Study of viral depuration in oysters and characterization of F-specific RNA phages

Enteric viruses, including noroviruses, are a major cause of gastroenteritis associated with the consumption of contaminated shellfish. These viruses mainly originate from fecal pollution, often linked to wastewater discharges into the sea. Purification processes are commonly used in shellfish farming to reduce microbial contamination before commercialization. However, these methods are slow to eliminate viral genomes. In the absence of a culture method for noroviruses, only viral genome detection is currently possible. As no routine culture method is available for noroviruses, virological indicators have been proposed to assess the efficiency of oyster purification processes. In this context, the use of F-specific RNA bacteriophages (FRNAPH), which are naturally present in wastewater in high prevalence and concentration, structurally similar to enteric viruses, and easily cultivable in the laboratory, has been described.

The aim of the present study was to understand the mechanisms of viral particle inactivation and persistence in oysters during depuration. To this end, the presence of infectious and genomic FRNAPH was monitored in two oyster compartments (hepatopancreas and hemolymph) and in seawater.

Phage behavior in oysters was assessed in two depuration trials mimicking conditions of shellfish farms by using natural seawater continuously treated by UV light and maintained at about 15°C. FRNAPH used to contaminate oysters had been isolated from raw wastewater and replicated on E. coli to obtain sufficient concentrations for our experiments. The experimental contamination was performed by spiking the FRNAPH in the seawater and applying a contact time of 24h. FRNAPH were detected in the two compartments just after the accumulation phase. During the depuration, the FRNAPH were monitored in hepatopancreas and in hemolymph (over 14 days and 26 days in experiments 1 and 2, respectively). Infectious particles and genome of FRNAPH were predominantly detected in the hepatopancreas. A significant difference was observed between concentrations of infectious phages (-1.5 log10) and viral genomes (no change) in this organ (p < 0.01), highlighting that viral inactivation predominated on elimination and the central role of the hepatopancreas in viral retention. Although no release of phages from the hepatopancreas into the hemolymph was observed during depuration, this compartment be involved in viral inactivation. To explore this hypothesis, phage persistence in hemolymph was evaluated in vitro at 4°C. This experiment revealed prolonged stability of both infectious and genomic viral particles, suggesting that at this temperature, hemolymph does not contribute to phage inactivation.

These findings support the hypothesis that viral inactivation occurs within oysters, rather than elimination into the seawater during depuration. They also highlight the importance of simultaneously measuring infectious viruses and genomes to effectively assess purification processes. Further experiments are underway to reproduce phage persistence in hemolymph at a temperature similar to that used during depuration in shellfish farming.