Ostreid herpesvirus 1 (OsHV-1) infections have been reported around the world and are associated with high mortalities of the Pacific oyster (Crassostrea gigas). In the summer 2008, abnormal mortality rates ranging from 80% to 100% were reported in France and affected only Pacific oysters. Analyses of oyster samples collected during mortality outbreaks demonstrated a significant detection of OsHV-1 (75% of analysed batches), which appeared stronger than previous years. DNA sequencing based on C and IA regions was carried out on 28 batches of OsHV-1 infected Pacific oysters collected in 2008. Polymorphisms were described in both the C and IA regions and characterized a genotype of OsHV-1 not already reported and termed OsHV-1 microVar. A microsatellite zone present in the C region showed several deletions. Additionally, 44 isolates collected in France and in the USA, from 1995 to 2007 were sequenced and compared to the 2008 sequences. The analyses of 76 sequences showed OsHV-1 microVar detection only in 2008 isolates. These data suggest that OsHV-1 microVar can be assumed as an emergent genotype.

Ostreid herpesvirus 1 (OsHV-1) is the only member of the Herpesviridae that has an invertebrate host and is associated with sporadic mortality in the Pacific oyster (Crassostrea gigas) and other bivalve species. Cryo-electron microscopy of purified capsids revealed the distinctive T=16 icosahedral structure characteristic of herpesviruses, although the preparations examined lacked pentons. The gross genome organization of OsHV-1 was similar to that of certain mammalian herpesviruses (including herpes simplex virus and human cytomegalovirus), consisting of two invertible unique regions (U L , 167?8 kbp; U S , 3?4 kbp) each flanked by inverted repeats (TR L /IR L , 7?6 kbp; TR S /IR S , 9?8 kbp), with an additional unique sequence (X, 1?5 kbp) between IR L and IR S . Of the 124 unique genes predicted from the 207 439 bp genome sequence, 38 were members of 12 families of related genes and encoded products related to helicases, inhibitors of apoptosis, deoxyuridine triphosphatase and RING-finger proteins, in addition to membrane-associated proteins. Eight genes in three of the families appeared to be fragmented. Other genes that did not belong to the families were predicted to encode DNA polymerase, the two subunits of ribonucleotide reductase, a helicase, a primase, the ATPase subunit of terminase, a RecB-like protein, additional RING-like proteins, an ion channel and several other membrane-associated proteins. Sequence comparisons showed that OsHV-1 is at best tenuously related to the two classes of vertebrate herpesviruses (those associated with mammals, birds and reptiles, and those associated with bony fish and amphibians). OsHV-1 thus represents a third major class of the herpesviruses. INTRODUCTIONViruses are assigned to the family Herpesviridae on the basis of morphological criteria and have been identified in a wide range of vertebrates and one invertebrate, the Pacific oyster, Crassostrea gigas (Minson et al., 2000). The vertebrate herpesviruses fall into two major phylogenetic groups. Those in the first group have mammalian or avian hosts and are classified into three subfamilies (Alphaherpesvirinae, Betaherpesvirinae and Gammaherpesvirinae) that share extensive genetic relationships . Reptilian herpesviruses probably also belong among the Alphaherpesvirinae (Nigro et al., 2004;Quackenbush et al., 1998;Une et al., 2000;Yu et al., 2001). Viruses in the second group infect amphibians or bony fish and again are interrelated (Bernard & Mercier, 1993;Davison, 1998; Davison et al., 1999). Genetic evidence for a common evolutionary origin for the two groups is tenuous, however, since not a single herpesvirus-specific gene is detectably conserved in both. The only completely sequenced lower vertebrate herpesvirus, channel catfish virus (CCV), does share a few genes with the higher vertebrate group, but these have counterparts in other organisms (Davison, 1992). The conserved gene that comes closest to being herpesvirus specific encodes the putative ATPase subunit of the terminase, an enzyme complex involved in p...

Herpes- and herpes-like viruses are known to infect a wide range of bivalve mollusc species throughout the world. Abnormal summer mortalities associated to the detection of ostreid herpesvirus 1 (OsHV-1) have been currently reported in France among larvae and spat of the Pacific cupped oyster Crassostrea gigas. In the present work, we have developed an experimental protocol of horizontal transmission based on the cohabitation between healthy and experimentally infected oysters. Through a cohabitation trial, the kinetics of OsHV-1 detection in different oyster organs and seawater samples were investigated and characterized for the first time using real time quantitative PCR.

Since 1972, several herpes-like virus infections have been reported among different bivalve species around the world. Most of these reports involved larvae or juveniles presenting high mortalities. Two case reports of herpes-like viruses concerned adult oysters, Crassostrea virginica in USA and Ostrea angasi in Australia. Molecular techniques including PCR and in situ hybridization (ISH) have been recently developed to detect the oyster herpesvirus genome. In the present study, 30 Pacific oyster, Crassostrea gigas, adults have been analyzed using three different techniques: PCR, ISH and immunochemistry, in order to detect herpesviruses in asymptomatic individuals. PCR and ISH allowed detection of oyster herpesvirus DNA in 93.3 and 86.6%, respectively, of analyzed oysters while polyclonal antibodies allowed detection of viral proteins in 76.6% of analyzed adult oysters. These results suggest that oyster herpesvirus infects adult oysters with high prevalence and that the virus may persist in its host after primary infection. The detection of viral DNA and viral proteins in the gonad of several individuals supports the hypothesis of a possible vertical transmission of the infection. Lastly, concordance among the three techniques used in this study is discussed.

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((R)) Green chemistry with specific primers C(9)/C(10) targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75+/-0.1 degrees C). The assay had a detection limit of 4 copies/microL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C(2)/C(6) primers. The assay was applied successfully for rapid diagnosis (100 min) and quantitation of OsHV-1 in different developmental stages of Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.

Virus-induced genes were identified using suppression subtractive hybridisation (SSH) from Pacific cupped oyster, Crassostrea gigas, haemocytes challenged by OsHV-1. A total of 304 clones from SSH forward library were sequenced. Among these sequences, some homologues corresponded to (i) immune related genes (macrophage express protein, IK cytokine, interferon-induced protein 44 or multicopper oxidase), (ii) apoptosis related genes (Bcl-2) and (iii) cell signalling and virus receptor genes (glypican). Molecular characterization and phylogenic analysis of 3 immune-related genes (macrophage expressed protein, multicopper oxidase and immunoglobulin domain cell adhesion molecule) were performed. Finally, quantitative PCR revealed significant changes in the expression of immune related genes (multicopper oxidase, macrophage expressed protein, myeloid differentiation factor 88 and interferon-induced protein 44) in oysters experimentally challenged with OsHV-1. These findings provide a first basis for studying the role of innate immunity in response to viruses in bivalves and identified genes may serve as markers of interest in breeding programs in order to obtain selected oysters presenting OsHV-1 resistance.

International audienceSummer mortality of Pacific oysters is known in several countries. However no specific pathogen has been systematically associated with this phenomenon. A complex combination of environmental and biological parameters has been suggested as the cause and is now starting to be identified. A high genetic basis was found for survival in oysters when a first generation (G1) was tested in three sites during summer. This paper presents a synthesis on physiological characteristics of two selected groups (‘R' and ‘S', from families selected for resistance and susceptibility to summer mortality respectively), of the second and third generations. R and S showed improvement or reduction of survival compared with the control in both field and laboratory trials confirming the high heritability of survival of juveniles <1 year old. Interestingly, no correlation was observed between growth and survival. Comparison between the two selected groups showed that S oysters invested more energy in reproduction and stayed a longer time without spawning than R oysters which had high synchronous spawning. This was mainly shown with high rather than low dietary rations (respectively 12% and 4% DW algae/DW oyster) in a controlled experiment. Moreover, early partial spawning was detected in S oysters and not R ones in the high dietary ration. S showed a higher respiration rate and an earlier decrease in absorption efficiency than R during gametogenesis, but they were not significantly different in glycogen or ATP utilisation. Two months before a mortality episode, hemocytes from S oysters had a higher adhesive capacity than R hemocytes and significantly higher reactive oxygen species production capacity. One month before mortality, S oysters had the highest hyalinocyte concentration and their expression of genes coding for glucose metabolism enzymes (Hexokinase, GS, PGM, PEPCK) was significantly lower in the labial palps. After a thermal increase from 13 °C to 19 °C, during 8 days in normoxia, S oysters showed a large HSP70 increase under hypoxia contrary to R oysters, suggesting their high susceptibility to stress. Their catalase activity was lower than in R oysters and showed no further change to subsequent hypoxia and pesticide stresses, in contrast to R oysters. These observations suggest possible links between higher reproductive effort in S oysters, their specific stress response to temperature and hypoxia, ROS production, partial spawning, hyalinocyte increase and the infection process. To compare R and S oysters in a more integrated way, a suppression subtractive hybridisation (SSH) library and a micro-array strategy are being undertaken

BackgroundMassive mortality outbreaks affecting Pacific oyster (Crassostrea gigas) spat in various countries have been associated with the detection of a herpesvirus called ostreid herpesvirus type 1 (OsHV-1). However, few studies have been performed to understand and follow viral gene expression, as it has been done in vertebrate herpesviruses. In this work, experimental infection trials of C. gigas spat with OsHV-1 were conducted in order to test the susceptibility of several bi-parental oyster families to this virus and to analyze host-pathogen interactions using in vivo transcriptomic approaches.ResultsThe divergent response of these oyster families in terms of mortality confirmed that susceptibility to OsHV-1 infection has a significant genetic component. Two families with contrasted survival rates were selected. A total of 39 viral genes and five host genes were monitored by real-time PCR. Initial results provided information on (i) the virus cycle of OsHV-1 based on the kinetics of viral DNA replication and transcription and (ii) host defense mechanisms against the virus.ConclusionsIn the two selected families, the detected amounts of viral DNA and RNA were significantly different. This result suggests that Pacific oysters are genetically diverse in terms of their susceptibility to OsHV-1 infection. This contrasted susceptibility was associated with dissimilar host gene expression profiles. Moreover, the present study showed a positive correlation between viral DNA amounts and the level of expression of selected oyster genes.