Retention of Virus Versus Surrogate, by Ultrafiltration in Seawater: Case Study of Norovirus Versus Tulane.

In the field of chemical engineering and water treatment, the study of viruses, included surrogates, is well documented. Often, surrogates are used to study viruses and their behavior because they can be produced in larger quantities in safer conditions and are easier to handle. In fact, surrogates allow studying microorganisms which are non-infectious to humans but share some properties similar to pathogenic viruses: structure, composition, morphology, and size. Human noroviruses, recognized as the leading cause of epidemics and sporadic cases of gastroenteritis across all age groups, may be mimicked by the Tulane virus. The objectives of this work were to study (i) the ultrafiltration of Tulane virus and norovirus to validate that Tulane virus can be used as a surrogate for norovirus in water treatment process and (ii) the retention of norovirus and the surrogate as a function of water quality to better understand the use of the latter pathogenic viruses. Ultrafiltration tests showed significant logarithmic reduction values (LRV) in viral RNA: around 2.5 for global LRV (i.e., based on the initial and permeate average concentrations) and between 2 and 6 for average LRV (i.e., retention rate considering the increase of viral concentration in the retentate), both for norovirus and the surrogate Tulane virus. Higher reduction rates (from 2 to 6 log genome copies) are obtained for higher initial concentrations (from 101 to 107 genome copies per mL) due to virus aggregation in membrane lumen. Tulane virus appears to be a good surrogate for norovirus retention by membrane processes.

Adsorption of norovirus and ostreid herpesvirus type 1 to polymer membranes for the development of passive samplers.

AIMS: This study was performed to develop a passive sampling methodology for the detection of two viruses in seawater in the area of shellfish production, the norovirus (NoV), a human pathogen implicated in gastroenteritis outbreaks linked to oyster consumption and the ostreid herpesvirus type 1 (OsHV-1), a virus associated with mass mortalities of Pacific oysters. METHODS AND RESULTS: Commercially, membranes were tested for their capacity to adsorb virus: zetapor, gauze, nylon, low-density polyethylene (LDPE) and polyvinylidene difluoride (PVDF). Laboratory exposures of membranes to contaminated water samples (stool, sewage, seawater) were performed. Our data show that the amount of NoV GII genome per membrane measured with qRT-PCR increased with the time of exposure up to 24 h, for all types of membranes except gauze. After 15 days of exposure, the amount of NoV GII per membrane continued to increase only for nylon and LDPE. The amount of OsHV-1 per zetapor membrane was significantly increased as soon as 4 h of exposure, and after 24 h of exposure for all types of membranes. Exposure of membranes to serial dilutions of various samples revealed that the amount of NoV GII and OsHV-1 per membrane is significantly higher in diluted samples. The detection of NoV and OsHV-1, respectively, with zetapor and PVDF membranes was found to be more efficient than the direct analysis of sewage and seawater. CONCLUSIONS: All membranes immersed in contaminated samples adsorbed NoV GII and OsHV-1. The amount of both viruses increased with the time of exposure. Zetapor and PVDF membranes seem to be more adapted to NoV GII and OsHV-1 detection respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Membranes tested will be used as passive samplers to improve the detection of virus in oyster production areas. Also, passive samplers could be a valuable tool for microbiome analysis with new generation sequencing.

Faecal contamination of echinoderms: first report of heavy Escherichia coli loading of sea urchins from a natural growing area.

UNLABELLED: Although little evidence existed to support that view, European countries and in particular France, have regarded echinoderms, including sea urchins, as low risk in terms of feacal contamination. It is hypothesized that the sea urchins mode of feeding, which is based on grazing and differs from bivalve molluscs, would prevent it from concentrating high levels of Escherichia coli. Here, we monitored E. coli levels in sea urchins (Paracentrotus lividus) and in filter-feeder mussels (Mytillus galloprovincialis), collected concurrently from the same natural area over a 1-year period to verify this assumption. Sea urchins were collected on the seafloor, whereas mussels were collected from the water column at a depth of 4 m. Our results showed heavy bacterial loading of sea urchins in a natural growing environment. Moreover, we highlighted that E. coli contamination of sea urchins could, in certain conditions, be higher than those detected in filter-feeding mussels collected at the same location. Finally, the results showed a significant correlation between rainfall and E. coli concentrations in sea urchins, suggesting that the bacterial safety of sea urchin could be linked to the quality of the surrounding water. SIGNIFICANCE AND IMPACT OF THE STUDY: The European regulation requires competent authorities to monitor the sanitary status of shellfish, including live echinoderms, through faecal indicator organisms. In the French Mediterranean, sea urchin production is significant. Until now, as no data showed significant E. coli contamination levels, no monitoring programs focused on this species. This study demonstrates that sea urchins are more vulnerable to faecal contamination than previously hypothesized, especially during heavy rainfall. In consequence, the European authority general approach to microbiological management of shellfish should be applied to sea urchins.

A norovirus oyster-related outbreak in a nursing home in France, January 2012.

The presence of norovirus in shellfish is a public health concern in Europe. Here, we report the results of an investigation into a norovirus gastroenteritis outbreak following a festive lunch which affected 84 (57%) residents and staff members of a nursing home in January 2012 in France. Individuals who had eaten oysters had a significantly higher risk of developing symptoms in the following 2·5 days than those who had not, the risk increasing with the amount eaten [relative risk 2·2 (1·0-4·6) and 3·3 (1·6-6·6) for 3-4 and 5-12 oysters, respectively]. In healthy individuals during those days, 29 (32%) subsequently became ill, most of whom were staff members performing activities in close contact with residents. Genogroup II noroviruses were detected in faecal samples, in a sample of uneaten oysters and in oysters from the production area. Identifying a norovirus's infectious dose may facilitate the health-related management of contaminated shellfish.

An oyster-associated hepatitis A outbreak in France in 2007.

Following the notification of nine hepatitis A cases clustered in the Cotes d Armor district in northwestern France, epidemiological, environmental and microbiological investigations were set up in order to identify the source and vehicle of contamination and implement control measures. In total, 111 cases were identified in the outbreak, all of whom lived or had stayed as tourists in the Cotes d Armor district. Of the cases, 87% had eaten raw shellfish, and 81% specifically oysters. Traceback investigations carried out on raw shellfish consumed by the cases showed that the raw shellfish originated from a single shellfish farm. The shellfish were probably contaminated either in the submersible tanks or in a depuration land-based tank where they were stored. The source of contamination was not identified but shellfish could have been tainted by sewage overflows or by wastewater releases from a polluted storm sewer close to the shellfish farm or from on-site sanitation facilities. To prevent future hepatitis A outbreaks due to shellfish consumption from this area, hazards specific to each farm should be analysed. Timely information on sewage overflows should also be part of communities efforts regarding sewage collection and treatment.

Detection of human enteric viruses in shellfish collected in Tunisia.

AIMS: The aim of this study was to detect the main pathogenic human RNA enteric viruses able to persist in the environment such as astrovirus, enterovirus, norovirus and hepatitis A virus (HAV) in shellfish collected from two locations in northern Tunisia. METHODS AND RESULTS: Viruses were eluted from digestive tissues and concentrated by polyethylene glycol precipitation before nucleic acid extraction and purification. After checking for inhibitors, all viruses were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by hybridization. Overall, 83% of the samples were found positive for at least one virus. Astrovirus was detected in 61% of the samples, norovirus in 35% and HAV in 26%. Surprisingly, only one sample was found positive for enterovirus. CONCLUSIONS: The mean number of positive samples found in this study is in accordance with the data found in the literature, indicating that no real difference exists in this respect among countries studied. A notable exception is HAV, which reflects the epidemiological status of the population. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the interest to analyse shellfish samples from different production areas. These data will be helpful to understand virus circulation and to improve shellfish safety. The results, which confirm contamination, necessitate the development of appropriate studies and monitoring in all shellfish-producing countries.

Characterization of new recombinant noroviruses.

Noroviruses are important etiologic agents of acute gastroenteritis and show great genetic diversity. To characterize more fully previously detected strains that could not be assigned unequivocally to one particular genotype based on the RNA polymerase, we have sequenced a region in the capsid gene and, in some cases, in the junction between open reading frame 1 (ORF 1) and ORF 2. The results allowed us to identify several recombinant noroviruses: GGIIb viruses were detected for the first time in France in August 2000 and then spread through France and to Europe during the following winter. Here we present the characterization of three other probable GII recombinants which showed different phylogenetic positions depending on their ORF 1 and ORF 2 sequences. Analysis of the region located between ORF 1 and ORF 2 by a nucleotide identity window search showed a sudden shift in similarities. Moreover, recombination breakpoints were identified upstream and downstream of the beginning of ORF 2 by using a statistical test, thus confirming the involvement of this region in recombination. Unlike GGIIb, the three recombinants described here do not seem to have diffused widely in the community: one was found in a waterborne outbreak, and the other two were found in sporadic cases. Recombination is important for the evolution of RNA viruses and has already been described for noroviruses. Our results suggest that recombination is not a rare phenomenon among noroviruses, but not all these presumed recombinants that formed during RNA replication are able to spread widely.

Real-time RT-PCR for norovirus screening in shellfish.

Real-time RT-PCR, combining amplification and detection of virus-specific amplicons, is a promising tool for norovirus detection in environmental or food samples such as shellfish. We developed a real-time RT-PCR assay based on one-step detection using single primer sets and probes for norovirus genogroups I and II. Seventy and seven RT-PCR units of genogroup I and II reference norovirus strains, respectively, were detected in artificially contaminated oysters. Validation of the new method on 150 archived naturally contaminated shellfish confirmed the utility of the genogroup II primer set to detect a large range of different strains circulating in France since 1995, but genogroup I strains were detected infrequently.

Sewage impact on shellfish microbial contamination.

Coastal areas are frequently contaminated by microorganisms of human origin, due to high population density and low seawater renewal. To evaluate the impact of wastewater input on shellfish quality, a study was conducted in Brittany (France) over a period of 20 months. A hydrodynamic model was used to simulate wastewater impact on microbial water quality. To validate the model, wastewater from the three main sewage treatment plants and shellfish from three sites were sampled monthly. Bacterial indicators (E. coli), F-RNA phages were searched for by culture and noroviruses by RT-PCR and hybridisation. These microorganisms were detected in the three effluents and clams, with no marked seasonal variation. The microbial concentrations in the two oyster beds, distant from the effluent outfall, were low, and only three of the samples were positive for norovirus. For simulation, the winter wastewater inputs of E. coli and phages were calculated and an estimation for norovirus flux was made from the epidemic situation in the population. The microbial behaviour was included in the model by a decay-rate factor. Results from the model calculations were found to be very similar to E. coli and phage concentrations observed in shellfish. For noroviruses, the model indicated that shellfish distant from the wastewater input were under the detection limit of the RT-PCR method. This study demonstrated the use of modelisation to interpret norovirus contamination in various areas.

Human enteric viruses in Coquina clams associated with a large hepatitis A outbreak.

An outbreak of hepatitis A, affecting 183 people, occurred in Valencia (Spain). Epidemiological evidence pointed to an association of the outbreak with consumption of Coquina clams (Donax sp), imported frozen from Peru. Shellfish were analysed for the presence of hepatitis A virus (HAV), enteroviruses, rotaviruses, astroviruses, caliciviruses and hepatitis E virus. HAV was detected in 75% of assayed shellfish samples. Other enteric viruses were occasionally found in the same samples. Molecular epidemiological analysis of fragments of the VP1/2A and the 5' end of the genome from shellfish and sera isolates, revealed the presence of six variants belonging to a single genotype.

Astrovirus detection in wastewater samples.

Procedures for the detection of astroviruses in wastewater samples have been developed and evaluated. Following these methodologies, we investigated the occurrence of astroviruses in wastewater samples from three different sewage treatments plants located in Southern France and two in the Barcelona area. Some positive samples were genotyped by analysis of a fragment of the ORF1a by restriction fragment length polymorphism (RFLP) with endonuclease DdeI. The amplimers generated contain several sites for the DdeI restriction endonuclease, being the number and location of sites different between strains.

An improved method for the detection of Norwalk-like caliciviruses in environmental samples.

An original magnetic beads RNA capture was developed for the detection of Norwalk-like virus by RT-PCR. The same oligonucleotide was used both for capture and reverse transcription of the viral RNA. The optimization studies showed that the most important parameter for sensitivity is the biotin-binding capacity of the beads. This method was found to be efficient for eliminating inhibitors in sewage samples compared with the classic RT-PCR. Moreover, the sensitivity was greatly enhanced, allowing the detection of 42% positive sample after gel electrophoresis, which is fourfold greater than classic RT-PCR (11%). Beads-RT-PCR sensitivity is the same as classic RT-PCR and hybridization. Thus, this method, which is easy to perform, should be of particular interest for developing quantitative RT-PCR and sequencing.

[Magnitude of rainfall on viral contamination of the marine environment during gastroenteritis epidemics in human coastal population]. / Importance de la pluviométrie sur la contamination virale du milieu littoral lors de phénomènes épidémiques dans la population.

BACKGROUND: Sewage treatments are not efficient to eliminate enteric microorganisms. Viruses are able to persist and are discharged into the marine environment with treated effluents. Few data are now available on the magnitude and the contributive processes of marine viral contamination. This work evaluates the relationship between the magnitude of rainfall and the viral contamination of the marine environment during winter epidemics of gastroenteritis in human coastal populations. METHODS: A RT-PCR method was used to detect enterovirus, hepatitis A virus, Norwalk-like virus, astrovirus and rotavirus in shellfish, harvested monthly between August 1995 and July 1998. The frequency of virus detection in shellfish was expressed as an Index of Viral Contamination. Acute gastroenteritis in the population was estimated using the French Sentinel System for Monitoring of Communicable Diseases. Rainfall effects on the efficiency of sewage treatment were assessed using an estimated staying time of sewage effluents in the plant. RESULTS: The results indicate that the highest viral contamination occurs in winter. Maximal indexes of viral contamination were respectively 70% in January 1996, 100% in January 1997, but only 31% in January 1998. Viral contamination variations seemed to follow the pattern of the winter epidemic of acute gastroenteritis in the local population in 1996 and 1997. These observations should be linked to the winter rainfalls. Heavy rains on short periods of time could create an hydraulic overload in the sewage treatment plant, reducing the staying time of the sewage effluents and thus the efficiency of the disinfection process. CONCLUSION: The magnitude of the viral contamination of shellfish seems to result from the simultaneity between the winter epidemics of acute gastroenteritis in the coastal population and heavy rainfall. To prevent public health hazards associated with shellfish consumption, the monitoring of microbiological quality in shellfish harvesting areas should include accompagning survey of viral epidemic in the coastal population, and of sewage outputs in the coastal environment.