Detection and quantification of hepatitis A virus and norovirus in Spanish authorized shellfish harvesting areas

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The prevalence of human forms of Sapovirus, an emerging pathogen of human gastroenteritis, was investigated in an 18-month survey from class B mollusc-harvesting areas in two Galician rias (northwest Spain). The detection and quantification of Sapovirus was performed by reverse transcription-real-time PCR, according to the recently developed standard method ISO/TS 15216-1:2013, and genotyping by reverse transcription-nested PCR. The bivalve species studied were wild and cultured mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and Venerupis decussata), and cockles (Cerastoderma edule). Sapovirus was detected in 30 out of 168 samples (17.9%), with cockles being the species with the highest prevalence of positives (28.1%), followed by clams (22.6%), wild mussels (14.3%), and cultured mussels (12.9%). The estuary in the south of the region demonstrated a higher percentage of positive samples (21.8%) than the one in the north (14.4%). Viral contamination levels for the positive samples ranged between 1.9 ؋ 10 3 and 1.4 ؋ 10 5 RNA copies/g of digestive tissue. Thirteen Sapovirus sequences could be obtained based on partial capsid gene sequence and were classified into four genotypes: GI.1 (2 samples), GI.2 (8 samples), GIV.1 (2 samples), and GV.1 (1 sample). Sapovirus (SaV), within the family Caliciviridae, is an etiological agent of human gastroenteritis, primarily in children (1, 2). In 1977 its type strain, Sapporo virus (Hu/SV/GI/Sapporo/77/JP), was first detected during an outbreak in a Japanese orphanage (3). Since then, five genogroups have been identified based on complete capsid gene sequences (4, 5). Genogroups I, II, IV, and V comprise human strains, whereas genogroup III was detected only in swine strains. The SaV genome is a single-stranded positivesense RNA molecule of approximately 7.3 to 7.5 kb with two or three open reading frames (ORF), depending on the genogroup (4, 5). ORF1 encodes nonstructural proteins such as viral protein genome-linked (VPg), protease, RNA-dependent RNA polymerase (RdRp), and a capsid protein (VP1), while ORF2 and ORF3 encode proteins of unknown function (5, 6). The development of molecular techniques became crucial for the study of SaV, as it cannot be cultivated in the laboratory. Reverse transcription-PCR (RT-PCR) is the method used worldwide to detect SaV. It has a high sensitivity and can be used to analyze the virus genetically when it is coupled with the sequencing of amplicons (7-10). Reverse transcription-quantitative real-time PCR (RT-qPCR) also is widely used to detect the virus. It is highly sensitive and useful for determining quantitative results (11).SaV is transmitted via the fecal-oral route, either through person-to-person contact or from contaminated food and water (5, 12). SaV outbreaks are less common than those caused by human norovirus (NoV), the major cause of human gastroenteritis, being detected principally in Southeast Asia. However, SaV recently was included in the U.S. Environmental Protection Agency's Candidate List (CCL3) as an...

Section: Discussioncontrasting

confidence: 59%

“…Samples were collected from 10 authorized production points in two Galician estuaries, 5 in Ría de Ares-Betanzos in the north and 5 in Ría de Vigo in the south (19). All sampling points are class B according to EU regulations (20).…”

Section: Methodsmentioning

confidence: 99%

Prevalence and Genetic Diversity of Human Sapoviruses in Shellfish from Commercial Production Areas in Galicia, Spain

Varela

Polo

Romalde

2016

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“…Fifty microliters of heat-treated or untreated TV, HuNoV GII.4, or HuNoV GII. 4 VLPs was added to 100 l of PGM-MBs, and the volume was brought up to 1 ml by adding 850 l of PBS in a low-adhesion 1.5-ml centrifuge tube. The mixture was shaken for 30 min at room temperature.…”

Section: Preparation Of Pgm-mbsmentioning

confidence: 99%

“…Viral titers were 400 times higher in the shellfish samples than in the growing water, indicating high levels of natural bioaccumulation (3). In the Galician Rias area, the largest shellfish production area in the European Union, 55% of mussel, clam, and cockle samples were contaminated by HuNoV genogroup I (GI) and GII and HAV (4). Thus, understanding of the ecology and persistence of enteric viruses in shellfish is needed to help prevent future outbreaks.…”

mentioning

confidence: 99%

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Araud

DiCaprio

et al. 2016

f Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin-magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.A pproximately 7.6 million to 14.5 million illnesses in the United States are attributed to the consumption of contaminated seafood each year, and enteric viruses are responsible for more than 50% of these cases (1). In a review of the available epidemiological evidence, human norovirus (HuNoV) and hepatitis A virus (HAV) were the leading viruses associated with shellfish, accounting for 83.7% and 12.8% of outbreaks, respectively (2). The type of shellfish most frequently associated with viral outbreaks was oysters, which were the vehicle in 58.4% of outbreaks (2). In some regions, human enteric viruses are practically ubiquitous in harvested shellfish. Keller et al. (3) showed that 100% of shellfish samples collected from Vitória Bay, Espírito Santo, Brazil, were positive for rotavirus (RV) and adenovirus. However, only 80% of the growing water samples were positive for these pathogens. Viral titers were 400 times higher in the shellfish samples than in the growing water, indicating high levels of natural bioaccumulation (3). In the Galician Rias area, the largest shellfish production area in the European Union, 55% of mussel, clam, and cockle samples were contaminated by HuNoV genogroup I (GI) and GII and HAV (4). Thus, understanding of the ecology and persistence of enteric viruses in shellfish is needed to help prevent future outbreaks.The consumption of uncooked contaminated bivalve shellfish continues to pose a public ...

“…Thereby, the resulting inactivated viral particles should still be able to accumulate in shellfish, and short RNA fragments targeted by molecular techniques remain detectable (26)(27)(28), which can explain the high prevalence of HuNoV genomes detected in shellfish. In this context, the 5.0% to 76.2% positive results for the detection of HuNoV genomes in shellfish intended for commercial use (17,(29)(30)(31)(32)(33)(34)(35)(36)(37) are difficult to interpret in terms of infectious risk. Although the dose-response concept is often observed (3,18), HuNoV genome levels may be sometimes low in shellfish commodities involved in HuNoV gastroenteritis outbreaks (19) and may be high in nonoutbreak-related samples (18).…”

mentioning

confidence: 99%

Relevance of F-Specific RNA Bacteriophages in Assessing Human Norovirus Risk in Shellfish and Environmental Waters

Hartard

Banas

Loutreul

et al. 2016

Human noroviruses (HuNoVs) are the main cause of shellfish-borne gastroenteritis outbreaks. In the absence of routine technical approaches allowing infectious particles to be detected, this viral pathogen is currently targeted by genome research, leading to difficult interpretations. In this study, we investigated the potential of F-specific RNA bacteriophages (FRNAPH) as fecal and viral contamination indicators in shellfish and water from a local harvesting area. FRNAPH were also used as microbial source tracking tools. Constraints imposed by detection limits are illustrated here by the detection of infectious FRNAPH in several samples in the absence of FRNAPH genomes. The opposite situation was also observed, likely explained by the persistence of the genomes being greater than infectivity. Similar considerations may be applied to HuNoVs, suggesting that HuNoV genome targeting is of limited relevance in assessing infectious risks. While FRNAPH did not provide any benefits compared to Escherichia coli as fecal pollution indicators in water, novel observations were made in shellfish: contrary to E. coli, a seasonal trend of infectious FRNAPH concentrations was observed. These concentrations were higher than those found in water, confirming bioaccumulation in shellfish. This study also underlines a relationship between the presence of HuNoV genomes and those of human- specific FRNAPH subgroup II (FRNAPH-II) in shellfish collected throughout Europe. Further research should be undertaken to evaluate FRNAPH potential as an indicator of the presence of infectious HuNoVs. To this end, shellfish involved in HuNoVcaused gastroenteritis outbreaks should be analyzed for the presence of infectious FRNAPH-II. IMPORTANCEThis work provides new data about the use of F-specific RNA phages (FRNAPH) as a tool for evaluating fecal or viral contamination, especially in shellfish. In our case study, FRNAPH did not provide any benefits compared to E. coli as fecal pollution indicators in water but were found to be very useful in shellfish. Their concentrations in shellfish were higher than those found in the surrounding water, confirming bioaccumulation. This study also underlines a relationship between the presence of human norovirus genomes (HuNoVs) and those of FRNAPH subgroup II (FRNAPH-II). Considering that the two virus types have similar behaviors and since FRNAPH infectivity can be investigated, the specific detection of infectious FRNAPH-II could be regarded as an indication of the presence of infectious HuNoVs. The contribution of infectious human FRNAPH targeting for assessing the viral risk associated with HuNoVs in shellfish should thus be investigated.