David Polo

Imported mollusks and dissemination of human enteric viruses.

To the Editor: The globalization of food production and trade has increased the potential risk for infectious foodborne diseases. Hepatitis A virus (HAV) and norovirus (NoV) constitute the most important foodborne pathogens of humans in terms of numbers of outbreaks and persons affected in industrialized countries (1,2). In these countries, improvement of health conditions and development of specific vaccines are changing the epidemiologic pattern of diseases such as hepatitis A, decreasing their prevalence and increasing the susceptibility of the unvaccinated adult population (1). In recent years, numerous cases of gastroenteritis caused by NoV and hepatitis A linked to imported shellfish have been reported (2–5). In Spain, 2 notable hepatitis A outbreaks associated with clams (Donax sp.) imported from Peru occurred in 1999 and 2008. In both situations, the Spanish Ministry of Health activated the National System of Epidemiologic Surveillance and the European Community Rapid Alert System for Foodstuffs. The implicated shellfish batches were immobilized or removed, and all the shellfish from Peru were banned from the European Union (6). We present further evidence that imported shellfish from developing countries, where these pathogens are endemic, can be a vehicle for viral gastroenteritis and HAV infections in areas where they are not endemic. Fifty mollusk samples imported into Spain during September 2006–March 2009 were analyzed for NoV genotype I (GI) and GII, HAV and astrovirus (AsV). Countries of origin were Morocco, Peru, Vietnam, and South Korea (Table). The species studied were clams (Callista chione, n = 25; Transanella pannosa, n = 6; Meretrix lyrata, n = 3; and Donax sp., n = 5), oysters (Crassostrea angulata, n = 1), cockles (Cerastoderma edule, n = 1), and razor clams (Solen marginatus, n = 1 and Ensis sp., n = 8). Digestive tissue was dissected from duplicated samples (10–20 individual mollusks) and homogenized with 0.1% peptone water (pH 7.4), centrifuged at 1,000 × g for 5 min, and supernatant recovered. RNA was extracted by using both Total Quick RNA extraction Cells and Tissue kit (Talent, Trieste, Italy) and Nucleospin RNA Virus Kit (Macherey-Nagel, Duren, Germany). Table Viral detection and quantification in imported mollusk samples* NoV and HAV were detected by real-time reverse transcription–PCR (RT-PCR) by using the Platinum Quantitative RT-PCR Thermoscript 1-step system (Invitrogen, Carlsbad, CA, USA) (25 µL final volume) with 5 µL of template RNA, and primers, probes, and conditions as described (7). A sample that displayed a cycle threshold value <41 was considered positive. AsV was detected by standard RT-PCR (7), coupled with hybridization by using specific biotin-labeled probes with the commercial Kit Hybridowell universal (Argene, Varilhes, France). Negative and specific positive controls for HAV, NoV, and AsV were introduced in each run. Real-time RT-PCR included appropriate external controls in each analysis to avoid underestimation of viral load. A mutant, nonvirulent, infective strain of mengovirus (vMC0) (103 PFU) was used as control for extraction. To calculate the real-time RT-PCR efficiencies, external viral RNA (HAV, 103 copies) or synthetic DNA (NoV, 105 copies) controls for the respective virus were co-amplified with each template viral RNA as described (8). The number of RNA viral genome copies per gram of digestive tissue (RNA copies/g digestive tissue) was estimated by using standard curves generated from RNA transcripts and synthetic DNA (8) and corrected with the extraction and real-time RT-PCR efficiencies. Twenty (40%) of 50 samples were contaminated by >1 virus (Table), although all the mollusk imports complied with the current sanitary standards. NoV GI was most prevalent, detected in 24% of samples, followed by AsV (18%), NoV GII (8%), and HAV (4%). One sample showed a low extraction efficiency ( 10%) (9). Six samples (30% of positive samples) were positive for >1 virus. Thus, 2 samples from Morocco showed contamination with NoV GI and AsV. From Peru, 1 sample was contaminated with both genogroups of NoV and another with NoV GI, NoV GII, and AsV. Samples from Vietnam (n = 2) were contaminated with HAV–NoV GI and NoV GI–AsV. Co-infection with different viruses or multiple virus strains could lead to more severe symptoms and the occurence of 2 episodes of the same or different diseases and also be a way to facilitate emergence of new recombinant strains (10). Contamination levels for NoV GI ranged from 3.5 × 104 to 7.7 × 107 RNA copies/g digestive tissue; for NoV GII, from 1.03 × 105 to 8.9 ×105 RNA copies/g digestive tissue; and for HAV, from 4.7 × 103 to 4.4 × 107 RNA copies/g digestive tissue (Table). For HAV, these values are in the same range or even higher than in the coquina clams from Peru implicated in the outbreak in Spain in 2008, which is noteworthy because the attack rate for different batches of shellfish is dose dependent (6). Determining the association of a viral infection with a particular contaminated product is often complicated, and the epidemiologic investigations necessary for finding this association are time consuming and allow the virus to spread before the problem is recognized. Furthermore, there are analytical difficulties in detecting and quantifying virus in shellfish samples and in monitoring them; other problems include ascertaining the representativeness of the sample (2,6) and the high cost of applying the technique in areas with extensive mollusk production. The inadequacy of the bacterial indicators makes it necessary to develop new prevention strategies, based on microbial risk assessment, to ensure the sanitary quality of shellfish, both in production areas and in international trade operations. Implementing these methods and providing training to laboratories in developing countries are essential to achieving these objectives.

Genotyping of hepatitis A virus detected in bivalve shellfish in Galicia (NW Spain)

Hepatitis A virus (HAV) represents a significant public health problem due to its high persistence in the environment and its transmission through contaminated water and food. Bivalve shellfish are filter feeders that can bioaccumulate human pathogens found in contaminated waters, their consumption being a potential cause of hepatitis A outbreaks. In this work, cultured and wild bivalve shellfish from the Ría de Vigo (Galicia, NW Spain) were analysed for the presence and genotyping of HAV. A total of 160 shellfish samples were collected between March 2004 and December 2006, including 68 samples from cultured mussels (Mytilus galloprovincialis), 30 from wild clams (Rupitapes decussatus), 31 from wild cockles (Cerastoderma edule) and 31 from wild mussel. HAV detection, carried out by quantitative RT-PCR, was positive for 29 (42.6%) cultured and 40 (43.5%) wild samples, with levels ranging from 3.1 x 10(2) and 1.4 x 10(10) RNA copies/g of shellfish digestive tissue. The phylogenetic analysis of VP1-P2A and VP3-VP1 regions, separately or as concatenated sequences, revealed that all HAV strains analysed belong to subgenotype IB. These results indicate a high prevalence of this subgenotype in the area studied.

Digital PCR for Quantifying Norovirus in Oysters Implicated in Outbreaks, France

Using samples from oysters clearly implicated in human disease, we quantified norovirus levels by using digital PCR. Concentrations varied from 43 to 1,170 RNA copies/oyster. The analysis of frozen samples from the production area showed the presence of norovirus 2 weeks before consumption.

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

ABSTRACT 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 × 103 and 1.4 × 105 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).

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.

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.

Depuration kinetics of murine norovirus in shellfish

This study evaluates and compares the uptake rates and depuration kinetics of murine norovirus (MNV-1), as a human norovirus surrogate, in Manila clams (Venerupis philippinarum) and Mediterranean mussels (Mytilus galloprovincialis). Ten trials of 70kg/trial (five with each mollusk) were performed. Mollusks were subjected to a controlled bioaccumulation step of 24h with 102pfu MNV-1/mL seawater. Then, mollusks were relocated in an experimental depuration system for 7days. Viral contamination was quantified after bioaccumulation and then daily during depuration by reverse transcription-real time PCR (RT-qPCR) with TaqMan probes. Infectivity assays were conducted to test the presence of infectious viral particles at the end of the depuration period. Results showed significant differences in the uptake and removal viral rates between molluscan species. The average viral uptake for clams and mussels were 5.4×106 and 4.0×105RNA copies/g digestive tissue respectively, representing an uptake rate >90% higher in clams. The average reductions with regard to the initial levels were 60.5% for clams and 91.6% for mussels. On the other hand, a similar logarithmic trend line in MNV-1 depuration kinetics was observed in both bivalves, with two differentiated phases: an initial rapid reduction of viruses during the first 24-72h of depuration, and a subsequent stabilization with a slower depuration rate. All trials with clams and mussels showed significant viral reductions but remaining virus were still infectious at the end of the process.

Solar water disinfection (SODIS): Impact on hepatitis A virus and on a human Norovirus surrogate under natural solar conditions.

This study evaluates the effectiveness of solar water disinfection (SODIS) in the reduction and inactivation of hepatitis A virus (HAV) and of the human Norovirus surrogate, murine Norovirus (MNV-1), under natural solar conditions. Experiments were performed in 330 ml polyethylene terephthalate (PET) bottles containing HAV or MNV-1 contaminated waters (10(3) PFU/ml) that were exposed to natural sunlight for 2 to 8 h. Parallel experiments under controlled temperature and/or in darkness conditions were also included. Samples were concentrated by electropositive charged filters and analysed by RT-real time PCR (RT-qPCR) and infectivity assays. Temperature reached in bottles throughout the exposure period ranged from 22 to 40ºC. After 8 h of solar exposure (cumulative UV dose of ~828 kJ/m2 and UV irradiance of ~20 kJ/l), the results showed significant (P<0.05) reductions from 4.0 (+/-0.56)x10(4) to 3.15 (+/-0.69)x10(3) RNA copies/100ml (92.1%, 1.1 log) for HAV and from 5.91 (+/-0.59)x10(4) to 9.24 (+/-3.91)x10(3) RNA copies/100 ml (84.4%, 0.81 log) for MNV-1. SODIS conditions induced a loss of infectivity between 33.4% and 83.4% after 4 to 8 h in HAV trials, and between 33.4% and 66.7% after 6 h to 8 h in MNV-1 trials. The results obtained indicated a greater importance of sunlight radiation over the temperature as the main factor for viral reduction.

Bioaccumulation and Removal Dynamics of Murine Norovirus in Manila Clams (Venerupis philippinarum) and Mussels (Mytilus galloprovincialis)

This study evaluates and compares the effectiveness of depuration in clams and mussels subjected to bioaccumulation with murine norovirus (MNV-1), as a surrogate for human norovirus. Ten experiments with artificially contaminated shellfish were performed with clams and mussels (five with each species) using 102 pfu of MNV-1/ml seawater. After 24 h of bioaccumulation, molluscs were relocated in an experimental depuration system (with mechanical and biological static filter system, water disinfection by ozone and UV radiation) under an exhaustive control of the physicochemical parameters. Viral contamination was quantified after bioaccumulation and then daily during the depuration period (7 day) by reverse transcription-real time PCR (qRT-PCR) with TaqMan probes. Results showed significant differences in uptake and removal viral rates between clams and mussels. After 24 h of bioaccumulation the average viral uptake for clams and mussels were 6.2 and 5.4 log RNA copies/g digestive tissue (DT) respectively, representing an uptake rate 73.8 % higher in clams. Only three out of five depuration trials with clams showed some reduction in viral quantification. The average reduction in these three trials was 0.5 log units (41.4 % reduction in RNA copies/g DT). Mussels showed viral reduction in all the depuration trials with an average reduction of 0.8 log units (74 % reduction in RNA copies/g DT).