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Crassostrea gigas is increasingly being cultured in a great number of countries from animaIs originally introduced from Japan, and is one of the most exploited bivalve species in the EU (FAO, 1989). Although no serious pathogen was detected previously in Japanese oysters in Europe, mortalities have been observed since the introduction of this bivalve species into the EU. Thus, oyster cultivation may be endangered by the occurrence of epizootics, especially virus diseases, which are considered one of the maj or risks to production. Indeed, mortalities have been described among different species of ostreids and are associated with the presence of viruses belonging to various families. The first description of a virus was reported in adult Eastern oyters, Crassostrea virginica, with the detection of particles indicating membership of the Herpesviridae (Farley & al. , 1972). Mass mortalities of adult Portuguese oysters, C. angulata, among French livestocks (between 1967 and 1973) were associated with iridovirus infections (Comps & al., 1976; Comps & Bonami, 1977; Comps & Duthoit, 1979). Other viruses described in ostreids are members of the Iridoviridae, Papovaviridae, Togaviridae, Retroviridae and Reoviridae (Elston, 1979; Farley, 1976; Farley, 1978; Meyers, 1979; Elston and Wilkinson, 1985). Recently, in 1991, viruses interpreted as belonging to the Herpesviridae were associated with high mortality rates of hatchery- reared larval Crassostrea gigas in France (Nicolas & 1992) and in New Zealand (Hine & al., 1992). Since 1992 sporadic high mortalities of larval C. gigas are regularly observed in some private French hatcheries, occurring each year during summer period in association with a herpes-like virus (Renault & al. , 1994b). The pathogenicity of the virus was demonstrated by experimental transmission of the infection to healthy C. gigas larvae (Le Deuff & al., 1994). Since 1993, sporadic high mortalities occur also in sorne batches of Pacific oyster spat cultured in different French locations (Renault & al, 1 994b). ln addition, herpesvirus infections were also reported in spat and larvae of the European flat oyster (Ostrea edulis) in France (Comps & Cochennec, Renault & al. , in press). Concomitant mortalities were observed among larvae and spat of Crassostrea gigas and 0. edulis, in 1994 and 1995, with the detection of herpes-like virus particles by transmission electron microscopy (Renault & al. , in press). Replication of herpes-like viruses was also described in 0. angasi adults in Australia (Hine & Thome, 1997), in larval Tiostrea chilensis in New Zealand (Hine, 1997; Hine & al. , 1998) and in larval Ruditapes philippinarum in France (Renault, 1998). Unexplained mortalities were observed in recent years among larvae in the United Kingdom and Spain, although samples were not examined. High losses were reported among spat in Ireland in 1994 and 1995. No obvious cause of mortalities was determined (Culloty & Mulcahy, 1995). However, screening using conventional light microscopy yielded little apart from sorne cell damage most noticeably enlarged cell nuclei and marginated chromatin. Results would now indicate that herpes-like virus is present in at least one site on the south coast of lreland (Culloty & Mulcahy, unpublished data). Herpes-like virus infections in bivalves belonging to the genera Crassastrea, Ostrea and Ruditapes seem to be ubiquitous and are associated with substantial mortalities. These observations highlight the importance of testing a range of efficient diagnostic methods in order to assess the causative role of the herpesviruses in bivalve mortalities. To diagnose herpes-like virus infections, the basic method for examination of suspect samples is still light microscopy. This method appears poorly adapted to viral diseases and needs to be improved upon by other techniques such as transmission electron microscopy. Both techniques are time consuming and inadequate for epidemiological surveys. In addition, research into virus cytopathogenic effects in cell cultures is impossible because the lack of bivalve cell lines. Serological methods are also not available due to the absence of immunoglobulin production in molluscs. However, a breakthrough was achieved recently in the development of a protocol, based on sucrose gradient centrifugation, for purifying oyster herpes-like virus particles from fresh infected larval Crassastrea gigas (Le Deuff & Renault, 1999). This advance has served as an appropriate platform for generating molecular biological reagents to diagnose virus infections (Renault & Lipart; 1998, Renault & al., 2000). A procedure to detect herpes-like virus in French oysters using the polymerase chain reaction (PCR) (Saiki & al., 1985; Mullis & al., 1986) was developped (Renault & al. , 2000). PCR offers many advantages for disease diagnosis (Henson & French, 1993; Jones & Bej, 1994; Martin, 1994). With regard to herpes-like viruses from oysters, important advantages include its extreme sensitivity, pathogen specificity, ease of sample processing, and availability of reagents. Research objectives : The observed assciation between oyster mortality and herpes-like virus infection provides an imperative to determining the extent to which the virus is involved as a causative agent of massive mortalities in different European countries. Il appears essential to survey in fections epidemiologically in di fferent European shellfish hatcheries. The aim of the work proposed is to use molecular reagents (PCR) specific for oyster herpes-like viruses in order to analyse larval samples from different origins. Thus, two tasks have been defined : - Analys is by PCR technique of bivalve larval samples originating from different European hatcheries in order to detect herpes-like virus DNA - Validating new PCR primer pairs for herpes-like virus diagnosis.
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