Miguel F. Varela

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).

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.

Low prevalence of Aichi virus in molluscan shellfish samples from Galicia (NW Spain)

The aim of this study was to detect and quantify Aichi virus (AiV) in shellfish from three estuaries in Galicia, the main producer of molluscs in Europe.

An overview of 20 years of studies on the prevalence of human enteric viruses in shellfish from Galicia, Spain.

Galicia (NW Spain) has 1490 km of coastline, and its particular topography, characterized by the presence of fiord‐like inlets, called rías, with an important primary production, makes this region very favourable for shellfish growth and culture. In fact, Galicia is one of the most important mussel producers in the world. Due to its proximity to cities and villages and the anthropogenic activities in these estuaries, and despite the routine official controls on the bivalve harvesting areas, contamination with material of faecal origin is sometimes possible but, current regulation based on Escherichia coli as an indicator micro‐organism has been revealed as useful for bacterial contaminants, this is not the case for enteric viruses. The aim of this review is to offer a picture on the situation of different harvesting areas in Galicia, from a virological standpoint. A recompilation of results obtained in the last 20 years is presented, including not only the data for the well‐known agents norovirus (NoV) and hepatitis A virus (HAV) but also data on emerging viral hazards, including sapovirus (SaV), hepatitis E virus (HEV) and aichivirus (AiV). Epidemiological differences related to diverse characteristics of the harvesting areas, viral genotype distribution or epidemiological links between environmental and clinical strains will also be presented and discussed. The presentation of these historical data all together could be useful for future decisions by competent authorities for a better management of shellfish growing areas.

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.

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

ABSTRACT 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.