Norovirus in bivalve molluscs: development of a Real Time RT PCR protocol and its application for a viral contamination monitoring and for a bioaccumulation study

The Food Hygiene Package is a body of European Regulations laying down hygiene rules for foodstuffs produced in the EU and non-EU countries exporting to the EU. The pursuit of a high level of protection of human life and health is one of the fundamental objectives of this laws package. Some points of EC Regulations undergo to critical review by the Commission, and particular attention was given to fishery products. Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs, in Annex I, Chapter 1, lays down the food safety criteria for live bivalve molluscs and live echinoderms, tunicates and gastropods and sets sampling-plans, limits and analytical reference methods only concerning bacterial micro-organisms of Salmonella spp. and Escherichia coli genera. Epidemiological data of the last years reported the consumption of live bivalve shellfish infected by enteric viruses as common cause of human gastroenteritis. Norovirus resulted the leading cause of all human gastroenteritis outbreaks worldwide. For this reason, in the first part of Reg. (EC) No 2073/2005, in the whereas at point 12, it’s written that the Scientific Committee on Veterinary Measures relating to Public Health (SCVPH) issued an opinion on Norwalk-like viruses (NLVs, noroviruses) on 30-31 January 2002. In that opinion it concluded that the conventional fecal indicators are unreliable for demonstrating the presence or absence of NLVs and that the reliance on fecal bacterial indicator removal for determining shellfish purification times is unsafe practice. So Salmonella and Escherichia coli can not be used as only indicators of safety criteria in live bivalve molluscs. In particular, at following points we can read that it may be necessary to set harmonized sampling frequencies at Community level, particularly in order to ensure the same level of controls to be performed throughout the Community. Finally it’s confirmed that criteria for pathogenic viruses in live bivalve molluscs should be established when the analytical methods are developed sufficiently (at point 27). In response to concerns expressed by EU Regulations and taking into account the latest epidemiological data, the international networks dealing prevention, communication and control of human food borne illness diseases caused by enteric viruses have intensified their researches on norovirus. In the past few years, numerous research projects financed by government departments and international organizations have been implemented in order to get more information on these viral pathogens and check most hazardous areas. Among the areas traditionally known for a high fish consumption, there are the Southern Italy regions, where mussels are highly appreciated. Mytilus galloprovincialis accounts for over 22.4% of annual consumption of fish (data by E.U., Unimar, Ismea e Uniprom), particularly in Campania the mussels consumption was calculated to be about 41 000 tonnes each year. Since ancient times, in most coastal areas the custom of eating raw or undercooked bivalve shellfish remains common. In this way, however, the assumption of norovirus eventually present and bioaccumulated in shellfish makes easier. Noroviruses are highly resistant to adverse environmental conditions and a simple shellfish cleaning or the application of so-called "mild technologies", such as steaming, are unable to eliminate viral contamination. Among filter feeders bivalve, oysters are the species most common in France. In fact the French coastal areas produce more than 90% of oysters in the EU and France has the historic first for production and consumption of these molluscs. Oysters are traditionally eaten raw, still alive, mostly with a few drops of lemon juice. As for Mytilus galloprovincialis, oysters can represent an hazard for the occurrence of human food borne illness from enteric viruses. In the course of my PhD studies, I have dealt with norovirus in bivalve molluscs. During my first year of PhD study, I concurred to the development of a method for detection of norovirus in live bivalve molluscs working together with researchers of the ISS. The method is based on the use of Real Time RT PCR. During our researches it has been subjected to inner validation by the ISS and it resulted provided of efficiency to research norovirus in live bivalve molluscs. So the Italian Ministry of Health with Note of 24/11/2009 established the one-step Real Time RT-PCR protocol as the official method to research norovirus in live bivalve molluscs. After this period, during the second year of my PhD studies, I worked for a national monitoring to test norovirus presence in bivalve molluscs. The one-step Real Time RT PCR protocol was used again. Studies have been carried out within a project financed by Campania Region. Shellfish collected from harvesting areas and bought at retailers located on the three coastal districts of Naples, Caserta and Salerno have been tested for norovirus presence. During the last year, my scientific experience has been enriched by a collaboration with IFREMER researchers. IFREMER is one of the French national research centers taking part in FBVE-network. I have worked at the section of the Virology of Laboratory of Microbiology (MIC) sited in Nantes, that was indicated National Reference Laboratory (NRL) for the control of bacterial and viral contamination of bivalve molluscs in France by the French Ministry of Agriculture and Fisheries. IFREMER has the same functions of the Italian NRL concerning norvirus research and control. During my stage at IFREMER, bioaccumulation physiological activity was analyzed in oysters living in seawater contaminated by Norovirus spp. The research was carried out on samples of Crassostea gigas coming from different harvesting areas located along the Atlantic coast of Brittany, area known for oysters production worldwide. For bioaccumulation studies classified and quantized strains of norovirus GI.1 and GII.3 were used. At the IFREMER laboratory, the one-step RT Real Time PCR protocol was used and further validated.