Sapovirus in Wastewater Treatment Plants in Tunisia: Prevalence, Removal, and Genetic Characterization.

Sapovirus (SaV), from the Caliciviridae family, is a genus of enteric viruses that cause acute gastroenteritis. SaV is shed at high concentrations with feces into wastewater, which is usually discharged into aquatic environments or reused for irrigation without efficient treatments. This study analyzed the incidence of human SaV in four wastewater treatment plants from Tunisia during a period of 13 months (December 2009 to December 2010). Detection and quantification were carried out using reverse transcription-quantitative PCR (RT-qPCR) methods, obtaining a prevalence of 39.9% (87/218). Sixty-one positive samples were detected in untreated water and 26 positive samples in processed water. The Dekhila plant presented the highest contamination levels, with a 63.0% prevalence. A dominance of genotype I.2 was observed on 15 of the 24 positive samples that were genetically characterized. By a Bayesian estimation algorithm, the SaV density in wastewater was estimated using left-censored data sets. The mean value of log SaV concentration in untreated wastewater ranged between 2.7 and 4.5 logs. A virus removal efficiency of 0.2 log was calculated for the Dekhila plant as the log ratio posterior distributions between untreated and treated wastewater. Multiple quantitative values obtained in this study must be available in quantitative microbial risk assessment in Tunisia as parameter values reflecting local conditions.IMPORTANCE Human sapovirus (SaV) is becoming more prevalent worldwide and organisms in this genus are recognized as emerging pathogens associated with human gastroenteritis. The present study describes novel findings on the prevalence, seasonality, and genotype distribution of SaV in Tunisia and Northern Africa. In addition, a statistical approximation using Bayesian estimation of the posterior predictive distribution ("left-censored" data) was employed to solve methodological problems related with the limit of quantification of the quantitative PCR (qPCR). This approach would be helpful for the future development of quantitative microbial risk assessment procedures for wastewater.

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

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).

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.

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.

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

An 18-month survey was conducted in ten class "B" harvesting areas from two Galician Rias (NW of Spain), the most important bivalve production area in Europe, to determine the prevalence of hepatitis A virus (HAV) and human norovirus (NoV), including genogroups I (GI) and II (GII). Quantification was performed by reverse transcription real-time PCR (RT-qPCR), according to the recently developed standard method ISO/TS 15216-1:2013. Four bivalve species were studied, including wild and cultured mussels (Mytilus galloprovincialis), clams (Venerupis philippinarum and Venerupis decussata) and cockles (Cerastoderma edule). Overall, 55.4% of samples were contaminated by at least one of the studied viruses, being detected the simultaneous presence of two or three viruses in 11.3% of the cases. NoV GI was the most prevalent virus (32.1%), followed by NoV GII (25.6%) and HAV (10.1%). Cultured mussels showed the highest percentage of positive samples (61.4%), followed by cockles (59.4%), wild mussels (54.3%) and clams (38.7%). Viral contamination levels for most of the positive samples ranged from 10(2) to 10(3) RNA copies/g of digestive tissue (RNAc/g DT). The presence of viral contamination was statistically higher (P<0.0001) in warm months (April to September) than in cold months (October to March). The data presented here may contribute to the development of more representative sampling strategies, in monitoring and management of shellfish growing areas as well as being useful in a future scenario in which viral critical values are adopted in legislation.

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.