towards risk management for these hazards and a two-part technical specification for determination of Hepatitis A virus and norovirus in food matrices (ISO/TS 15216:2013) was published jointly by the European Committee for Standardisation and the International Organization for Standardization in 2013. As part of the European Mandate No. M381 to validate 15 standards in the field of food microbiology, an international validation study involving 18 laboratories from 11 countries in Europe was conducted between 2012 and 2014. This study aimed to generate method characteristics including limit of detection, limit of quantification, repeatability and reproducibility for ISO 15216 - Part 1, the method for quantification, in seven food matrices. The organization and results of this study, including observations that led to improvements in the standard method are presented here. After its conclusion, the method characteristics generated were added to the revised international standard, ISO 15216-1:2017, published in March 2017., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study investigated relationships between organohalogen compound (OHC) exposure, feeding habits, and pathogen exposure in a recovering population of Atlantic walruses (Odobenus rosmarus rosmarus) from the Svalbard Archipelago, Norway. Various samples were collected from 39 free-living, apparently healthy, adult male walruses immobilised at three sampling locations during the summers of 2014 and 2015. Concentrations of lipophilic compounds (polychlorinated biphenyls, organochlorine pesticides and polybrominated diphenyl ethers) were analysed in blubber samples, and concentrations of perfluoroalkylated substances (PFASs) were determined in plasma samples. Stable isotopes of carbon and nitrogen were measured in seven tissue types and surveys for three infectious pathogens were conducted. Despite an overall decline in lipophilic compound concentrations since this population was last studied (2006), the contaminant pattern was similar, including extremely large inter-individual variation. Stable isotope ratios of carbon and nitrogen showed that the variation in OHC concentrations could not be explained by some walruses consuming higher trophic level diets, since all animals were found to feed at a similar trophic level. Antibodies against the bacteria Brucella spp. and the parasite Toxoplasma gondii were detected in 26% and 15% of the walruses, respectively. Given the absence of seal-predation, T. gondii exposure likely took place via the consumption of contaminated bivalves. The source of exposure to Brucella spp. in walruses is still unknown. Parapoxvirus DNA was detected in a single individual, representing the first documented evidence of parapoxvirus in wild walruses. Antibody prevalence was not related to contaminant exposure. Despite this, dynamic relationships between diet composition, contaminant bioaccumulation and pathogen exposure warrant continuing attention given the likelihood of climate change induced habitat and food web changes, and consequently OHC exposure, for Svalbard walruses in the coming decades., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

There are many problems associated with extracting viral genetic material from contaminated samples of bivalve molluscs, specifically because the hepatopancreas has many PCR inhibitors. For this reason, nucleic acid extraction methods must consider a process control virus (PCV) that may help to measure extraction efficiency. In the market, there are many commercial kits to extract nucleic acid from RNA viruses, as well as others to perform one-step real time RT-PCR, but most of them have not been evaluated for bivalve molluscs. For this reason, the aim of this study was to evaluate the extraction efficiency of the PCV (Mengovirus), it was performed using 3 different RNA extraction kits and 2 one-step real time RT-PCR kits. 10 μL of Mengovirus at a concentration of 1.6 × 10 4 viral particles/μL was added to 29 samples of hepatopancreas of Donax sp. Sample processing was performed according to the ISO/TS 15216-2: 2013 standard. RNA was extracted from each sample with the kits: (1) BioMerieux NucliSens®system (BioMérieux SA, France), (2) PureLink™ RNA Mini Kit (Ambion-Life Technologies™, USA) and (3) Hugh Pure RNA Tissue Kit (Roche SA, Germany). Once RNA was extracted, one-step real time RT-PCR was conducted by using the following kits: (A) Ultrasense One-step qRT-PCR Kit (Invitrogen, USA) according to ISO/TS 15216-2:2013, and (B) Mengovirus@ceeramTools™ Kit (Ceeram, France) according to the manufacturer's specifications. The extraction efficiency of PCV when using the extraction kits 1, 2 and 3 combined with real time RT-PCR kit A were: 10.82, 1.90 and 0.64, respectively; and when using real time RT-PCR kit B were: 7.34, 0.97 and 0.47, respectively. It is concluded that the BioMerieux NucliSens®system RNA extraction kit was the most efficient and that the Ultrasense One-step qRT-PCR Kit performed better than the Mengovirus@ceeramTools™ RT-PCR kit., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We determined the abundance and virulence of Vibrio parahaemolyticus in seawater and bivalves from the Gyeongnam coast in Korea, a major area for the seafood industry, during 2004-2016. V. parahaemolyticus is one of the most common pathogen causing seafood-borne illnesses in Korea, and increases during the summer. Its occurrence in seawater and bivalve samples was seasonally dependent, with high levels during the summer to early autumn. There were more strains in the area of sea continually exposed to inland wastewater. Only 5.1% and 3.5% of V. parahaemolyticus isolates from seawater and bivalves, respectively, had the trh gene, and only the bivalve isolates produced the tdh gene at levels below 2%. Continuous monitoring is clearly needed to reduce seafood-borne outbreaks of disease caused by V. parahaemolyticus, and to reveal the occurrence patterns and the presence of toxic genes of the strains in different marine environments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The contamination of mussels and oysters by viruses and bacteria is often associated with water contamination and gastroenteritis in humans. The present study evaluated viral and bacterial contamination in 380 samples, from nine mollusk-producing regions in coastal water north of the Brazilian Amazon. Rotavirus contamination was studied for groups A to H, using a two-step SYBR Green RT-qPCR (quantitative reverse transcription polymerase chain reaction), and bacterial families Enterobacteriaceae, Vibrionaceae, and Aeromonadaceae by classical and molecular methods. From the 19 pools analyzed, 26.3% (5/19) were positive for group A Rotavirus, I2 genotype for VP6 region, without amplifications for groups B-H. Bacteriological analysis identified Escherichia coli isolates in 89.5% (17/19) with identification of atypical enteropathogenic E. coli aEPEC in 10.5% (2/19), Salmonella (Groups C1 and G) (10.5%, 2/19), Vibrio alginolyticus (57.9%, 11/19) V. parahaemolyticus (63.2%, 12/19), V. fluvialis (42.1%, 8/19), V. vulnificus (10.5%, 2/19), V. cholerae non-O1, non O139(10.5%, 2/19) and Aeromonas salmonicida (52.6%, 10/19). All the samples investigated presented some level of contamination by enterobacteria, rotavirus, or both, and these results may reflect the level of contamination in the Northern Amazon Region, due to the natural maintenance of some of these agents or by the proximity with human populations and their sewer.

Human noroviruses (NoVs) are responsible for 50% of food-related disease outbreaks and are notably associated with shellfish consumption. Despite the detrimental health impacts of human NoV-contaminated seafood to public health, there is a lack of knowledge on the physicochemical conditions that govern NoV transmission in aquatic ecosystems. In the present study, we investigated the propensity for NoVs to associate with aquatic aggregates, which have been shown to efficiently deliver nano-sized particles to shellfish. Specific physicochemical conditions characteristic of shellfish cultivation waters, specifically salinity and transparent exopolymer particles (TEP), were targeted in this study for investigating aggregate formation and NoV association dynamics. Murine norovirus (MNV) was used in aggregation experiments as a model surrogate for NoVs. Results demonstrate increased aggregate formation as a function of increasing salinity and TEP concentrations, as well as greater numbers of MNV genomes incorporated into aggregates under conditions that favor aggregation. As aggregate formation was enhanced in waters representing optimal conditions for shellfish production, specifically saline and high TEP waters, the implications to virus transport and shellfish food safety are profound: more aggregates implies increased scavenging of virus particles from surrounding waters and therefor greater risk for bivalve contamination with nano-sized pathogens. These novel data provide insight into where and when NoVs are most likely to be ingested by shellfish via contaminated aggregates, thereby informing best management and water quality monitoring practices aimed at providing safe seafood to consumers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

EC Regulation 854/2004 requires the classification of bivalve mollusc harvesting areas according to the faecal pollution status of sites. It has been reported that determination of Escherichia coli in bivalve shellfish is a poor predictor of norovirus (NoV) contamination in individual samples. We explore the correlation of shellfish E. coli data with norovirus presence using data from studies across 88 UK sites (1,184 paired samples). We investigate whether current E. coli legislative standards could be refined to reduce NoV infection risk. A significant relationship between E. coli and NoV was found in the winter months (October to February) using data from sites with at least 10 data pairs (51 sites). We found that the ratio of arithmetic means (log 10 E. coli to log 10 NoV) at these sites ranged from 0.6 to 1.4. The lower ratios (towards 0.6) might typically indicate situations where the contribution from UV disinfected sewage discharges was more significant. Conversely, higher ratios (towards 1.4) might indicate a prevalence of animal sources of pollution; however, this relationship did not always hold true and so further work is required to fully elucidate the factors of relevance. Reducing the current class B maximum (allowed in 10% of samples) from 46,000 E. coli per 100 g (corresponding NoV value of 75750 ± 103) to 18,000 E. coli per 100 g (corresponding NoV value of 29365 ± 69) reduces maximum levels of NoV by a factor of 2.6 to 1; reducing the upper class B limit to 100% compliance with 4,600 E. coli per 100 g (corresponding NoV value of 7403 ± 39) reduces maximum levels of NoV by a factor of 10.2 to 1. We found using the UK filtered winter dataset that a maximum of 200 NoV corresponded to a maximum of 128 ± 7 E. coli per 100 g. A maximum of 1,000 NoV corresponded to a maximum of 631 ± 14 E. coli per 100 g.

AiV-1 is considered an emerging human enteric pathogens and foodborne transmission has been documented as an important source of exposure for humans, chiefly in relation to non-safe, risky food habits. We surveyed the presence of AiV-1 in retail shellfish, including oysters and mussles, identifying the virus in 3/170 (1.8%) of the analysed samples. The AiV-1 positive samples were of different geographic origin. Upon sequence analysis of a portion of the 3CD junction region, two AiV strains identified from harvesting areas in Northern Italy were characterised as genotype B and displayed 99-100% identity at the nucleotide level to other AiV-1 strains detected in sewages in Central Italy in 2012, suggesting that such strains are stably circulating in Italian ecosystems. Interestingly, a strain identified from mussles harvested in Southern Italy could not be characterised firmly, as inferred in the Bayesian analysis and by sequence comparison, indicating that different AiV strains are also circulating in Italy. Viral contamination in retail shellfish challenges the microbiological guidelines for food control and requires the development and optimization of additional diagnostic and prevention strategies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Noroviruses and hepatitis A virus are the pathogens most frequently involved in non-bacterial gastroenteritis and hepatitis worldwide. They are mainly transmitted via the faecal-oral route, direct person-to-person contact or through the consumption of contaminated water and foods. In food virology, detection methods of these viruses are currently based on real-time reverse transcription-polymerase chain reaction (RT-PCR). A crucial step in this process is the acid nucleic extraction, since its performance can negatively influence viral detection and thus give false negative results. The aim of this study is to evaluate the efficiency of 2 automated extraction systems, MagMAX Express and EZ1 Advanced XL, in recovering hepatitis A virus and norovirus RNA from mussels. In the present study, we used mengovirus as a process control to assess the efficiency of the extraction process. Samples were tested for mengovirus, hepatitis A, and norovirus by real time one-step RT-PCR assay. Our data indicates the MagMax Express is the better system to extract hepatitis A and norovirus RNA from mussels since its extraction efficiency was higher (p<0.05) than EZ1 Advanced XL.

Shellfish samples (n = 310) purchased from local supermarkets were analysed for the presence of hepatitis E virus (HEV) by nested RT-PCR and real-time qRT-PCR. Overall, 2.9% of samples tested positive for the presence of HEV. Phylogenetic analysis of HEV sequences revealed all as being genotype 3 HEV. This is the first report of the detection of HEV in commercially sold shellfish in Scotland. These findings may encourage further research that will help address the gaps in the knowledge in respect to foodborne transmission of HEV in Scotland and the rest of the United Kingdom.

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty-eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000-2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs., (© 2017 John Wiley & Sons Ltd.)

Aim: To improve the efficacy of intercalating dyes to distinguishing between infectious and inactivated hepatitis A virus (HAV) in food., Methods and Results: Different intercalating dyes were evaluated for the discrimination between infectious and thermally inactivated HAV suspensions combining with the RT-qPCR proposed in the ISO 15216. Among them, PMAxx was the best dye in removing the RT-qPCR signal from inactivated HAV. Applied to lettuce and spinach, PMAxx-Triton pretreatment resulted in complete removal of the RT-qPCR signal from inactivated HAV. Likewise, this study demonstrates that this pretreatment is suitable for the discrimination of inactivated HAV in shellfish without further sample dilution. In mussels and oysters, the developed viability RT-qPCR method reduced the signal of inactivated HAV between 1·7 and 2·2 logs at high inoculation level, and signal was completely removed at low inoculation level., Conclusions: This study showed that the use of PMAxx is an important improvement to assess HAV infectivity by RT-qPCR. It was shown that PMAxx-Triton pretreatment is suitable for the analysis of infectious HAV in complex food samples such as vegetables and shellfish., Significance and Impact of the Study: The PMAxx-Triton pretreatment can be easily incorporated to the ISO norm for infectious virus detection., (© 2017 The Society for Applied Microbiology.)

Galicia (NW Spain) has 1490 km of coastline, and its particular topography, characterized by the presence of fiord-like inlets, called rías, with an important primary production, makes this region very favourable for shellfish growth and culture. In fact, Galicia is one of the most important mussel producers in the world. Due to its proximity to cities and villages and the anthropogenic activities in these estuaries, and despite the routine official controls on the bivalve harvesting areas, contamination with material of faecal origin is sometimes possible but, current regulation based on Escherichia coli as an indicator micro-organism has been revealed as useful for bacterial contaminants, this is not the case for enteric viruses. The aim of this review is to offer a picture on the situation of different harvesting areas in Galicia, from a virological standpoint. A recompilation of results obtained in the last 20 years is presented, including not only the data for the well-known agents norovirus (NoV) and hepatitis A virus (HAV) but also data on emerging viral hazards, including sapovirus (SaV), hepatitis E virus (HEV) and aichivirus (AiV). Epidemiological differences related to diverse characteristics of the harvesting areas, viral genotype distribution or epidemiological links between environmental and clinical strains will also be presented and discussed. The presentation of these historical data all together could be useful for future decisions by competent authorities for a better management of shellfish growing areas., (© 2017 The Society for Applied Microbiology.)

Worldwide, norovirus is one of the most associated causes of acute gastroenteritis, which leads to nearly 50 000 child deaths every year in developing countries. Therefore, there is great demand to develop a rapid, low-cost, and accurate detection assay for the foodborne norovirus infection to reduce mortality caused by norovirus. Considering the importance of norovirus, we have demonstrated a highly sensitive and specific colorimetric detection method for analysis of human norovirus genogroups I and II (HuNoV GI and GII) in oyster samples. This is the first report to employ colorimetric HRPzyme-integrated polymerase chain reaction (PCR) for direct norovirus detection from the real shellfish samples. We found that the HRPzyme-integrated PCR method is more sensitive than the gel electrophoresis approach and could detect the HuNoV GI and GII genome up to 1 copy/mL. The specificity of the proposed method was successfully demonstrated for HuNoV GI and GII. Further, we performed testing HuNoVs in the spiked oyster samples, and the HRPzyme-integrated PCR method proved to be an ultrasensitive and selective method for detecting HuNoVs in the real samples. By integration of the proposed method with the portable PCR machine, it would be more reliable to improve food safety by detecting HuNoVs in the different types of shellfish, such as oyster and mussel, at the production field.

Human bocavirus (HBoV) has been shown to be a common cause of respiratory infections and gastroenteritis in children. Recently, HBoVs have been detected in sewage and river waters in Italy and worldwide. However, studies on their presence in other water environments and in bivalve mollusks are not yet available. In this study, 316 bivalve shellfish samples collected in three Italian regions over a 6-year period (2012 to 2017) were analyzed by nested PCR and sequencing using broad-range primer pairs targeting the capsid proteins VP1 and VP2 of HBoV. The virus was detected in 27 samples (8.5% of the total samples), and a statistically significant difference was found within the three regions. A further 13 samples, collected in geographic and temporal proximity to positive samples, were included in the study to assess the spread of HBoV in shellfish production areas at the time of contamination. Twelve of these additional samples were found to be positive for HBoV. All positive samples in this study were characterized as HBoV species 2 (17 samples; 8 different sequences) or species 3 (22 samples; 4 different sequences). This study reports the occurrence of HBoV in bivalve shellfish and shows evidence of considerable spatial spread of the virus throughout shellfish production areas. Further studies are needed to elucidate both the role of HBoV as an agent of gastroenteritis and the risk for foodborne transmission of this virus. IMPORTANCE Human bocavirus is recognized as an important cause of acute respiratory tract infections and has recently been considered an etiological agent of gastroenteritis in the pediatric population. Our findings document that HBoVs are detected in bivalve shellfish with a relevant prevalence and suggest that an assessment of the risk for foodborne transmission of these viruses should be undertaken., (Copyright © 2018 American Society for Microbiology.)

Bivalve mollusks are filter feeders and may accumulate human pathogens in their tissues. Many studies demonstrated human diseases associated with bivalve consumption, especially oysters. Anomalocardia brasiliana clams are distributed along the Brazilian coastal area and are an exotic ingredient for some typical dishes in Brazil. Even though there are several reports describing the contamination of oysters and mussels with human pathogens, there is a lack of studies reporting contamination of A. brasiliana with human pathogens. An evaluation of natural microbiological contamination in A. brasiliana samples over a period of 18months (November 2014 to April 2016) showed that the bacteria indices were in accordance with Brazilian regulations (E. coli<230MPN and Salmonella sp. absent in 25g of meat). However, the enteric viruses evaluated were detected throughout the analysis period, with the highest result for the hepatitis A virus (HAV); followed by Rotavirus-A (RVA); Human Adenovirus (HAdV) and Norovirus GI (NoV GI). The bioaccumulation of enteric viruses by A. brasiliana during a period of 24h was performed using NoV GI and GII, HAV, RVA and HAdV as models. Interestingly the mollusk demonstrated different uptake behaviors in relation to these viruses throughout the time period. NoV GI was the most adsorbed virus after 24h. HAV concentration was <1% at 3h, but it increased to <10% at 8h, remaining unchanged until 12h, and decreasing to <3% at 24h; HAdV reached its highest concentration at 12h, being released by the animals and lowering to <3% at 24h. RVA bioaccumulation was unstable over time, reaching its highest values after 24h (<5%); NoV GII bioaccumulation remained <1%. Thermal inactivation of HAdV-2 in A. brasiliana was also evaluated. After the usual gentle cooking procedure using different times (0, 1, 1.5, 3 and 5mins), viral infectivity was evaluated using ICC-et-RT-qPCR. The temperature inside the DT remained <80°C over time and after 5min of cooking the HAdV reached a decay of 90% (1 log 10 ). The results showed a real warn to the consumers that can be exposed to infectious human viruses if they eat these clams improperly cooked. HAV was the most detected virus in these animals, which may lead to outbreaks. A. brasiliana exhibited distinct behavior in NoV GI bioaccumulation and persistence, pointing to the need for further studies about the cellular ligands used by these viruses to become attached to these clams., (Copyright © 2017. Published by Elsevier B.V.)

Using two serially executed PCRs, the discriminative multiplex two-step RT-PCR (DMT-2 RT-PCR) following the detection seminested two-step RT-PCR (DSN-2 RT-PCR), we found a high frequency presence of BFNNV genotype as well as RGNNV in various domestic and imported shellfish. This was definitely different from the previous reports of outbreaks and asymptomatic infection only by the RGNNV genotype in cultured finfish in Korea. Cultivation of NNV entrapped in shellfish was performed successfully by a blind passage. Thus, in an attempt to elucidate the epidemiology of betanodavirus, experiments conducted on 969 shellfish samples concluded that (i) distribution of NNV genotype, especially BFNNV, in shellfish is clearly different from that found in finfish of the world; (ii) unlike RGNNV, which showed a high rate in summer, BFNNV showed no seasonal variation and this result suggests BFNNVs in the marine environment remain fairly constant throughout the year; and (iii) the entrapped virus in shellfish was alive and culturable in vitro. These results are the first report of high level prevalence of in vitro culturable NNV in shellfish, for both BFNNV and RGNNV, which may present a potential risk in transmitting nodaviruses to host species in a marine environment., (© 2017 John Wiley & Sons Ltd.)

Norovirus (NoV) and Hepatitis A virus (HAV) are the most important viral hazards associated with human illness following consumption of contaminated bivalve molluscs. The effectiveness of the current EU criteria for heat processing of bivalve molluscs (i.e. raising the temperature of the internal mollusc flesh to at least 90°C for a minimum of 90 seconds) was evaluated using predictive microbiology. A HAV thermal inactivation model was developed based on literature data in mollusc matrices during isothermal heat treatment. Application of the developed model demonstrated that the 90°C-90 s requirement may lead to significantly different virus inactivation depending on the commercial process design. This shows the need for the establishment of a Performance Criterion for bivalve molluscs heat processing which will assure a common specified level of consumer protection. A risk-based approach is described that allows for an effective processing design providing a more transparent and objective relation between the thermal processing targets and public health. Model simulations demonstrate that the F-value is a more appropriate Process Criterion than a single time-temperature combination since it enables the food business operators to design a process that is compliant with the safety requirements while at the same time achieving a desired product quality.

Levels of Escherichia coli and male-specific bacteriophages (MSBs) were determined in the filter feeders obtained from retail markets, commercial farms, and wild beds in Korea. The accumulation and elimination of E. coli and MSBs were compared between ascidians and bivalves (oysters and mussels) during relaying and depuration. E. coli concentrations in ascidians from retail markets ranged between < 20 and 460 most probable number/100 g while MSBs were not detected. E. coli levels in bivalves from commercial farms and wild beds were not significantly different but bacterial levels in ascidians were consistently lower. Ascidians exhibited much lower ability than bivalves to accumulate E. coli and MSBs during relaying in a polluted coastal area. This study also shows that an equilibrium was developed between levels of microbes in water and ascidians and shellfish during relaying. E. coli and MSBs in ascidians decreased quickly during depuration in a clean seawater tank. However, after 1 day, E. coli in bivalves decreased by only 1.1-1.6 logs, and the elimination of MSBs was negligible. Therefore, depuration is an effective means to reduce the health risk of contaminated ascidians.

This study comprises the first systematic survey of the occurrence of Norovirus in Mediterranean mussels from harvesting areas in Montenegro coast of Adriatic Sea. Mussels may accumulate contaminants of public health concern, including pathogenic bacteria and viruses. Microbiological monitoring of harvesting areas is based on count of Escherichia coli in bivalve molluscs in the European Union. It is assumed that E. coli does not reflect contamination with enteric viruses. A structured field study was undertaken at six locations in Bay of Kotor, Montenegro, in order to investigate plausible influence of environmental factors on the variability of E. coli and norovirus (NoV). From July 2015 to July 2016, a total of 72 samples of mussels were collected in coastal harvesting areas of the Montenegro. The samples were screened for NoV of genogroups GI and GII using reverse transcription-qPCR (RT-qPCR). There were 43% NoV positive samples with higher presence of genogroup GII (74.2%). With regard to influence of environmental conditions on Norovirus presence, we have proved seasonal pattern of virus occurrence i.e., the largest number of positive samples was noticed during winter, while other physico-chemical factors were not of great significance. It was found that count of E. coli did not correlate with Norovirus prevalence. From the aspect of food safety, an upgrade of monitoring plans could lead to obtaining safer products.

The aim of this study was to assess the trend of hepatitis A virus (HAV) in a coastal zone impacted by a contamination event, providing data for the development of management strategies. A total of 352 samples, including four bivalve mollusc species (Mytilus galloprovincialis, Solen vagina, Venus gallina and Donax trunculus), were taken over a period of 6 months from 27 production areas of the coast and analysis were performed according to ISO/TS 15216-1:2013. HAV presence was detected in 77 samples from 11 production areas and all positive results were related to samples collected in the first 3 months of the surveillance, during which HAV prevalence was 39.9% and values as high as 5096 genome copies/g were detected. A progressive reduction of viral contamination was evident during the first trimester of the monitoring, with prevalence decreasing from 78.8% in the first month, to 37.8% in the second and 3.9% in the third and quantitative levels reduced from an average value of 672 genome copies/g to 255 genome copies/g over a period of 4 weeks (virus half-life: 21.5 days). A regression analysis showed that, during the decreasing phase of the contamination, the data fitted a reciprocal quadratic model (Ra 2  = 0.921) and, based on the model, a residual presence of HAV could be estimated after negativization of the production areas. The statistical analysis of the results per shellfish species and per production area showed that there were limited differences in contamination prevalence and levels among diverse bivalve species, while a statistically significant difference was present in quantitative levels of one production area. These data could be useful for the development of both risk assessment models and code of practice for the management of viral contamination in primary production.