Can shellfish be used to monitor SARS-CoV-2 in the coastal environment?

The emergence and worldwide spread of SARS-CoV-2 raises new concerns and challenges regarding possible environmental contamination by this virus through spillover of human sewage, where it has been detected. The coastal environment, under increasing anthropogenic pressure, is subjected to contamination by a large number of human viruses from sewage, most of them being non-enveloped viruses like norovirus. When reaching coastal waters, they can be bio-accumulated by filter-feeding shellfish species such as oysters. Methods to detect this viral contamination were set up for the detection of non-enveloped enteric viruses, and may need optimization to accommodate enveloped viruses like coronaviruses (CoV). Here, we aimed at assessing methods for the detection of CoV, including SARS-CoV-2, in the coastal environment and testing the possibility that SARS-CoV-2 can contaminate oysters, to monitor the contamination of French shores by SARS-CoV-2 using both seawater and shellfish. Using the porcine epidemic diarrhea virus (PEDV), a CoV, as surrogate for SARS-CoV-2, and Tulane virus, as surrogate for non-enveloped viruses such as norovirus, we assessed and selected methods to detect CoV in seawater and shellfish. Seawater-based methods showed variable and low yields for PEDV. In shellfish, the current norm for norovirus detection was applicable to CoV detection. Both PEDV and heat-inactivated SARS-CoV-2 could contaminate oysters in laboratory settings, with a lower efficiency than a calicivirus used as control. Finally, we applied our methods to seawater and shellfish samples collected from April to August 2020 in France, where we could detect the presence of human norovirus, a marker of human fecal contamination, but not SARS-CoV-2. Together, our results validate methods for the detection of CoV in the coastal environment, including the use of shellfish as sentinels of the microbial quality of their environment, and suggest that SARS-CoV-2 did not contaminate the French shores during the summer season.

Improving the efficacy of sewage treatment decreases norovirus contamination in oysters.

As human population increases worldwide, water quality will become increasingly problematic, and food consumed raw may be of higher risk. This is already evident for oysters grown in coastal areas - despite regulations based on bacterial indicators, oysters are still implicated in food-borne outbreaks worldwide. The pathogens most frequently detected are human noroviruses, which are shed at high concentrations in human excreta and are very resistant to environmental conditions. Sewage treatment plants usually apply a variety of steps such as activated sludge treatment, chlorine or UV disinfection to eliminate contaminants, these processes have variable efficacy. This study demonstrates the impact of replacing an old lagoon-based sewage treatment plant with a new membrane bioreactor sewage treatment plant on human norovirus levels in treated sewage and oysters. While comparable norovirus concentrations were detected in the influent samples, a clear difference was observed in effluent quality, as norovirus was only detected in one sample after treatment in the new membrane bioreactor system, confirming the efficiency of such technology. As a direct impact, oysters located close to the membrane bioreactor sewage outfall were less frequently contaminated by norovirus, and showed lower concentrations compared to the first period of the study when they were exposed to sewage effluent from the lagoon outfall. Shellfish located upstream showed comparable contamination levels suggesting that there are also other sources of norovirus contamination in the estuary. Considering the health benefits of shellfish consumption, improving wastewater quality will make an important contribution to enhancing the safety of shellfish and international food security.

Follow-Up of Norovirus Contamination in an Oyster Production Area Linked to Repeated Outbreaks.

A production area repeatedly implicated in oyster-related gastroenteritis in France was studied for several months over 2 years. Outbreaks and field samples were analyzed by undertaking triplicate extractions, followed by norovirus (NoV) detection using triplicate wells for genomic amplification. This approach allowed us to demonstrate that some variabilities can be observed for samples with a low level of contamination, but most samples analyzed gave reproducible results. At the first outbreak, implicated oysters were collected at the beginning of the contamination event, which was reflected by the higher NoV levels during the first month of the study. During the second year, NoV concentrations in samples implicated in outbreaks and collected from the production area were similar, confirming the failure of the shellfish depuration process. Contamination was detected mainly during winter-spring months, and a high prevalence of NoV GI contamination was observed. A half-life of 18 days was calculated from NoV concentrations detected in oysters during this study, showing a very slow decrease of the contamination in the production area. Preventing the contamination of coastal waters should be a priority.

Bioaccumulation efficiency, tissue distribution, and environmental occurrence of hepatitis E virus in bivalve shellfish from France.

Hepatitis E virus (HEV), an enteric pathogen of both humans and animals, is excreted by infected individuals and is therefore present in wastewaters and coastal waters. As bivalve molluscan shellfish are known to concentrate viral particles during the process of filter feeding, they may accumulate this virus. The bioaccumulation efficiencies of oysters (Crassostrea gigas), flat oysters (Ostrea edulis), mussels (Mytilus edulis), and clams (Ruditapes philippinarum) were compared at different time points during the year. Tissue distribution analysis showed that most of the viruses were concentrated in the digestive tissues of the four species. Mussels and clams were found to be more sensitive to sporadic contamination events, as demonstrated by rapid bioaccumulation in less than 1 h compared to species of oysters. For oysters, concentrations increased during the 24-h bioaccumulation period. Additionally, to evaluate environmental occurrence of HEV in shellfish, an environmental investigation was undertaken at sites potentially impacted by pigs, wild boars, and human waste. Of the 286 samples collected, none were contaminated with hepatitis E virus, despite evidence that this virus is circulating in some French areas. It is possible that the number of hepatitis E viral particles discharged into the environment is too low to detect or that the virus may have a very short period of persistence in pig manure and human waste.

Impact of xynthia tempest on viral contamination of shellfish.

Viral contamination in oyster and mussel samples was evaluated after a massive storm with hurricane wind named "Xynthia tempest" destroyed a number of sewage treatment plants in an area harboring many shellfish farms. Although up to 90% of samples were found to be contaminated 2 days after the disaster, detected viral concentrations were low. A 1-month follow-up showed a rapid decrease in the number of positive samples, even for norovirus.