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Ostreid herpesvirus 1 (OsHV-1) has caused mass mortalities in Pacific oysters (Crassostrea gigas) in Europe, Australia, and New Zealand. While aquaculture-associated movements of infected Pacific oysters are a well-known cause of OsHV-1 spread once established in a region, translocation via biofouling of aquaculture equipment or vessels needs further investigation to explain the more distant spread of OsHV-1. Laboratory experiments were designed to test for transmission of OsHV-1 between infected and naive Pacific oysters via a simulated biofouling translocation scenario. Three common biofouling species [Sydney rock oysters (Saccostrea glomerata), Mediterranean mussels (Mytilus galloprovincialis) and Pacific oysters] were tested as intermediaries using a cohabitation challenge with Pacific oysters infected by injection. Transmission occurred, albeit for one of eight replicates when Pacific oysters were the intermediary species. This demonstrated a possible pathway for pathogen spread via biofouling containing Pacific oysters while highlighting the complexity of OsHV-1 transmission. Such complexities require further investigation to inform future risk assessments and management of fouled aquaculture equipment and vessels.

Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.

WOS:000531794600001; Ostreid herpesvirus-1 (OsHV-1) is known to associate with particles in seawater, leading to infection and disease in the Pacific oyster Crassostrea gigas. The estuarine environment is highly complex and changeable, and this needs to be considered when collecting environmental samples for pathogen detection. The aims of this study were to (1) compare different aspects of collecting natural seawater and plankton samples for detection of OsHV-1 DNA and (2) determine whether detection of OsHV-1 DNA in such environmental samples has merit for disease risk prediction. The results of one experiment suggest that sampling on the outgoing tide may improve the detection of OsHV-1 DNA in seawater and plankton tow samples (odds ratio 2.71). This statistical comparison was not possible in 2 other experiments. The method (plankton tow or beta bottle) and depth of collection (range: 250-1250 mm) had no effect on the likelihood of detection of OsHV-1. OsHV-1 DNA was found at low concentrations in plankton tow and seawater samples, and only when outbreaks of mortality associated with OsHV-1 were observed in nearby experimental or farmed populations of C. gigas. This suggests that single point in time environmental samples of seawater or plankton are not sufficient to rule out the presence of OsHV-1 in an estuary. The association of OsHV-1 with particles in seawater needs to be better understood in order to determine whether more selective and sensitive methods can be devised to detect it, before environmental samples can be reliably used in disease risk prediction.

The emergence of the microvariant genotype of Ostreid herpesvirus 1 (OsHV-1 μVar) has caused mass mortalities of Pacific oysters Crassostrea gigas, resulting in significant economic losses in Europe, New Zealand and Australia. There is variability in the occurrence and severity of disease caused by OsHV-1, with the disease incompletely described by the known complex interactions between host, environment and pathogen. There is a need to evaluate the role of anthropogenic factors on this disease expression due to the number of interactions between humans and oysters. A controlled in vivo laboratory infection model was used to assess changes to the susceptibility of 6 mo old Pacific oysters to OsHV-1 challenge after pre-exposure to combinations of stressors. Pre-exposure of oysters to a concentration of the pesticide imidacloprid consistent with the higher range of environmental contamination in some estuaries had no impact on their survival or OsHV-1 viral load. Oysters pre-exposed to air for 24 h prior to OsHV-1 challenge by cohabitation were more resilient to infection. Moderate physical handling that simulated on-farm handling did not affect survival. This indicates that farm management practices implemented prior to OsHV-1 exposure might not specifically predispose oysters to more severe disease, and more complex confounding factors need to be considered. It is likely that changes in host physiology during emersion provide the host with increased resilience to disease caused by OsHV-1. Continued investigation of the effect of air exposure in the field will aid in validating the results from this laboratory experiment.

Mollusc farming is the third most productive aquaculture activity in the world, and the Pacific oyster (Crassostrea gigas) is one of the most important farmed species. Since 2008, mass mortalities in C. gigas due to ostreid herpesvirus 1 microvariants have challenged the viability of this industry in Europe, New Zealand and Australia. Ten years after the emergence of this disease, there is evidence that the industry has become consolidated into fewer, larger companies, with the displacement of small farming enterprises and loss of employment in coastal communities. Rather than seeking technical solutions, the industry has turned to compensatory production strategies, such as increasing the number of spat placed on farms, higher market prices for table oysters and direct marketing, which appear to have allowed profitability. Biosecurity policies and responses to outbreaks, including those from within the industry, have had unintended consequences for hatcheries and farmers in areas free of disease, mainly caused by restrictions on animal movements, and have not prevented global spread. There may be opportunities for better coordination of industry and government responses to epizootic disease emergence in aquaculture. There is certainly a need for increased adoption of technical advances from research, once these solutions have been adequately verified.

Microvariant genotypes of Ostreid herpesvirus 1 (OsHV-1) are associated with mass mortality events of Pacific oysters in many countries. The OsHV-1 microvariant (µVar) emerged in France 2008 and caused significant economic losses as it became endemic and displaced the previously dominant OsHV-1 reference genotype. Recently, considerable genotypic variation has been described for OsHV-1 microvariants, however, less is known about variation in viral phenotype. This study used an in vivo laboratory infection model to assess differences in total cumulative mortality, peak viral load, transmissibility, and dose-response for three OsHV-1 isolates obtained between 2011 and 2015 from endemic waterways in Australia. This followed field observations of apparent reductions in the severity of mass mortalities over this time. Significantly higher hazard of death and cumulative mortality were observed for an isolate obtained in 2011 compared to isolates from 2014–2015. In keeping with other studies, the hazard of death was higher in oysters challenged by injection compared to challenge by cohabitation and the mortality was higher when the initial dose was 1 × 104 OsHV-1 DNA copies per oyster injection compared to 1 × 102 DNA copies. There was no difference in the quantity of OsHV-1 DNA at time of death that could be related to isolate or dose, suggesting similar pathogenetic processes in the individual oysters that succumbed to end-stage disease. While the isolates examined in this study were biased towards pathogenic types of OsHV-1, as they were collected during disease outbreaks, the variation in virulence that was observed, when combined with prior data on subclinical infections, suggests that surveillance for low virulence genotypes of OsHV-1 would be rewarding. This may lead to new approaches to disease management which utilize controlled exposure to attenuated strains of OsHV-1.

Ostreid Herpesvirus 1 (OsHV-1) represents a serious threat to shellfish aquaculture worldwide. To minimise its impact, early warning systems able to detect the virus in seawater prior to infection of oysters are of utmost importance. However, monitoring OsHV-1 in seawater is challenging because of its presence at very low concentrations. Thus, a rapid and simple method to pre-concentrate the virus is needed to enable detection. Herein, magnetic beads (MBs) coated with an anionic polymer were used to pre-concentrate OsHV-1 from biological matrices including oyster homogenate and seawater samples. Following virus capture, OsHV-1 DNA detection was performed by quantitative PCR (qPCR). The MB-based approach combined with qPCR attained a limit of detection (LOD) as low as 0.1 viral copy/μL, which was 100 times lower than that of the qPCR alone. This approach was applied to the analysis of OsHV-1 in seawater from an Enhanced Hydro-Optic UV (HOD-UV) disinfection experiment operating at a UV dose of 1360 J/m2 in an open flow system. Our approach enabled detection of the virus in non-treated seawater and not in UV-treated seawater, discrimination that was not possible using qPCR alone. Moreover, the strategy provided data on the pattern of kinetics of the release of the virus in seawater. The approach could find applications in shellfish hatcheries and depuration plants to ensure biosecurity requirements. info:eu-repo/semantics/acceptedVersion

International audience; Significant mortality of Crassostrea gigas juveniles is observed systematically every year worldwide. Pacific Oyster Mortality Syndrome (POMS) is caused by Ostreid Herpesvirus 1 (OsHV-1) infection leading to immune suppression, followed by bacteraemia caused by a consortium of opportunistic bacteria. Using an in-situ approach and pelagic chambers, our aim in this study was to identify pathogen dynamics in oyster flesh and in the water column during the course of a mortality episode in the Mediterranean Thau lagoon (France). OsHV-1 concentrations in oyster flesh increased before the first clinical symptoms of the disease appeared, reached maximum concentrations during the moribund phase and the mortality peak. The structure of the bacterial community associated with oyster flesh changed in favour of bacterial genera previously associated with oyster mortality including Vibrio, Arcobacter, Psychrobium, and Psychrilyobacter. During the oyster mortality episode, releases of OsHV-1 and opportunistic bacteria were observed, in succession, in the water surrounding the oyster lanterns. These releases may favour the spread of disease within oyster farms and potentially impact other marine species, thereby reducing marine biodiversity in shellfish farming areas

In genomic selection (GS), genome-wide SNP markers are used to generate genomic estimated breeding values for selection candidates. The application of GS in shellfish looks promising and has the potential to help in dealing with one of the main issues currently affecting Pacific oyster production worldwide, which is the 'summer mortality syndrome'. This causes periodic mass mortality in farms worldwide and has mainly been attributed to a specific variant of the ostreid herpesvirus (OsHV-1). In the current study, we evaluated the potential of genomic selection for host resistance to OsHV-1 in Pacific oysters, and compared it with pedigree-based approaches. An OsHV-1 disease challenge was performed using an immersion-based virus exposure treatment for oysters for 7 days. A total of 768 samples were genotyped using the medium-density SNP array for oysters. A GWAS was performed for the survival trait using a GBLUP approach in blupf90 software. Heritability ranged from 0.25 ± 0.05 to 0.37 ± 0.05 (mean ± SE) based on pedigree and genomic information respectively. Genomic prediction was more accurate than pedigree prediction, and SNP density reduction had little impact on prediction accuracy until marker densities dropped below approximately 500 SNPs. This demonstrates the potential for GS in Pacific oyster breeding programmes, and importantly, demonstrates that a low number of SNPs might suffice to obtain accurate genomic estimated breeding values, thus potentially making the implementation of GS more cost effective.

The Ostreid herpesvirus 1 species affects shellfish, contributing significantly to high economic losses during production. To counteract the threat related to mortality, there is a need for the development of novel point-of-care testing (POCT) that can be implemented in aquaculture production to prevent disease outbreaks. In this study, a simple, rapid and specific colorimetric loop-mediated isothermal amplification (LAMP) assay has been developed for the detection of Ostreid herpesvirus1 (OsHV-1) and its variants infecting Crassostrea gigas (C. gigas). The LAMP assay has been optimized to use hydroxynaphthol blue (HNB) for visual colorimetric distinction of positive and negative templates. The effect of an additional Tte UvrD helicase enzyme used in the reaction was also evaluated with an improved reaction time of 10 min. Additionally, this study provides a robust workflow for optimization of primers for uncultured viruses using designed target plasmid when DNA availability is limited. info:eu-repo/semantics/publishedVersion

The microvariant genotype of Ostreid herpesvirus 1 (OsHV-1 μVar) has severely disrupted oyster production in Europe, New Zealand, and Australia by causing repeated and seasonal outbreaks of mass mortality in Pacific oysters (Magallana gigas). The virus was first detected in Tasmania, Australia, in January 2016, and mortalities of up to 87% were reported (de Kantzow et al., 2017). This study surveyed 95% of Tasmanian oyster farmers in OsHV-1 infected growing areas one year following initial detection, and recorded mortalities and associated farm management strategies in the 2016/2017 season, compared with the initial outbreak and before OsHV-1 occurrence. The survey was comprised of 37 open- and closed-ended questions, with data collected on background information, mortalities, environmental, genetic, and husbandry information. Perceived business viability was overall strong (75%), with changes to farm management occurring on 88% of leases in response to the virus. Commercial oyster farming businesses ranked handling regimes and stocking densities as the most important husbandry factors for influencing mortalities. Water temperature was ranked as the most important environmental factor, with 60% of businesses considering mean water temperature of 18–

New farm management practices are required to reduce the impact of disease caused by Ostreid herpesvirus-1 (OsHV-1) on commercial production of Crassostrea gigas. Both the age and size of oysters are thought to be correlated with survival after exposure to OsHV-1. It is important to distinguish between the protective effects of these factors because the phenotypic plasticity of C. gigas enables a wide range of oyster sizes at any given age. The purpose of this study was to: (1) determine if the age and size of C. gigas have independent effects on susceptibility to disease caused by OsHV-1; (2) determine whether deliberate manipulation of the size of oyster spat (defined as oysters ≤ 12 months old) or adults (oysters > 12 months old) would alter their susceptibility to disease; and (3) assess whether size manipulation could be used as a feasible management strategy to protect C. gigas from mortality due to OsHV-1. Two experiments were conducted, with replicated field and laboratory challenges. In Experiment 1, a protective effect of larger size, independent of age (spat v. adults), was shown for oysters that had grown to be larger under standard commercial farming conditions. After adjusting for clustering due to site and basket, the hazard of death in small oysters was 1.9 times that of large spat and adult oysters. In a second experiment, the size of oysters was deliberately manipulated by restricting the growth of a half of the oysters (independent of starting size); immersion time was limited using a higher longline height compared to oysters held at the optimal intertidal growing height for 6 months, in a region free of OsHV-1. After controlling for the effects of variation in exposure due to location in the field, size group did not impact mortality for either age group but the hazard of death for oyster spat (8 months old) was 5.5 times that of adult oysters (17 months old). Laboratory trials for these prospectively differentiated oysters indicated an interaction between age and size whereby smaller spat (growth restricted) were relatively protected (hazard ratio 0.6) compared to those grown to a larger size. For adults the hazard of death was higher (HR = 2.3) for smaller oysters. Further investigation is required before size manipulation of oysters can be effectively utilised as a management strategy to protect C. gigas from OsHV-1 associated mortality. A thorough understanding of the physiological and metabolic condition of oysters, produced under different grow-out conditions when challenged with OsHV-1 is required.

Ostreid herpesvirus 1 (OsHV-1) is a DNA virus of the genus Ostreavirus (Malacoherpesviridae family, Herpesvirales order). Worldwide, OsHV-1 and its microvariants have been associated with increased mortality of Pacific oysters, Crassostrea gigas. Adult asymptomatic oysters also have shown a high prevalence of viral infection. As a consequence, surveillance is needed to better describe OsHV-1 diversity, pathogenicity, clinical signs, and geographical distribution. We examined Crassostrea gigas sampled in October 2017 from the inner zone of the Bahía Blanca Estuary, Argentina, and found that 8 of 30 specimens (26.7%) presented macroscopic lesions in mantle tissues. Histological analysis revealed abnormal presentation of mantle epithelial cells and connective tissues. Conventional and real-time PCR conducted on the oyster samples revealed 70% to be positive for presence of OsHV-1 DNA. The nucleotide sequence of the amplicon obtained from one sample using the primer pair IA1/IA2 (targeting ORF 42/43) was 99% identical to OsHV-1 reference as well as µVar strains B and A (KY271630, KY242785.1), sequenced from France and Ireland. This finding represents the first detection of OsHV-1 DNA in a wild population of C. gigas in Argentina in association with gross mantle lesions. Fil: Barbieri, Elena Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentina Fil: Medina, Cintia Débora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentina Fil: Vázquez, Nuria Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentina Fil: Fiorito, Carla Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentina Fil: Martelli, Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentina Fil: Wigdorovitz, Andrés. CICVyA, Laboratorio de IncuINTA; Argentina Fil: Schwindt, Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto de Biología de Organismos Marinos; Argentina Fil: Morga, Benjamín. IFREMER, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins; Francia Fil: Renault, Tristan. IFREMER, SG2M-LGPMM, Laboratoire de Génétique et Pathologie des Mollusques Marins; Francia Fil: Parreño, Viviana. CICVyA, Laboratorio de IncuINTA; Argentina Fil: Barón, Pedro J.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Centro para el Estudio de Sistemas Marinos; Argentina

Seasonally recurrent outbreaks of mass mortality in Pacific oysters (Crassostrea gigas) caused by microvariant genotypes of ostreid herpesvirus 1 (OsHV-1) occur in Europe, New Zealand and Australia. The incubation period for OsHV-1 under experimental conditions is 48-72 hours and depends on water temperature, as does the mortality. An in vivo growth curve for OsHV-1 was determined by quantifying OsHV-1 DNA at 10 time points between 2 and 72 hours after exposure to OsHV-1. The peak replication rate was the same at 18 °C and 22 °C; however, there was a longer period of amplification leading to a higher peak concentration at 22 °C (2.34 × 107 copies/mg at 18 hours) compared to 18 °C (1.38 × 105 copies/mg at 12 hours). The peak viral concentration preceded mortality by 72 hours and 20 hours at 18 °C and 22 °C, respectively. Cumulative mortality to day 14 was 45.9% at 22 °C compared to 0.3% at 18 °C. The prevalence of OsHV-1 infection after 14 days at 18 °C was 33.3%. No mortality from OsHV-1 occurred when the water temperature in tanks of oysters challenged at 18 °C was increased to 22 °C for 14 days. The influence of water temperature prior to exposure to OsHV-1 and during the initial virus replication is an important determinant of the outcome of infection in C. gigas.

Background Variants of the Ostreid herpesvirus 1 (OsHV-1) cause high losses of Pacific oysters globally, including in Tomales Bay, California, USA. A suite of new variants, the OsHV-1 microvariants (μvars), cause very high mortalities of Pacific oysters in major oyster-growing regions outside of the United States. There are currently no known Pacific oysters in the United States that are resistant to OsHV-1 as resistance has yet to be evaluated in these oysters. As part of an effort to begin genetic selection for resistance to OsHV-1, 71 families from the Molluscan Broodstock Program, a US West Coast Pacific oyster breeding program, were screened for survival after exposure to OsHV-1 in Tomales Bay. They were also tested in a quarantine laboratory in France where they were exposed to a French OsHV-1 microvariant using a plate assay, with survival recorded from three to seven days post-infection. Results Significant heritability for survival were found for all time points in the plate assay and in the survival phenotype from a single mortality count in Tomales Bay. Genetic correlations between survival against the French OsHV-1 μvar in the plate assay and the Tomales Bay variant in the field trait were weak or non-significant. Conclusions Future breeding efforts will seek to validate the potential of genetic improvement for survival to OsHV-1 through selection using the Molluscan Broodstock Program oysters. The lack of a strong correlation in survival between OsHV-1 variants under this study’s exposure conditions may require independent selection pressure for survival to each variant in order to make simultaneous genetic gains in resistance.

The ostreid herpesvirus 1 microvariant (OsHV-1 µVar) causes mass mortality of Pacific oysters Crassostrea gigas. Water temperature can directly influence the incidence of disease or correlate with seasonal changes in the environment and oyster physiology that modify the susceptibility of the oysters to disease. The effect of water temperature was evaluated in con- trolled laboratory conditions by intramuscular injection of OsHV-1 µVar after acclimation of 8 mo old spat and 17 mo old adult oysters at 4 different temperatures (14, 18, 22 and 26°C). Mortality was 84 and 77% at 26 and 22°C, respectively, compared to 23% at 18°C and nil at 14°C. There was a statistically significant interaction between the dose of OsHV-1 µVar and water temperature. At 18°C, mortality occurred exclusively at a dose of 10 6 OsHV-1 µVar genome copies per oyster whereas at the higher temperatures, oysters challenged with 10 3 copies per oyster also died. Mor- tality did not occur at 14°C and OsHV-1 µVar was detected in tissues of only 1% of the oysters after 14 d. When accounting for temperature and dose, spat (8 mo) were 2.7 times more likely to die than adults (17 mo). Our study confirms a direct effect of water temperature on infection and dis- ease caused by OsHV-1 µVar. We identified a threshold water temperature of between 14 and 18°C below which productive infection does not occur and the requirement for a higher dose of OsHV-1 µVar to initiate infection at 18°C than at 22°C. These results have implications for predict- ing and managing disease outbreaks caused by OsHV-1 µVar.

High mortality rates are reported in spat and larvae of Pacific oyster Crassostrea gigas and associated with ostreid herpesvirus 1 (OsHV-1) detection in France. Although the viral infection has been experimentally reproduced in oyster larvae and spat, little knowledge is currently available concerning the viral entry and its distribution in organs and tissues. This study compares OsHV-1 DNA and RNA detection and localization in experimentally infected oysters using two virus doses: a low dose that did not induce any mortality and a high dose inducing high mortality. Real time PCR demonstrated significant differences in terms of viral DNA amounts between the two virus doses. RNA transcripts were detected in oysters receiving the highest dose of viral suspension whereas no transcript was observed in oysters injected with the low dose. This study also allowed observing kinetics of viral DNA and RNA detection in different tissues of oyster spat. Finally, viral detection was significantly different in function of tissues (p < 0.005), time (p < 0.005) with an interaction between tissues and time (p < 0.005) for each probe.

The Ostreid herpesvirus 1 (OsHV-1) and variants, particularly the microvariants (μVars), are virulent and economically devastating viruses impacting oysters. Since 2008 OsHV-1 μVars have emerged rapidly having particularly damaging effects on aquaculture industries in Europe, Australia and New Zealand. We conducted field trials in Tomales Bay (TB), California where a non-μVar strain of OsHV-1 is established and demonstrated differential mortality of naturally exposed seed of three stocks of Pacific oyster, Crassostrea gigas, and one stock of Kumamoto oyster, C. sikamea. Oysters exposed in the field experienced differential mortality that ranged from 64 to 99% in Pacific oysters (Tasmania>Midori = Willapa stocks), which was much higher than that of Kumamoto oysters (25%). Injection trials were done using French (FRA) and Australian (AUS) μVars with the same oyster stocks as planted in the field and, in addition, two stocks of the Eastern oyster, C. virginica. No mortality was observed in control oysters. One C. virginica stock suffered ~10% mortality when challenged with both μVars tested. Two Pacific oyster stocks suffered 75 to 90% mortality, while one C. gigas stock had relatively low mortality when challenged with the AUS μVar (~22%) and higher mortality when challenged with the French μVar (~72%). Conversely, C. sikamea suffered lower mortality when challenged with the French μVar (~22%) and higher mortality with the AUS μVar (~44%). All dead oysters had higher viral loads (~1000×) as measured by quantitative PCR relative to those that survived. However, some survivors had high levels of virus, including those from species with lower mortality. Field mortality in TB correlated with laboratory mortality of the FRA μVar (69% correlation) but not with that of the AUS μVar, which also lacked correlation with the FRA μVar. The variation in response to OsHV-1 variant challenges by oyster species and stocks demonstrates the need for empirical assessment of multiple OsHV-1 variants.

OsHV-1 is an epidemic pathogen of molluscs, and temperature has been recognized as a decisive environmental factor in its pathogenicity. In recent years, ark clam, Scapharca broughtonii, emerged as a host for OsHV-1. In the north of China, massive summer mortalities of ark clams infected with OsHV-1 have been continuously reported since 2012. However, the interaction between temperature and the pathogenicity of OsHV-1 was unknown in ark clams. In this study, the effect of temperature (10 °C to 18 °C stepped by 2 °C) on the occurrence of OsHV-1 disease in ark clams was analyzed. OsHV-1 infection led to gill erosion but not below the critical low temperature (between 12 °C and 14 °C). However, OsHV-1 persisted for more than 2 weeks at 12 °C post inoculation and replication was reactivated when the temperature was elevated to 18 °C. No significant reduction of OsHV-1 DNA load was found when the temperature descended to 12 °C from 18 °C, while the gill erosion remained unchanged. Ark clams failed to show the capability of effective clearance of OsHV-1 below the critical low temperature. Our results demonstrated that the pathogenicity of OsHV-1 was influenced significantly by temperature. Moreover, high temperature favored infection, which could provide more information to understand summer mortality of ark clams.

High levels of ostreid herpesvirus 1 (OsHV-1) were detected in hemocytes of OsHV-1 infected mollusks. Mollusk hemocytes are comprised of different cell types with morphological and functional heterogeneity. Granular cells are considered the main immunocompetent hemocytes. This study aimed to ascertain if OsHV-1 infects specific types of hemocytes in ark clams. Types of hemocytes were first characterized through microexamination and flow cytometry. In addition to a large group of red cells, there were three types of recognizable granular cells in ark clams. Type II granular cells were mostly found with OsHV-1 infection by transmission electron microscope (TEM) examination, and represented the hemocyte type that was susceptible to OsHV-1 infection. The subcellular location of OsHV-1 particles in apoptotic type II granular cells was further analyzed. Some OsHV-1 particles were free inside the apoptotic cells, which may contribute to OsHV-1 transmission among cells in the host, some particles were also found enclosed inside apoptotic bodies. Apoptosis is an important part of the host defense system, but might also be hijacked by OsHV-1 as a strategy to escape host immune attack. Following this investigation, a primary culture of type II granular cells with OsHV-1 infection would facilitate the research on the interaction between OsHV-1 and mollusk hosts.

Since November 2017, mass mortalities of larvae of bay scallop (Argopecten irradians) were reported in hatcheries located at the southern area of Republic of Korea. Over 90% of larvae aged 5-10 days sank to the bottom of the tank and died. The hatcheries could not produce spat, and thus artificial seed production industry incurred huge losses. We identified Ostreid Herpesvirus-1 μVar (OsHV-1 μVar) associated with mass mortality by PCR, sequencing and transmission electron microscopy (TEM). All the samples were positive for OsHV-1 μVar with 99% sequence identity to previously reported OsHV-1 μVar sequences. Partial sequence of ORF-4 of OsHV-1 detected in this study was more closely related to sequences isolated from Europe. This is the first report to confirm the mortality caused by an OsHV-1 infection in the bay scallop.

An understanding of husbandry strategies and any associated risk factors is important for designing management control measures that can reduce mortality in Pacific oysters, Crassostrea gigas, caused by ostreid herpesvirus 1 (OsHV‐1). The type of culture facility can be considered in relation to the potential pathways that could lead to the entry of a pathogen and its survival. In addition, the animal host (e.g. age, physiological state, selective breeding programmes), husbandry procedures (e.g. stocking density), the pathogen itself (e.g. pathogenicity, virulence) and environmental effects (e.g. temperature) represent other relevant interconnected factors. However, all these factors provide valuable background information for outlining the mitigation strategies needed by the industry, as well as in the context of surveillance and biosecurity programmes. These control mechanisms for hatchery or nursery areas are related to movement restrictions, water treatment, virus inactivation, the production calendar and practical farm management decisions. This comprehensive literature review compiles information related to such approaches and also includes the different existing guidelines suggested for control of OsHV‐1. Therefore, the review represents a solid foundation for a more critical appraisal currently being developed to support recommendations for disease management strategies. info:eu-repo/semantics/publishedVersion

The acute course of disease in young oysters infected by OsHV-1 and the rapid tissue degradation often preclude histological examination of specimens collected during outbreaks in field. Herein, live spat originated from two geographical areas were sampled just at the onset of a mortality event that occurred in Normandy (France) in June 2016. The lesions, associated with high OsHV-1 DNA quantities, were characterized by severe and diffuse haemocytosis mainly involving blast-like cells, myocyte degeneration and large, irregularly shaped degenerate eosinophilic cells in the connective tissue. The herpesvirus was identified by negative staining TEM and real-time PCR. Sequencing of the C region and ORFs 42/43 confirmed that the variants met the definition of OsHV-1 μVar. We sequenced 30 other ORFs in twenty OsHV-1-positive individuals and compared them to the μVar specimens isolated between 2009 and 2011. The ORFs encoding putative membrane proteins showed the highest number of variations. Seven different genotypes were identified, confirming the presence of relevant genetic diversity. Phylogenetic analysis provided evidence for a well-separated μVar new group, with an evolutionary divergence estimated at 0.0013 from the other μVar variants. The geographical distribution of these newly described variants and their effective virulence should be investigated in future.

Mortalities of oyster Crassostrea gig-as seed associated with ostreid herpesvirus OsHV-1 mu Var have been observed in many oyster-producing countries since 2008. The present study, comprised of 4 complementary experiments, aimed to identify factors associated with disease-induced oyster mortality in order to propose mitigation strategies. Our first experiment compared survival of oysters from natural spatfall with others sampled from nurseries, after thermal elevation in the laboratory from

In Australia, the spread of the ostreid herpesvirus-1 microvariant (OsHV-1 µVar) threatens the Pacific oyster industry. There is an urgent need to develop an experimental infection model in order to study the pathogenesis of the virus under controlled laboratory conditions. The present study constitutes the first attempt to use archived frozen oysters as a source of inoculum, based on the Australian OsHV-1 µVar strain. Experiments were conducted to test (1) virus infectivity, (2) the dose-response relationship for OsHV-1, and (3) the best conditions in which to store infective viral inoculum. Intramuscular injection of a viral inoculum consistently led to an onset of mortality 48 h post-injection and a final cumulative mortality exceeding 90%, in association with high viral loads (1 × 105 to 3 × 107 copies of virus mg-1) in dead individuals. For the first time, an infective inoculum was produced from frozen oysters (tissues stored at -80°C for 6 mo). Storage of purified viral inoculum at +4°C for 3 mo provided similar results to use of fresh inoculum, whereas storage at -20°C, -80°C and room temperature was detrimental to infectivity. A dose-response relationship for OsHV-1 was identified but further research is recommended to determine the most appropriate viral concentration for development of infection models that would be used for different purposes. Overall, this work highlights the best practices and potential issues that may occur in the development of a reproducible and transferable infection model for studying the pathogenicity of the Australian OsHV-1 strain in Crassostrea gigas under experimental conditions.

Generalised reviews of RNA interference (RNAi) in invertebrates, and for use in aquaculture, have taken for granted that RNAi pathways operate in molluscs, but inspection of such reviews show little specific evidence of such activity in molluscs. This review was to understand what specific research had been conducted on RNAi in molluscs, particularly with regard to aquaculture. There were questions of whether RNAi in molluscs functions similarly to the paradigm established for most eukaryotes or, alternatively, was it more similar to the ecdozoa and how RNAi may relate to disease control in aquaculture? RNAi in molluscs appears to have been only investigated in about 14 species, mostly as a gene silencing phenomenon. We can infer that microRNAs including let-7 are functional in molluscs. The genes/proteins involved in the actual RNAi pathways have only been rudimentarily investigated, so how homologous the genes and proteins are to other metazoa is unknown. Furthermore, how many different genes for each activity in the RNAi pathway are also unknown? The cephalopods have been greatly overlooked with only a single RNAi gene-silencing study found. The long dsRNA-linked interferon pathways seem to be present in molluscs, unlike some other invertebrates and could be used to reduce disease states in aquaculture. In particular, interferon regulatory factor genes have been found in molluscs of aquacultural importance such as Crassostrea, Mytilus, Pinctada and Haliotis. Two possible aquaculture scenarios are discussed, zoonotic norovirus and ostreid herpesvirus 1 to illustrate the possibilities. The entire field of RNAi in molluscs looks ripe for scientific exploitation and practical application.

International audience; Ostreid herpesvirus 1 (OsHV-1 μvar) infection has caused significant mortalities in juvenile oysters (Crassostrea gigas). In contrast to the practices of other animal production industries, sick and dead oysters are not separated from live ones and are left to decay in the surrounding environment, with unknown consequences on fluxes of dissolved materials. A laboratory approach was used in this study to test the influence of oyster mortality episode on dissolved inorganic fluxes at the oyster interface, dissociating (i) the effect of viral infection on metabolism of juvenile oysters and (ii) the effect of flesh decomposition on oxygen consumption and nutrient releases at the individual scale. Nine batches of juvenile oysters (Individual Total wet weight 1 g) were infected via injection of OsHV-1 enriched inoculums at different viral loads (108 and 109 OsHV-1 DNA copies per oyster) to explore infection thresholds. Oysters injected with filtered seawater were used as controls (C). Oysters were maintained under standard conditions to avoid stress linked to hypoxia, starvation, or ammonia excess. Before, after the injection and during the mortality episode, i.e. at days 1, 3, 7, 10 and 14, nine oysters per treatment were incubated in individual metabolic chambers to quantify oxygen, ammonium and phosphate fluxes at the seawater-oyster interface. Nine empty chambers served as a reference. Injections of the two viral loads of OsHV-1 induced similar mortality rates (38%), beginning at day 3 and lasting until day 14. The observed mortality kinetics were slower than those reported in previous experimental pathology studies, but comparable to those observed in the field (Thau lagoon, France). This study highlights that oxygen and nutrient fluxes significantly varied during mortality episode. Indeed (i) OsHV-1 infection firstly modifies oyster metabolism, with significant decreases in oxygen consumption and ammonium excretion, and (ii) dead oysters lead to a strong increase of ammonium (6 fold) and phosphate (41 fold) fluxes and a decrease in the N/P ratio due to mineralisation of their flesh. The latter may modify the structure of the planktonic community in the field during mortality episode. This study is a first step of the MORTAFLUX program. The second step was to in situ confirm this abnormal nutrient loading during a mortality episode and show its impact on bacterio-, phyto- and protozoo-plankton.

Mortality associated with ostreid herpesvirus 1 (OsHV-1) has significantly impacted Crassostrea gigas farmers all around the world. Although selective breeding programs to improve OsHV-1 resistance can significantly reduce the mortality rate below 20%, husbandry practices have also been one way to limit or decrease mortality. The main objectives of this study were to describe the influence of two husbandry practices on OsHV-1-induced mortality in C. gigas spat and how different family lines respond to husbandry practices to gain a better understanding of the potential benefits of breeding management as a tool to adapt to improved practices. The first analysis investigated the importance of the size of C. gigas when challenged with OsHV-1 for the first time. For each of the 40 families of C. gigas evaluated, small (S) and large (L) oyster groups were tested for two years in the field. A significant mortality outbreak associated with OsHV-1 occurred during the first spring. At the end of the trial, the mean cumulative mortality of the L group (54.1%) was significantly lower than the mean cumulative mortality of the S group (74.8%), indicating the importance of size in OsHV-1-associated mortality. However, the overall effect of size was not consistent for all families: in 11 of the 40 families tested, there appeared to be no difference in mortality between the S and L groups. Observations at the family level allowed the identification of a large range of susceptibility to OsHV-1 regardless of the size group, with highly resistant families (80%). The second investigation compared different growing heights. Each of the 40 families was grown at three heights (high Hi, medium Me, and low Lo) corresponding to mean emersion times of 25, 12 and 2%, respectively. After an OsHV-1-related mortality outbreak, the mean mortality among the families was 91.6%, 88.0% and 86.9% for the Lo, Me and Hi conditions, respectively. Growing height had no effect in 31 of the 40 families tested, and these families showed no potential for adaptation to improve growing height practices. Among them, 24 families showed extremely high mortality (>95%) and 2 families showed the lowest mortality (

Double-stranded RNA (dsRNA)-mediated genetic interference (RNAi) is a widely used reverse genetic tool for determining the loss-of-function phenotype of a gene. Here, the possible induction of an immune response by long dsRNA was tested in a marine bivalve (Crassostrea gigas), as well as the specific role of the subunit 2 of the nuclear factor kappa B inhibitor (I kappa B2). This gene is a candidate of particular interest for functional investigations in the context of oyster mass mortality events, as Cg-I kappa B2 mRNA levels exhibited significant variation depending on the amount of ostreid herpesvirus 1 (OsHV-1) DNA detected. In the present study, dsRNAs targeting Cg-I kappa B2 and green fluorescent protein genes were injected in vivo into oysters before being challenged by OsHV-1. Survival appeared close to 100% in both dsRNA-injected conditions associated with a low detection of viral DNA and a low expression of a panel of 39 OsHV-1 genes as compared with infected control. Long dsRNA molecules, both Cg-I kappa B2- and GFP-dsRNA, may have induced an anti-viral state controlling the OsHV-1 replication and precluding the understanding of the specific role of Cg-I kappa B2. Immune-related genes including Cg-I kappa B1, Cg-Rel1, Cg-IFI44, Cg-PKR and Cg-IAP appeared activated in the dsRNA-injected condition, potentially hampering viral replication and thus conferring a better resistance to OsHV-1 infection. We revealed that long dsRNA-mediated genetic interference triggered an anti-viral state in the oyster, emphasizing the need for new reverse genetics tools for assessing immune gene function and avoiding off-target effects in bivalves.

Monthly Screening of Pacific oyster, Crassostrea gigas, was surveyed by PCR for Ostreid herpesvirus(OsHV-1) infections from 2012 to 2013 in Korea. It was showed positive results in one hatchery in April and May 2012 and in oyster farming areas from August to November in 2013. The obtained sequences showed 99.3~99.5% homology with OsHV-1 μvar(Accession Number: HQ842610) and 96.2~96.3% homology with OsHV isolated from Korea(Accession Number: AY509253).

In 2010 Ostreid herpesvirus-1 (OsHV-1) was detected in Australia and had a disastrous impact on Pacific oyster Crassostrea gigas aquaculture and coastal communities. The acronym POMS (Pacific Oyster Mortality Syndrome) was created in Australia to refer to mass mortalities due to OsHV-1. While management of this disease mainly involves active surveillance, rigorous biosecurity protocols and mollusc breeding programs targeting production of resistant animals, the effects of aquaculture practices on mortality outbreaks are still poorly understood. The present study aimed to determine the effect of growing heights on OsHV-1 associated mortality in C. gigas in Woolooware Bay (Australia) during the summer 2011/2012. Growing height influences the immersion time of inter-tidally cultivated oysters during each tide cycle, and could be an important risk factor for OsHV-1 exposure and mortality. Pacific oysters of different ages (2 and 12 month) were placed in intertidal rearing structures at three different sites in the bay. Mortality and growth rates, infection prevalence and seawater parameters (temperature and salinity) were monitored over 7 months. The outbreak started in November 2011 and mortalities were observed until late April 2012. The pattern of disease expression was time and site dependent as the mortalities started immediately after infection with OsHV-1 at two of the three sites, while the infection preceded the onset of mortality by two months at the third site. No clear difference in salinity or temperature of water was observed among sites suggesting that other environmental features influence the onset of the disease. Extreme mortalities were observed in the younger class of oysters, while the modification of growing height led to a significant increase in survival of adult oysters. Infection prevalence and intensity decreased in surviving oysters suggesting that some individuals may be able to clear the virus. Differences in mortality among sites and growing heights are discussed in relation to OsHV-1 infection intensity and prevalence in oysters, and the environmental data recorded during the outbreak. Keywords Summer mortality; Crassostrea gigas; Ostreid herpesvirus 1; Aquaculture practices; Environmental factors fisheries research and development corporation

The presence of ostreid herpesvirus 1 (OsHV-1) and the percentage of viral DNA detected in Pacific oyster Crassostrea gigas adults were investigated monthly between May and November 2012 at three locations along the southern coast of Korea. Among 210 oysters examined by polymerase chain reaction (PCR) analysis, OsHV-1 DNA was detected in only one oyster collected in August. The low detection rate of OsHV-1 DNA was consistent with the lack of reported OsHV-1-associated disease in C. gigas cultured in Korea. The sequence of the present PCR product amplified with the C2/C6 primer pair was identical to that of OsHV-1 μVar except for one nucleotide, and the sequence amplified with Del36-37F2/Del36-37R showed a 605-bp deletion as in OsHV-1 μVar. Although these sequence data are insufficient to determine genotype, the results suggest that the herpesvirus detected was similar to OsHV-1 μVar. This is the first report on the presence of OsHV-1 in adult Pacific oysters cultured in Korea.

In order to determine if ostreid herpesvirus 1 (OsHV-1) can be vertically transmitted, 9 full-sib families of the Portuguese oyster Crassostrea angulata were produced using a factorial mating design with 3 males and 3 females. The parents were survivors from an OsHV-1 mortality outbreak. OsHV-1 DNA was not detected by conventional PCR in the mantle of parents, gametes or 3 day-old larvae. However, viral DNA was detected by real-time PCR in all gametes and larvae samples. These results show that C. angulata that have survived an OsHV-1 mortality outbreak can carry the virus and vertically transmit it to their offspring.

Cytogenetic abnormalities associated with viral infections, including from viruses of the Herpesvirales order, have been reported in vertebrate species. Ostreid herpesvirus 1 (OsHV-1) has been detected worldwide during mortality outbreaks of the Pacific oyster Crassostrea gigas. On the other hand, a high proportion of aneuploid cells in somatic tissues have been observed in C. gigas. In this study, we analysed the putative association between aneuploidy levels and the detection of OsHV-1 in gills of C. gigas, the Portuguese oyster C. angulata and their F1 hybrids cultured in Ria Formosa (Portugal). OsHV-1 was detected by PCR in 5.4% of the total of oysters analysed (n = 111) namely in 11.1%, 8.0% and 1.7% of C. gigas, C. angulata and F1 hybrid respectively. Sequencing analysis of a viral fragment amplified with the C2/C6 primer pair revealed a high similarity with the OsHV-1 reference type. Moreover, in situ hybridization confirmed the presence of OsHV-1 in gill tissue. Oysters where OsHV-1 was detected had a significantly higher mean percentage of aneuploid cells (25%) than the ones where the virus was not detected (18%). However, the overall low percentage of positive samples contrasted with the high mean percentage of aneuploidy observed, with 50% of the oysters analysed showing a percentage of aneuploid cells between 20% and 30%. We hypothesize that somatic aneuploidy may adversely affect oysters making them more prone to OsHV-1 infection, but the virus is unlikely to be the cause of somatic aneuploidy. FCT. Grant Number: SFRH/BD/8972/2002 Marie Curie Training fellowship. Grant Numbers: PLUDAMOR, QLK5-CT-2000-60036 Wiley Online Library

Since the summer of 2008, the mortality of young Pacific oysters Crassotrea gigas has been greatly increased on the French coast and may be linked to a microvariant of the Ostreid Herpesvirus 1 (OsHV-1), named OsHV-1 μVar. This variant is mainly characterized by a deletion of 12 consecutive nucleotides followed by a deletion of one adenine in a microsatellite area located in the C region. The present study is the continuity of a previous study that was conducted on oysters collected from July 2008 to June 2010 (Martenot et al., 2011). The purpose of the current survey is to determine the genotype of oyster samples collected from July 2010 to August 2011 and samples taken before 2008. The OsHV-1 μVar was detected in 98% of characterized samples and the two new forms of microvariants were found in the remaining 2%. The first genotype has 15 bp successive deletions and the second genotype is similar to the OsHV-1 reference with an insertion of three nucleotides. The OsHV-1 μVar Δ9 previously described in the literature was detected in a sample originating in Brittany. These observations reinforce the possibility of the emergence of different OsHV-1 variants.

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.

Early detection of Pacific oyster spat infected with ostreid herpesvirus 1 (OsHV-1) could prevent introduction of OsHV-1-infected individuals into farming areas or onshore rearing facilities, thus reducing the risk of infection of naïve oysters in such production systems. Experiments were conducted on several hundred oyster spat provided by producers in order to examine whether early rearing practices could be considered as potential risk factors for (1) OsHV-1 infection as detected by molecular methods and (2) spat mortality experimentally induced through thermal challenge. Spat groups collected on oyster beds and hatchery spat reared in growout areas during summer exhibited higher viral DNA contamination and mortalities during the trial than spat kept in onshore rearing facilities. Quantification of viral DNA before and during the trial showed that infection prevalence and intensity changed over time and revealed latent infection initially unsuspected in 3 of 10 groups. Thermal challenge induced a clear increase in the probability of detecting infected individuals, particularly for groups exhibiting significant prevalence of OsHV-1-contaminated spat prior to the challenge. The use of detection methods are discussed in relation to early rearing practices and disease control strategies., La détection précoce des lots de naissain d'huître creuse porteurs du virus OsHV-1 pourrait permettre d'éviter leur introduction dans des zones ou des installations d'élevage (en mer, ou à terre) jusque là épargnées par les mortalités de naissain. L'étude rapportée ici s'intéressait à l'effet des pratiques d'élevage "précoces" sur : 1) la probabilité de détection de l'OsHV-1 par les méthodes moléculaires de diagnostic et 2) la probabilité de mortalité révélée au moyen d'un protocole expérimental de conditionnement zootechnique du naissain. Les résultats montrent que les lots de naissain gardés isolés dans des structures d'élevage à terre présentent une probabilité de détection de l'OsHV-1 moindre que les lots issus de captage naturels, ou que les lots d'écloserie prégrossis en milieu naturel. L’utilisation conjointe du protocole expérimental de conditionnement, et des méthodes moléculaires destinées à quantifier l'ADN viral, a permis la révélation d'une infection latente, jusque-là insoupçonnée, chez 3 des 10 lots testés ici. Le conditionnement des naissains a ainsi induit une augmentation nette de la prévalence (ou de la fréquence de détection d'individus infectés) à l'échelle du groupe de naissain suivi, ainsi que du niveau moyen de contamination individuelle. La discussion de l'article portera sur ces aspects, et sur l'emploi des méthodes de détection précoce appliquée à l'amélioration des pratiques d'élevage et du contrôle des maladies infectieuses.

Using Ostreid herpesvirus-1 (OsHV-1) as a case study, this paper considers a survey design methodology for an aquatic animal pathogen that incorporates the concept of biologically independent epidemiological units. Hydrodynamically-modelled epidemiological units are used to divide marine areas into sensible sampling units for detection surveys of waterborne diseases. In the aquatic environment it is difficult to manage disease at the animal level, hence management practices are often aimed at a group of animals sharing a similar risk. Using epidemiological units is a way to define these groups, based on a similar level of probability of exposure based on the modelled potential spread of a viral particle via coastal currents, that can help inform management decisions.

Mortality of farmed triploid Pacific oysters (Crassostrea gigas) associated with Ostreid herpesvirus-1 (OsHV-1) was first recorded in Australia in the Georges River/Botany Bay estuary (New South Wales) in late 2010. Two years later, the first sign of possible inter-estuarine spread was observed when commercial triploid Pacific oysters in the Hawkesbury River estuary, located 50 km north of Botany Bay, were affected by mass mortality. The aim of this study was to describe the epidemiological features of the Hawkesbury outbreak via a formal investigation which was conducted in real time and comprised: an assessment of stock levels, past oyster acquisitions and a trace forward investigation to identify stock at greatest risk due to transfers of oysters; passive surveillance of the spread of mortalities in the estuary; active surveillance using PCR to identify the distribution of OsHV-1 infection on farms in the estuary and mortality estimates to identify age and size classes affected; identification of the time of first infection using data from sentinel oysters; and assessment of environmental risk factors. Mortalities were recorded in all age classes but were greater in spat and juveniles than in adults. The incubation period for mass mortality was < 4 days, however subclinical OsHV-1 infection was detected three months prior to the first signs of mortality in the index case (first location affected), which suggests that low viral loads of OsHV-1 are insufficient to induce the disease. While inefficient oyster-to-oyster transmission occurred at two locations, a synchronous infection arising from a common environmental source was required to explain the mass mortalities at the index case (Mullet Creek). Estuarine hydrodynamics then assisted rapid dispersal of viral particles throughout the estuary. Seawater temperatures were consistently above 24°C during the month preceding mass mortalities with variations (± 3°C) being observed over a few days during this period; however this did not necessarily lead to mortality events when the virus was present. There was no evidence of prior movement of potentially infected oysters or farming equipment into the Hawkesbury River estuary to explain the outbreak. Key words: Disease outbreak investigation; Pacific oysters; Ostreid herpesvirus-1; disease transmission; incubation period; water temperature; epidemiology Funded by the Fisheries Research Development and Corporation (FRDC), The University of Sydney and the Sydney Metropolitan Catchment Management Authority

The ostreid herpesvirus (OsHV-1) has the potential to devastate Crassostrea gigas culture in Australia as it has done in many other countries, highlighting the need for a better understanding of disease expression and transmission. The aim of this study was to assess the spatial distribution of OsHV-1 associated mortalities in one of only two infected areas in Australia, Woolooware Bay (Botany Bay New South Wales). In October 2011, healthy sentinel Pacific oysters were placed in three different locations at three different tidal levels and OsHV-1 associated mortalities were closely monitored over 7 months. The outbreak started in November 2011 and the disease remained active until April 2012. Three major mortality events were detected. Rather than being a propagating epizootic, it appeared that most oysters were infected from the same environmental source. The distribution of OsHV-1 associated mortalities was spatially clustered, highly variable and clearly dependent on the age of oysters and their position in the water column. Non-random distribution of mortalities at macro scale (sites several km apart) and micro scale (within rearing trays), and vertical clustering patterns in the water column are discussed in relation to mechanisms of transmission in water. We hypothesise that OsHV-1 may be carried through water by particles, possibly plankton. Key words: Crassostrea gigas, Ostreid herpesvirus 1, summer mortalities, spatial distribution, plankton, disease transmission Funded by the Fisheries Research and Development Corporation, the University of Sydney and the Sydney Metropolitan Catchment Management Authority

International audience; Since 2008, mass mortalities of 1-yr-old Crassostrea gigas associated with the ostreid herpesvirus OsHV-1 μVar have occurred along all the coasts of France when seawater temperature reaches 16 to 17°C. The present study aimed to characterize the effect of temperature on oyster survival in combination with OsHV-1 DNA quantification by standard real-time PCR and total vibrio population levels in oyster tissues. To examine the effect of seawater temperature on disease transmission and related mortality of oysters, cohabitation experiments were conducted between healthy naïve oysters and oysters previously exposed to field conditions in areas where mortalities were occurring. Oysters initially maintained in controlled conditions (free of mortality and negative for OsHV-1), and then transferred to an area where high mortalities were occurring among farmed stocks, became infected with OsHV-1 and exhibited high loads of vibrios followed by significant mortalities. When previously exposed oysters were maintained indoors at 13.0°C for 40 d and then at 20.6°C, they exhibited no mortality, were negative for OsHV-1 detection, and did not transmit the disease to healthy oysters. Survival of previously exposed oysters maintained indoors at 8 temperatures ranging from 13.4 to 29.0°C varied from 25 to 48% and was negatively correlated with holding temperature. Concomitantly, survival of naïve cohabiting animals (62 to 98%) decreased with increasing seawater temperature until a plateau was reached between 16.2 and 21.9°C, and increased at higher temperatures. Therefore, the optimal temperature range for disease transmission from field-exposed to naïve animals was between 16.2 and 21.9°C. Our results suggest that a long-term period (40 d) at low temperature (13°C) may offer a method of mitigating mortalities in oysters that have been exposed to an infective environment.

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 “specimens” collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.

Although viruses have been associated with mortality outbreaks in different mollusc species, little information is available on their exact affiliation and taxonomic position. Herpes viruses have attracted particular attention because of their economic and ecological impact on both farmed and wild marine molluscs over the past 20 years. Investigations based on molecular approaches have been carried out on the herpes virus infecting Pacific oysters in France. Molecular and ultra-structural data were used to classify the virus under the name ostreid herpesvirus 1 (OsHV-1), as the first known species in the family Malacoherpesviridae. Since 2008, massive mortality outbreaks were reported in several Crassostrea gigas oyster farms in France, Ireland, and the United Kingdom. The outbreaks were attributed to adverse environmental conditions associated with a particular variant of OsHV-1, called OsHV-1 μVar., Bien que des épisodes de mortalité associés à la détection de virus aient été rapportés chez différentes espèces de mollusques, peu d’informations sont aujourd’hui disponibles concernant l’affiliation exacte de ces virus et leur position taxinomique. Parmi les virus infectant les coquillages, les herpèsvirus ont fait l’objet d’une attention particulière du fait de leurs impacts économiques et écologiques sur les animaux en élevage et sauvages. Des recherches par des approches de biologie moléculaire ont été menées en France sur le virus infectant les huîtres creuses, Crassostrea gigas. Sur la base des données moléculaires et ultra-structurales, ce virus a été classé, sous le nom d’ostreid herpesvirus 1 (OsHV-1), comme première espèce virale de la famille Malacoherpesviridae. Depuis 2008, des épisodes de surmortalité ont été rapportés dans différentes régions de production des huîtres creuses C. gigas, en France, en Irlande et au Royaume Uni. Ils ont été attribués à des conditions environnementales défavorables associées à la découverte d’un variant génotypique viral particulier, appelé OsHV-1 μVar., Renault Tristan. Les virus infectant les mollusques marins: un exemple d'actualité, les herpèsvirus. In: Bulletin de l'Académie Vétérinaire de France tome 164 n°4, 2011. pp. 359-364.

Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.

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.

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.

Background Ostreid herpesvirus-1 (OsHV-1) is the major bivalve pathogen associated with severe mortality events in a wide host range. In the early summer of 2012 and 2013, mass mortalities of blood clam (Scapharca broughtonii) broodstocks associated with a newly described variant of OsHV-1 (OsHV-1-SB) were reported. Methods In this study, the complete genome sequence of the newly described variant was determined through the primer walking approach, and compared with those of the other two OsHV-1 variants. Results OsHV-1-SB genome was found to contain 199, 354 bp nucleotides with 38.5 % G/C content, which is highly similar to those of acute viral necrosis virus (AVNV) and OsHV-1 reference type. A total of 123 open reading frames (ORFs) putatively encoding functional proteins were identified; eight of which were duplicated in the major repeat elements of the genome. The genomic organization of OsHV-1-SB could be represented as TRL-UL-IRL-IRS-US-TRS, which is different from that of OsHV-1 reference type and AVNV due to the deletion of a unique region (X, 1.5Kb) between IRL and IRS. The DNA sequence of OsHV-1-SB is 95.2 % and 97.3 % identical to that of OsHV-1 reference type and AVNV respectively. On the basis of nucleotide sequences of 32 ORFs in OsHV-1-SB and the other nine OsHV-1 variants, results from phylogenetic analysis also demonstrated that OsHV-1-SB is most closely related to AVNV. Conclusions The determination of the genome of OsHV-1 with distinguished epidemiological features will aid in our better understanding of OsHV-1 diversity, and facilitate further research on the origin, evolution, and epidemiology of the virus. Electronic supplementary material The online version of this article (doi:10.1186/s12985-015-0334-0) contains supplementary material, which is available to authorized users.