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

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

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.

A Comprehensive Review on Human Aichi Virus.

Although norovirus, rotavirus, adenovirus and Astrovirus are considered the most important viral agents transmitted by food and water, in recent years other viruses, such as Aichi virus (AiV), have emerged as responsible for gastroenteritis outbreaks associated with different foods. AiV belongs to the genus Kobuvirus of the family Picornaviridae. It is a virus with icosahedral morphology that presents a single stranded RNA genome with positive sense (8280 nucleotides) and a poly (A) chain. AiV was first detected from clinical samples and in recent years has been involved in acute gastroenteritis outbreaks from different world regions. Furthermore, several studies conducted in Japan, Germany, France, Tunisia and Spain showed a high prevalence of AiV antibodies in adults (between 80% and 99%), which is indicative of a large exposure to this virus. The aim of this review is to bring together all the discovered information about the emerging pathogen human Aichi virus (AiV), discussing the possibles routes of transmission, new detection techniques and future research. Although AiV is responsible for a low percentage of gastroenteritis outbreaks, the high seroprevalence shown by human populations indicates an evident role as an enteric agent. The low percentage of AiV detection could be explained by the fact that the pathogen is more associated to subclinical infections. Further studies will be needed to clarify the real impact of AiV in human health and its importance as a causative gastroenteritis agent worldwide.

Comprehensive comparison of chemically enhanced primary treatment and high-rate activated sludge in novel wastewater treatment plant configurations.

Novel wastewater treatment plants (WWTPs) are designed to be more energy efficient than conventional plants. One approach to becoming more energy efficient is the pre-concentration of organic carbon through chemically enhanced primary treatment (CEPT) or high-rate activated sludge (HRAS). This study compares these approaches in terms of energy demand, operational costs, organic micropollutants (OMP), and virus removal efficiency. A CEPT pilot-scale plant was operated at a hydraulic retention time (HRT) of 30 min, and a lab-scale HRAS reactor was operated at an HRT of 2 h and a solid retention time (SRT) of 1 d in continuous mode. A minimum dose of 150 mg/L ferric chloride (FeCl3) was required to achieve a threshold chemical oxygen demand (COD)-to-ammonium ratio below 2 g COD to 1 g of NH4+ -N (fulfilling the requirement for a partial nitritation-anammox reactor), reaching high phosphate (PO43-)-removal efficiency (>99%). A slightly lower COD recovery was attained in the HRAS reactor, due to the partial oxidation of the influent COD (15%). The lower PO43- removal efficiency achieved in the HRAS configuration (13%) was enhanced to a comparable value of that achieved in CEPT by the addition of 30 mg/L FeCl3 at the clarifier. The CEPT configuration was less energy-intensive (0.07 vs 0.13 kWh/m3 of wastewater) but had significantly higher operational costs than the HRAS-based configuration (6.0 vs 3.8 c€/m3 of wastewater). For OMPs with kbiol > 10 L/gVSS·d, considerably higher removal efficiencies were achieved in HRAS (80-90%) than in CEPT (4-55%). For the remaining OMPs, the biotransformation efficiencies were generally higher in HRAS than in CEPT but were below 55% in both configurations. Finally, CEPT was less efficient than HRAS for virus removal. HRAS followed by FeCl3 post-treatment appeared to be a more effective alternative than CEPT for COD pre-concentration in novel WWTPs.

Human Sapovirus among Outpatients with Acute Gastroenteritis in Spain: A One-Year Study.

Viral agents of human gastroenteritis affect people of all ages across the globe. As a mainly self-limiting disease, it is difficult to evaluate the real prevalence of etiological agents circulating in each region. Many of the analyzed outbreaks are caused by viruses of the family Caliciviridae, especially the genus Norovirus (NoV). Most studies have focused on other enteric viruses, leaving sapovirus (SaV) underestimated as an important emerging human threat. This one-year study analyzed clinical samples from hospital outpatients with acute gastroenteritis in Spain, with the aim of revealing the importance of human SaV as an emerging viral pathogen. A total of 2667 stools were tested using reverse transcription (RT)-qPCR to detect and quantify SaV. Sapovirus was detected in all age groups, especially in infants, children, and the elderly. The prevalence was 15.64% (417/2667), and was slightly higher in 0⁻2- and 3⁻5-year-olds (19.53% and 17.95%, respectively) and much lower in 13⁻18-year-olds (9.86%). Positive samples were detected throughout the year, with peaks of detection during autumn and the late winter to early spring months. The mean value for the quantified samples was 6.5 × 105 genome copies per gram of stool (GC/g) (range 2.4 × 10³â»6.6 × 1011 GC/g). RT-nested PCR and sequencing were used for further genotyping. Genetic characterization showed a predominance of genogroup I (GI), followed by GII and GIV. The detection of multiple genotypes suggests the circulation of different strains without any clear tendency. The results obtained suggest SaV as the second major gastroenteritis agent after NoV in the region.

Epidemiology of Aichi virus in fecal samples from outpatients with acute gastroenteritis in Northwestern Spain.

BACKGROUND: In recent years, Aichi virus (AiV) has been involved in acute viral gastroenteritis outbreaks. However, the common pathogenesis of AiV releases more in subclinical infections underestimating the impact of AiV in human health. OBJECTIVES: The present study describes the presence and genetic diversity of AiV in patients with gastroenteritis in Northwestern Spain. STUDY DESIGN: A total of 2667 stool samples, obtained between July 2010 and June 2011, from diarrheic outpatients were studied for detection and molecular characterization of AiV using PCR techniques followed by sequencing and phylogenetic analyses. RESULTS: The virus was detected in 124 (5.0%) of the samples among all age groups. Coinfections were also detected, from the 124 positive samples, 72 (58.1%) were positive only for AiV, whereas mixed contaminations with Norovirus genogroup I or genogroup II, Sapovirus, or other enteric pathogens were detected in 52 (41.9%) samples. A total of 70 positive samples could be genotyped, being characterized as genotype A (58.6%) or B (41.4%). AiV was detected from August to April, being the highest number of AiV positive samples detected during autumn and winter seasons. CONCLUSIONS: This survey remarks the importance of emerging enteric viruses in patients who require medical assistance, and offers more information about the real importance of AiV as gastroenteritis agent.

Detection of Hepatitis E Virus in Shellfish Harvesting Areas from Galicia (Northwestern Spain).

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 (<102 copies of the RNA genome (RNAc)/g tissue) to 1.1 × 105 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.

Development of a novel digital RT-PCR method for detection of human sapovirus in different matrices.

A new nanofluidic digital RT-PCR method was developed for sapovirus (SaV) using control material obtained according to standards for enteric viruses. Primers employed amplify a fragment of 112 bp of the polymerase capsid junction, allowing the detection of human genogroups I, II and IV. Analytical validation was performed in clinical, shellfish and environmental water samples. This novel protocol rendered great effectiveness and repetitiveness, as well as higher sensitivity than real time RT-PCR assay, with differences in quantification ranging from 0.1 to 2.6 log-units. The method described here can constitute a promising tool for standardizing SaV quantification.

Human Sapovirus in Mussels from Ría do Burgo, A Coruña (Spain).

The prevalence and genetical diversity of human Sapovirus were studied during an 18-month study in Ría do Burgo, an estuary nearby the city of A Coruña in Galicia (NW Spain). Sapovirus was detected using RT-qPCR procedure in 30 out of 80 mussel samples (37.5 %). Quantifications ranged from 2.2 × 10(3) to 2.1 × 10(5) RNA copies per gram of digestive tissue. Detection occurred mainly during the cold and rainy seasons of the period studied. Sequences obtained could be distributed into 5 genotypes being the most abundant GI.1 and GI.3. Results obtained indicate that the hydrodynamic characteristics of the harvesting area and the proximity of population density clearly influence the presence of the virus in shellfish.