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Herpes-like viral infections have been reported in different bivalve mollusc species throughout the world. High mortalities among hatchery-reared larvae and juveniles of different bivalve species have been associated often with such infections. The diagnosis of herpes-like viruses in bivalve molluscs has been performed traditionally by light and transmission electron microscopy. The genome sequencing of one of these viruses, oyster herpesvirus 1 (OsHV-1), allowed the development of DNA-based diagnostic techniques. The polymerase chain reaction (PCR) has been used for the detection of OsHV-1 DNA in bivalve molluscs at different development stages. In addition, the PCR used for detection of OsHV-1 has also allowed the amplification of DNA from an OsHV-1 variant. The literature on DNA extraction methods, primers, PCR strategies, and confirmatory procedures used for the detection and identification of herpesviruses that infect bivalve molluscs are reviewed. (c) 2006 Elsevier B.V. All rights reserved.

Mortalities related to the detection of Ostreid Herpesvirus 1 (OsHV-1) have been previously reported in France among larvae and spat of the Pacific oyster Crassostrea gigas. Adult oysters appear less sensitive to herpesvirus infections, although OsHV-1 has been detected in adults without signs of disease or mortality. This suggests that the virus is able to persist in its host and that adult oysters may be able to control OsHV-1 infection. Little is known about antiviral substances in invertebrates. The present work concerns the research of antiviral substances in adult oyster C. gigas, where putative antiviral activities were monitored using 3 strategies: (1) in metabolites with variable polarity, (2) in peptidic extracts and (3) in crude haemolymph. In vitro antiviral assays were based on inhibition of Herpes simplex virus type 1 (HSV-1) replication in Vero cell monolayers. All extracts presented no cytotoxicity. Antiviral activity was detected in the fresh filtered haemolymph (EC50:425 microg ml(-1)) and seasonal variation of the haemolymph antiviral activity was monitored.

Sporadic high mortalities were reported among larval French scallops (Pecten maximus). Electron microscopy of moribund larvae revealed particles with the characteristics of a herpesvirus in association with cellular lesions. PCR and DNA sequencing showed that the virus is a variant of ostreid herpesvirus-1 that has already been described in clams and oysters. This is the first description of a herpesvirus infection of a scallop species. The virus was transmitted successfully from an extract of infected scallop larvae to uninfected scallop or oyster (Crassostrea gigas) larvae, demonstrating that it is able to infect both species. Detection of viral DNA in asymptomatic adult scallops by in situ hybridisation indicates that the herpesvirus may have been transmitted from adults to larvae. It is notable that, unlike most herpesviruses, this virus has a wide host range reflected by its ability to infect several species of marine bivalve.

Ostreid herpes virus 1 (OsHV-1) infections, notably reported in Europe and the USA, are closely associated with significant mortalities of the Pacific oyster, Crassostrea gigas, especially during its early stages of life. In summer 2006, we monitored mortality by strict daily verification of three full-sib families of oysters reared under common conditions. We quantified OsHV-1 using real-time PCR in dead and living individuals during and after a mortality event. Mortality events were severe and brief, but significantly different between tested families (cumulative mortality ranging from 1.2 to 49%). Real-time PCR assays revealed different viral DNA loads in dead individuals from different families (P < 0.001). Moreover, the mean level of infection among families was correlated with mortality (P < 0.05). Living oysters showed a significantly lower amount of viral DNA compared with dead ones. This is the first experiment showing the daily changes of individual OsHV-1 DNA load during a mortality outbreak. Our results also support the previously reported high genetic basis underlying the variance of resistance of Pacific oyster to summer mortality, suggesting that there might be a possibility to improve resistance to OsHV-1 by selective breeding.

Herpes-like viruses have been reported last decades in various marine mollusc species in association with mortality outbreaks throughout the world (Renault & Novoa 2004). One of these viruses isolated during French outbreaks has been characterised as an unassigned member of the Herpesviridae family and named ostreid herpes virus 1 (OsHV-1) or Pacific oyster herpes virus (Davison et al. 2005). Studying anti-viral non-specific defence mechanisms (innate immunity) is of great interest, because they constitute the only one in molluscs. Therefore, innate immunity has be investigated in the Pacific cupped oyster, Crassostrea gigas. The main aim of the work was focused on the identification and the characterisation of genes induced by OsHV-1 in the Pacific cupped oyster. In turn, this could be of benefit to the control of viral diseases of mollusc species. Suppression Subtractive Hybridisation (SSH) has been used to characterize genes involved in anti-viral response using OsHV-1 and Pacific oyster as a disease model. The subtracted library was obtained using RNA from oyster haemocytes (control and after virus contact). Clones identified as differentially expressed using SSH were sequenced and compared to sequences available in databases. Of the clones showing homology with known genes, several of them have functions connected to the innate immune response : macrophage expressed protein, molluscan defense molecule precursor or laccase 1. The full-length cDNA of these three genes was obtained using 5' and 3' RACE PCR. The predicted amino sequences were analysed for domain features and conserved signature motifs (Smart). Multiple alignment (ClustalW) and homology analysis of the deduced amino acid sequences of laccase 1, molluscan defence molecule precursor and macrophage expressed protein gene homologues were carried out. A phylogenic analysis was performed with MEGA program (version 3.1) based on amino acids alignment. The expression pattern of transcripts in healthy and OsHV-1 challenged oysters was studied by RT qPCR for laccase, molluscan defence molecule precursor and macrophage expressed protein gene homologues. The expression of the three selected genes was up-regulated and reached a maximum 48 hours after OsHV-1 challenge.

OsHV-1 was the first herpesvirus to be identified in an invertebrate host and is associated to mortalities in the Pacific oyster (Crassostrea gigas) and other economically important bivalve species. Infections due to OsHV-1 are frequently reported and cause high mortality rates in larvae and spat of C. gigas. Detection of this pathogen is usually performed by PCR and/or in situ hybridization. This presentation will focus on the development of a new molecular diagnostic tool for OsHV-1 (and OsHV-1 variant 1) detection: the mini-array. This detection system, which allows multiple pathogens detection, combines viral DNA amplification by PCR and a specific hybridisation on a nylon membrane, revealed by a colorimetric reaction (DNA chip). Multiplex PCR involves specific primers amplifying sequences from OsHV-1. Hybridisation involves probes designed to specifically match to the PCR products. Detection of the obtained hybrids is performed using enzyme-linked antibodies producing a dark blue precipitate following addition of an appropriate substrate. Interpretation of the results is performed by the naked eye. Mini-array's main features include: double specificity, high sensitivity and standardisation of the process and reagents. Moreover, internal controls ensure limited risk of false positives & negatives samples. Subsequently, PCR, qPCR and mini-array methods have been submitted to a validation protocol (based on OIE recommendations and experts' advices) including rough estimation of analytical sensitivity and specificity, as well as repeatability. Two oyster populations with different prevalence of infection by OsHV-1 (high and low prevalence) have been tested to obtain rough estimations of diagnostic sensitivity & specificity. Inter-laboratory tests with reference samples should be the following step to confirm validation of these methods.

Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR® Green chemistry with specific primers C9/C10 targeting the C region, which is present in two copies in the OsHV-1 genome and encodes two proteins of unknown function. The assay was applied successfully to rapid diagnosis (100 minutes) of OsHV-1 in different developmental stages and tissue samples of Crassostrea gigas. This capability is important because no cell culture or in vitro system is available for quantitation of virus replication. The quantitative data that will emerge in future using the new assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues. This poster presents real-time PCR assay and first data with field and experimental material.

A DNA extraction procedure was developed for the detection of ostreid herpesvirus-1 (OsHV-1) using the polymerase chain reaction (PCR) in oyster larvae. The DNA extraction procedure developed was tested on 8 larval samples. Abnormal nuclei with characteristic features associated with OsHV-1 infections were only observed in samples in which the viral DNA was detected by PCR. A previously described competitive PCR method was applied to detect inhibition during PCR reactions. The results show that the method can be used on small amounts of oyster larvae (3 mg) for the detection of OsHV-1 DNA by PCR.