Jean-Francois Pepin

Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpesvirus 1: demonstration of oyster spat susceptibility

In 2008 and 2009, acute mortalities occurred in France among Pacific cupped oyster, Crassostrea gigas, spat. Different hypothesis including the implication of environmental factors, toxic algae and/or pathogens have been explored. Diagnostic tests indicated that OsHV-1 including a particular genotype, termed OsHV-1 μVar, was detected in most of samples and especially in moribund oysters with the highlighting of virus particles looking like herpes viruses by TEM examination. In this study, an experimental protocol to reproduce OsHV-1 infection in laboratory conditions was developed. This protocol was based on the intramuscular injection of filtered (0.22 μm) tissue homogenates prepared from naturally OsHV-1 infected spat collected on French coasts during mortality outbreaks in 2008. Results of the experimental trials showed that mortalities were induced after injection. Moreover, filtered tissue homogenates induced mortalities whereas the same tissue homogenates exposed to an ultraviolet (UV) treatment did not induce any mortality suggesting that oyster spat mortalities require the presence of a UV sensitive agent. Furthermore, analysis of injected oyster spat revealed the detection of high amounts of OsHV-1 DNA by real-time quantitative PCR. Finally, TEM analysis demonstrated the presence of herpes virus particles. The developed protocol allowed to maintain sources of infective virus which can be useful for the development of further studies concerning the transmission and the development of OsHV-1 infection.

Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea-bass (Dicentrarchus labrax) larvae and juveniles

Abstract Histopathological lesions of the central nervous system are described hatchery-reared larval seabass during mass mortalities. Lesions are consistent with an extensive vacuolation of the brain and retina, associated with intracytoplasmic inclusions, containing RNA as demonstrated with acridineorange epifluorescence. Electron microscope observations revealed numerous cytoplasmic, non-enveloped particles about 22–23 nm in diameter. Isopycnic centrifugation in a CsCl gradient produced iwo bands of virus particles showing buoyant densities of 1.30 to 1.34 g/cm 3 , respectively. Negatively stained preparations exhibited full and empty icosahedral particles about 26 nm in diameter from side to side and 29 nm from point to point. By their characteristics, these particles are closely related to the Picornaviridae family.

Experimental ostreid herpesvirus 1 infection of the Pacific oyster Crassostrea gigas: kinetics of virus DNA detection by q-PCR in seawater and in oyster samples.

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.

Rapid and sensitive detection of ostreid herpesvirus 1 in oyster samples by real-time PCR.

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.

Dual transcriptomics of virus-host interactions: comparing two Pacific oyster families presenting contrasted susceptibility to ostreid herpesvirus 1

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.

Transcriptomic study of 39 ostreid herpesvirus 1 genes during an experimental infection.

Massive mortality outbreaks have been reported in France since 2008 among Pacific oysters, Crassostrea gigas, with the detection of a particular OsHV-1 variant called μVar. Virus infection can be induced in healthy spat in experimental conditions allowing to better understand the disease process, including viral gene expression. Although gene expression of other herpesviruses has been widely studied, we provide the first study following viral gene expression of OsHV-1 over time. In this context, an in vivo transcriptomic study targeting 39 OsHV-1 genes was carried out during an experimental infection of Pacific oyster spat. For the first time, several OsHV-1 mRNAs were detected by real-time PCR at 0 h, 2 h, 4 h, 18 h, 26 h and 42 h post-injection. Several transcripts were detected at 2h post-infection and at 18 h post-infection for all selected ORFs. Quantification of virus gene expression at different times of infection was also carried out using an oyster housekeeping gene, Elongation factor. Developing an OsHV-1-specific reverse transcriptase real time PCR targeting 39 viral gene appears a new tool in terms of diagnosis and can be used to complement viral DNA detection in order to monitor viral replication.

New Insight for the Genetic Evaluation of Resistance to Ostreid Herpesvirus Infection, a Worldwide Disease, in Crassostrea gigas

The Pacific oyster, Crassostrea gigas, is the most important commercial oyster species cultivated in the world. Meanwhile, the ostreid herpesvirus 1 (OsHV-1) is one of the major pathogens affecting the Pacific oyster, and numerous mortality outbreaks related to this pathogen are now reported worldwide. To assess the genetic basis of resistance to OsHV-1 infection in spat C. gigas and to facilitate breeding programs for such a trait, if any exist, we compared the mortality of half- and full-sib families using three field methods and a controlled challenge by OsHV-1 in the laboratory. In the field, three methods were tested: (A) one family per bag; (B) one family per small soft mesh bag and all families inside one bag; (C) same as the previous methods but the oysters were individually labelled and then mixed. The mean mortality ranged from 80 to 82% and was related to OsHV-1 based on viral DNA detection. The narrow-sense heritability for mortality, and thus OsHV-1 resistance, ranged from 0.49 to 0.60. The high positive genetic correlations across the field methods suggested no genotype by environment interaction. Ideally, selective breeding could use method B, which is less time- and space-consuming. The narrow sense heritability for mortality under OsHV-1 challenge was 0.61, and genetic correlation between the field and the laboratory was ranged from 0.68 to 0.75, suggesting a weak genotype by environment interaction. Thus, most of families showing the highest survival performed well in field and laboratory conditions, and a similar trend was also observed for families with the lowest survival. In conclusion, this is the first study demonstrating a large additive genetic variation for resistance to OsHV-1 infection in C. gigas, regardless of the methods used, which should help in selective breeding to improve resistance to viral infection in C. gigas.

Resistance to OsHV-1 Infection in Crassostrea gigas Larvae

The ostreid herpesvirus (OsHV-1) is one of the major diseases that affect the Pacific oyster Crassostrea gigas. Selective breeding programs were recently shown to improve resistance easily to OsHV-1 infections in spat, juvenile and adult oysters. Nevertheless, this resistance has never been investigated in larvae, whereas this developmental stage has crucial importance for the production of commercial hatcheries, as well as explaining the abundance of spatfall. A first trial tested several viral suspensions at several concentrations using contaminated water with OsHV-1 in four-day-old and ten-day-old larvae that were produced from an unselected broodstock. In follow up on the results, one viral suspension at a final concentration of 10+6 OsHV-1 DNA copies per L was used to assess resistance to OsHV-1 infection in C. gigas larvae that were produced from selected and unselected broodstock. A second trial evaluated OsHV-1 resistance in larvae from both broodstocks in trials 2a, 2b and 2c with 4, 10 and 16-day-old larvae for 7 days, which corresponded to post D larvae, umbo larvae and eyed larvae, respectively. The mortality of unchallenged larvae for both stocks were low (<15%) at day 7 in trials 2a and 2b, whereas it ranged from 48 to 56% in trial 2c. More interestingly, selected larvae had significantly lower mortality than unselected larvae when exposed to OsHV-1 in all of the trials. Thus, the mortality was 11% and 49% for the selected larvae at day 7 post-exposure in trials 2a and 2c, respectively, in comparison with 84% and 97% for the unselected larvae. Although this difference in mortality was observed at day 5 in trial 2b, it was reduced at day 7, to 86% and 98% for the selected and unselected larvae, respectively. For the first time in the literature, the difference in mortality or the delayed onset of mortality between selected and unselected larvae have indicated a genetic resistance to OsHV-1 infection at the larval stage. Such finding should facilitate the selective breeding programs focusing on resistance to OsHV-1 infection by reducing the span of the genetic evaluation, and thus decreasing its cost