Accumulation and Depuration Kinetics of Rotavirus in Mussels Experimentally Contaminated.

Bivalve mollusks as filter-feeders concentrate in their digestive tissue microorganisms likely present in the harvesting water, thus becoming risky food especially if consumed raw or poorly cooked. To eliminate bacteria and viruses eventually accumulated, they must undergo a depuration process which efficacy on viruses is on debate. To better clarify the worth of the depuration process on virus elimination from mussels, in this study we investigated rotavirus kinetics of accumulation and depuration in Mytilus galloprovincialis experimentally contaminated. Depuration process was monitored for 9 days and virus residual presence and infectivity were evaluated by real time quantitative polymerase chain reaction, cell culture and electron microscopy at days 1, 2, 3, 5, 7, 9 of depuration. Variables like presence of ozone and of microalgae feeding were also analyzed as possible depuration enhancers. Results showed a two-phase virus removal kinetic with a high decrease in the first 24 h of depuration and 5 days necessary to completely remove rotavirus.

A relationship between environmental pollutants and enteric viruses in mussels(Mytilus galloprovincialis).

Mussels can be affected by environmental contaminants, as non-dioxin-like polychlorinated biphenyls (NDL-PCBs), polycyclic aromatic hydrocarbons (PAHs) and cadmium (Cd). Moreover, mussels may concentrate human enteric viruses, like noroviruses (NoVGI/GII), astrovirus (AsV) and rotavirus (RV). Herein, to establish a relationship between environmental and viral contamination, with the aim to ensure human food safety, both chemical and microbiological analysis were carried out in mussels Mytilus galloprovincialis, farmed in Campania region (Italy). Chemical analysis revealed ranges below the European maximum limits, and were: ∑6 NDL-PCBs (28, 52, 101, 138, 153, 180) 0.579-16.857 ng g-1 wet weight (ww); BaP LOQ (<0.2 µg kg-1)- 2.9 µg kg-1 ww, and ∑4 PAHs (BaA, CHR, BbFA, BaP) 0.7-23.7 µg kg-1 ww; Cd LOQ (<0.005 mg kg-1)- 0.078 mg kg-1 ww. In addition, mussels exhibited a prevalence of NoVGI (18%), NoVGII (43%), AsV (40%), RV (30%) and the presence of more than one viruses resulted in about 35%. Overall, a simultaneous contamination, both chemical and viral, was found in 62% of samples. Interestingly, virus-positive mussels showed levels of chemicals higher than negative ones.

The role of the mussel Mytilus spp. in the transmission of ostreid herpesvirus-1 microVar.

The Pacific oyster Crassostrea gigas contributes significantly to global aquaculture; however, C. gigas culture has been affected by ostreid herpesvirus-1 (OsHV-1) and variants. The dynamics of how the virus maintains itself at culture sites is unclear and the role of carriers, reservoirs or hosts is unknown. Both wild and cultured mussels Mytilus spp. (Mytilus edulis, Mytilus galloprovincialis and hybrids) are commonly found at C. gigas culture sites. The objective of this study was to investigate if Mytilus spp. can harbour the virus and if viral transmission can occur between mussels and oysters. Mytilus spp. living at oyster trestles, 400-500 m higher up the shore from the trestles and up to 26 km at non-culture sites were screened for OsHV-1 and variants by all the World Organization for Animal Health (OIE) recommended diagnostic methods including polymerase chain reaction (PCR), quantitative PCR (qPCR), histology, in situ hybridization and confirmation using direct sequencing. The particular primers that target OsHV-1 and variants, including OsHV-1 microVar (µVar), were used in the PCR and qPCR. OsHV-1 µVar was detected in wild Mytilus spp. at C. gigas culture sites and more significantly the virus was detected in mussels at non-culture sites. Cohabitation of exposed wild mussels and naïve C. gigas resulted in viral transmission after 14 days, under an elevated temperature regime. These results indicate that mussels can harbour OsHV-1 µVar; however, the impact of OsHV-1 µVar on Mytilus spp. requires further investigation.

Detection and molecular characterization of betanodaviruses retrieved from bivalve molluscs.

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.

Application of next-generation sequencing to investigation of norovirus diversity in shellfish collected from two coastal sites in Japan from 2013 to 2014.

A better understanding of the role played by shellfish regarding the manner of pathogen contamination, persistence, and selection may help considering epidemiology of noroviruses. Thus, norovirus genotype profiles in shellfish (Crassostrea gigas and Mitilus galloprovincialis) were investigated by using Next-generation sequencing (NGS) technology. In genogroup I (GI), 7 genotypes (abbreviated as GI.2 to GI.7, and GI.9) were detected from C. gigas, whereas 9 genotypes (GI.1 to GI.9) were detected from M. galloprovincialis. The genotype with the highest proportion found in both C. gigas and M. galloprovincialis was GI.4, and the second highest was GI.3. In genogroup II (GII), 17 genotypes (GII.1 to GII.9, GII.11 to GII.17, GII.21 and GI.22) were detected from C. gigas, whereas 16 genotypes (GII.1 to GII.8, GII.11 to GII.17, GII.21 and GI.22) were detected from M. galloprovincialis. The genotype with the highest proportion in both C. gigas and M. galloprovincialis was GII.4, the next highest differed between C. gigas and M. galloprovincialis. To our knowledge, this study may be the first trial to utilize the latest technology in this field, and reveal the diversity of norovirus genotypes present in shellfish.

Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain.

Coastal waters can become contaminated with both human waste from sewage treatment plants and runoff following manure application. Thus, shellfish produced close to land can bioaccumulate enteric viruses of human and animal origin, including zoonotic hepatitis E virus that infect both human and swine. The goal of this study was to evaluate the presence of HEV in shellfish from Galicia (NW Spain), a densely populated region with a strong tradition of swine farming, and one of the most important regions in the world for mussel production. We tested 81 mussel batches by RT-qPCR followed by conventional broad-spectrum nested RT-PCR and phylogenetic analysis. We have obtained 12 positive samples by RT-qPCR (14.81%) with HEV contamination levels ranging from 6.7 × 10(1) to 8.6 × 10(4) RNA copies/g digestive tissue. Phylogenetic analysis based on a 330 nt region of the ORF 1 showed that all sequenced isolates belonged to the zoonotic genotype 3 subgenotype e, being closely related to strains of human and swine origin. Results show that shellfish may be a potential route for HEV transmission to humans.

Molecular characterisation of noroviruses detected in mussels (Mytilus galloprovincialis) from harvesting areas in Slovenia.

Noroviruses are a leading cause of viral gastroenteritis in humans and are responsible for many outbreaks worldwide. Mussels are one of the most important foodstuffs connected with norovirus outbreaks, also resulting in multinational dimensions. Two hundred and thirty-eight (238) samples of mussels (Mytilus galloprovincialis) were collected in periods between the years 2006-2008 and 2010-2012 to study the prevalence of noroviruses (NoVs) from harvesting areas along the Adriatic coast of Slovenia. Between 2006 and 2008, 9.1% to 24.6% of mussel samples tested by specific GI and/or GII real-time RT-PCR methods were found to be positive for NoVs while between 2010 and 2012 the percentage of NoV positive samples varied from 12.5% to 22.2%. At the nucleotide level within the RdRp gene fragment the genetic diversity of NoVs detected in mussels ranged between 78.8-81.0% nucleotide identity among GII strains (92.1-99.6% within the GII.P4 genotype), 100% nucleotide identity among GI and 58.4-60.2% among GI and GII strains. Nine of the NoV strains detected from mussels were genotyped as GII.4, while two samples were within GI.P2 and one was a positive sample within genotype GII.P21. This study confirmed that mussels are a potential source of the NoV infection. The detected NoVs share the same topology on the phylogenetic tree within the NoV strains detected in water samples and human patients, not only from Slovenia but also from many different countries worldwide. We can assume that mussels in harvesting areas are not only contaminated from the surrounding area but also by contaminated water and sewage from large transport ships, which are regularly present in the area.

Mathematical model for viral depuration kinetics in shellfish: an useful tool to estimate the risk for the consumers.

Enteric virus depuration from shellfish is a complex biological process that may be influenced by biological properties of the mollusc and/or virus species. On the basis of previous experimental data, a mathematical model was developed to characterize the kinetics of viral elimination during the depuration process. The experimental data consisted on twenty depuration trials, each with 60 kg of Manila clams (Venerupis philippinarum) and mediterranean mussels (Mytilus galloprovincialis) previously subjected to bioaccumulation with HAV or MNV-1 (as a surrogate for human norovirus), that were performed in an experimental depuration system during 7 days. It was observed that although viral loads decay along depuration, a residual viral load remains at the end of the process suggesting a decomposition of viral load in a diluted load (susceptible of depuration) and a non-diluted load (unavailable to depurate). The model yielded a general equation, which can predict the viral load at any depuration time knowing the specific filtration rate, dependent on the bivalve species, and specific viral properties. The mathematical model can be combined with quantitative risk assessment calculations to determine the safety of the depurated shellfish, which can be very helpful not only for shellfish producers but also to public health authorities.

Evaluation of Immunomagnetic Separation Method for the Recovery of Hepatitis A Virus and GI.1 and GII.4 Norovirus Strains Seeded on Oyster and Mussel.

Outbreaks of viral diseases are frequently associated with the consumption of minimally processed shellfish. Among the viruses in these outbreaks, hepatitis A virus (HAV) and human norovirus (NoV) have been increasingly reported as the most common food-borne pathogens. These viruses must be concentrated in tested samples in order to be detected. In this study, a method for the detection of NoV and HAV in shellfish using an immuno-magnetic separation (IMS) procedure combined with reverse transcriptase (RT)-PCR was developed. The IMS/RT-PCR method was applied to investigate the recovery rates of HAV, NoV GI.1, and GII.4 from oyster and mussel. Based on IMS/RT-PCR results, recovery rates for HAV from oyster and mussel test samples were 2.4 and 1.1%, respectively. The NoV GI.1 recovery rates from oyster and mussel samples were 4.9-9.2% (mean 6.9%) and 4.3-8.6% (mean 6.2%), respectively, and the NoV GII.4 recovery rates were 8.8 and 8.5%, respectively. These results verified that HAV, NoV GI.1, and GII.4 can be detected in all the test samples using the IMS/RT-PCR method, although the three inoculated viruses were recovered with low efficiency. In conclusion, the IMS/RT-PCR method can be used to efficiently and rapidly detect viruses such as HAV and NoV in shellfish such as oyster and mussel.

Effectiveness of depuration for hepatitis A virus removal from mussels (Mytilus galloprovincialis).

The efficacy and kinetic of depuration of hepatitis A virus (HAV) were evaluated under experimental conditions with Mediterranean mussels (Mytilus galloprovincialis) subjected previously to bioaccumulation processes. Seven independent trials (70kg of mussels each) were performed in a closed experimental system using two different water temperatures (13 and 17°C) during 7days. The real time RT-PCR technique with TaqMan probes was used for viral quantification. Qualitative infectivity assays were conducted to test the presence of infectious viral particles at the end of the depuration period. The depuration trials showed an average reduction of HAV levels of aproximately 1.1 Log units (>90%). However, the average final viral loads in shellfish samples remain at relatively high levels (6.5×10(3) RNA copies/g digestive tissue) and still infectious. A positive correlation between the initial and the final numbers of the viral RNA copies was observed. The reduction of HAV showed a two-phase removal kinetic, an initial logarithmic trendline, with a rapid reduction of viruses during the first 24-48h of depuration, and a subsequent stabilization with a slower depuration rate until the end of the process.

Noroviruses in seafood: a 9-year monitoring in Italy.

Norovirus (NoV) are increasingly important as etiological agents of gastrointestinal infections. Consumption of bivalve molluscs and ready-to-eat fishery products is one of the most common ways of acquiring NoV foodborne infections, and the rise of outbreaks of viral gastroenteritis represents an important health problem that is also responsible for economic losses. The aim of this work was to define the prevalence of NoV contamination in preserved fishery products and in shellfish commercialized in Italy, taking into account the results obtained during 9 years of survey (2003-2011) and paying special attention to the regions more involved in national production. A total of 4463 samples were examined (2310 mussels, 1517 clams, 510 oysters, 22 other shellfish species, 104 preserved seafood products) and the average positivity rate for NoV presence was 4.1% and ranged from 0.6% in 2007 to 9.8% in 2003 and from 1.9% in preserved seafood products to 4.7% in mussels. Genetic characterization of circulating strains showed a prevalence of genogroup II genotypes, including GII.b and GII.e polymerase types and different GII.4 variants. This information could contribute to the optimization of risk-based sampling strategies for NoV contamination in seafood, taking into account variability in different species and from year to year.

Prevalence of hepatitis A virus in bivalve molluscs sold in Granada (Spain) fish markets.

Viruses are the leading cause of foodborne illness associated with the consumption of raw or slightly cooked contaminated shellfish. The aim of this study was to evaluate the prevalence of hepatitis A virus in molluscs. Standard and real-time reverse transcription-polymerase chain reaction procedures were used to monitor bivalve molluscs from the Granada fish markets (southern Spain) for this human enteric virus. Between February 2009 and October 2010, we collected a total of 329 samples of different types of bivalve molluscs (mussels, smooth clams, striped venus, and grooved clams). The results showed the presence of hepatitis A virus in 8.5% of the 329 samples analyzed. We can therefore confirm that conventional fecal indicators are unreliable for demonstrating the presence or absence of viruses. The presence of hepatitis A virus in molluscs destined for human consumption is a potential health risk in southern Spain.

Presence of hepatitis E RNA in mussels used as bio-monitors of viral marine pollution.

Mussels (Mytilus galloprovincialis), collected from a harvesting area approved by European Community Regulation, were transplanted to four polluted sites located in the Northwestern Mediterranean area (Tuscany). They were used as bio-monitors to test the quality of the marine water pollution. At different times after the transplantation, mussels were withdrawn and tested for presence of phages and enteric viruses by molecular tests. 52.4% of the transplanted mussel samples were positive for at least one enteric virus. Hepatitis A virus (HAV) was identified in each site (17/37; 45.9%). Three samples were positive for hepatitis E virus (HEV) (8.1%) and two (5.4%) for norovirus (NoV) genogroup I. Coliphages and RYC 2056 phages were detected in all sites, while HSP 40 phages were detected in three sites. Results demonstrate the ability of transplanted mussels in accumulating and retaining different species of enteric microorganisms. Their utility as bio-monitor organisms enables testing for viral marine pollution.

Duplex Real Time PCR for the detection of hepatitis A virus in shellfish using Feline Calicivirus as a process control.

The consumption of bivalve shellfish is a common cause of foodborne outbreaks of viral origin. The evaluation of the sanitary quality of these products, however, is still based on bacterial indicators of fecal contamination (Reg. (EC) No. 2073/2005 and No.1441/2007) even if it is known that they are not reliable indicators of viral contamination. In this study a duplex Real Time PCR method for quantitative detection of hepatitis A (HAV) in shellfish was developed. Feline Calicivirus (FCV) was used as a control for assessing the effectiveness of the concentration and extraction process and the ability to eliminate PCR inhibitors present in the food matrix. The specific primers and probes for detection of HAV and FCV, chosen respectively from the 5'-UTR region and in the ORF1 region, were labeled with two different dyes and detected simultaneously. The method was applied on spiked and non-spiked shellfish from a local market. The amplification of HAV in the presence of FCV showed good linearity (R(2)=0.994) and the sensitivity limit of the reaction was at least 5 x 10(2)TCID(50)g(-1) of an hepatopancreas extract.

HSP70 gene expression in Mytilus galloprovincialis hemocytes is triggered by moderate heat shock and Vibrio anguillarum, but not by V. splendidus or Micrococcus lysodeikticus.

Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.