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Ostreid herpesvirus 1 (OsHV-1) has caused a serious economic loss to the global bivalve aquaculture industry. Given its wide host range and the frequent emergence of mutated strains, OsHV-1 infection threatens mollusk production. Unlike the common vertebrate herpesviruses that generally exhibit high host specificity, more than ten bivalve species have been identified as potentially susceptible to OsHV-1 infection, including members of Ostreoida, Pterioida, Arcoida, and Veneroida. A variety of OsHV-1 detection methods have been developed, such as PCR, in situ hybridization, in situ PCR, histopathology, transmission electron microscopy, ring-mediated isothermal nucleic acid amplification (LAMP), and recombinase polymerase amplification (RPA). The sensitivity and specificity of transmission electron microscopy detection are low, and specific complementary detection methods such as PCR are needed to confirm the occurrence of OsHV-1. In situ hybridization, although highly specific, has the disadvantages of low sensitivity, complicated procedures, and high investment of effort. PCR methods are still the most widely used for epidemiological investigation of OsHV-1. However, PCR cannot alone confirm infection, which has led to the emergence of unconfirmed cases of OsHV-1 infection in many species and regions. According to the World Organization for Animal Health´s (OIE) Aquatic Animal Manual, positive nucleic acid-specific detection combined with histopathology and transmission electron microscopy is required for the confirmed diagnosis of OsHV-1 infection. Other detection methods, such as LAMP and RPA, which use nucleic acid amplification alone to detect OsHV-1, can not confirm infection as the presence of nucleic acids is not equivalent to viral infection. As a result, the susceptibility of several bivalve species to OsHV-1 infection has not been evaluated. Instead, several obstacles remain with regards to the development of epidemiological surveillance programs and the implementation of quarantine, prevention, and control measures for OsHV-1 infection. Compared with traditional in situ hybridization and in situ PCR assays, in situ LAMP has the advantages of low nucleic acid amplification reaction temperature, less damage to tissue, constant temperature amplification that does not require special experimental equipment, and more convenient and efficient experimental design. In this study, we selected a set of LAMP primers designed for specific detection of OsHV-1, and a pair of loop primers were designed to improve the specificity and stability of the LAMP reaction on slides. An optimized in situ LAMP method for OsHV-1 detection was developed, which provides a rapid diagnostic method with high specificity and sensitivity to identify hosts that are potentially susceptible to OsHV-1 infection, and to characterize the distribution pattern and tissue affinity of the virus in new hosts. The optimized protocol and quantitative PCR (qPCR) were then used to detect OsHV-1 infection in bivalve samples (Crassostrea gigas, C. angulata, Chlamys farreri, Mizuhopecten yessoensis, Ruditapes philippinarum, and Scapharca subcrenata) collected from 2019 to 2021. OsHV-1 hybridization signals were observed in S. subcrenata samples alone, although positive qPCR results were obtained in more species. Further investigation of pathological characteristics and associated viral hybridization signals indicated that OsHV-1 infection always occurred in fibroblasts and hemocytes in the connective tissues of the mantle and hepatopancreas, nucleus of muscle cells in the foot, and adductor muscle. Signals were also occasionally observed in infiltrated hemocytes between and within the gill filaments. OsHV-1 hybridization signals were observed within the hemocytes infiltrating several organs of the furcula visceral cluster. The hemocytes of S. subcrenata seem to be particularly susceptible to OsHV-1 infection. Histopathological lesions and viral hybridization signals in the hepatopancreas organ were consistently observed in individuals with clinical signs, and we recommend the hepatopancreas as the preferred target organ for histopathological and in situ LAMP assays for OsHV-1 infection in S. subcrenata. In situ LAMP-based detection indicated that S. subcrenata was a susceptible host for OsHV-1, and the connective tissue, muscle tissue, and hemolymph cells had a strong affinity for the virus. At present, the susceptibility of many cultured and wild shellfish to OsHV-1 infection is still unknown. Since the emergence of mollusk mortalities associated with OsHV-1 infection occurred in China in the 1990s, its host range has expanded and changed along with the environment, which has complicated its prevention and control. In recent years, the scale of artificial breeding and cultivation of triploid C. gigas has expanded in China, and larval mortalities are frequently associated with OsHV-1 infection. The in situ LAMP detection method for OsHV-1 infection developed in this study proved to be convenient and fast and could be a valuable tool for the rapid detection and confirmation of OsHV-1 infection.

Abstract 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.

Abstract Competition between organisms interfere in host and pathogen dynamics in ways that are difficult to predict. By one side, competitors can reduce the food supply and cause nutritional stress. Such stress could further modulate the susceptibility to infection by altering immune response or metabolic rate of the host. Alternatively, competitors may trap pathogens before they reach the focal host, and therefore reduce, enhance, or have no effect on infection according to the competitor's susceptibility to the infection. To better understand how competition influences host and pathogen interactions, we experimentally assessed the relative importance of competition for pathogens and resources on the severity of a viral disease infecting the Pacific oyster Crassostrea gigas. We designed an open‐flow system where food enriched seawater flowed to filter‐feeding competitors (or empty controls) before being delivered to recipient oysters. We tested a range of competing species that exhibit both low (ascidians, European oysters, mussels) and high (Pacific oysters) susceptibility to the virus. We assessed the physiological condition of the recipient oysters during acclimation, we added virus‐contaminated seawater upstream of the distribution system, and we monitored host and pathogen dynamics. We found that the presence of competitors, regardless of susceptibility to the virus, indirectly reduced the infection rate of hosts by decreasing their food ingestion and growth rates. Although competitors can reduce viral particles from the seawater, this had no effect on the host population. Our data suggest that the effect of competition for food overwhelmed that of competition for pathogens, thus emphasizing the importance of considering resource availability in host and pathogen dynamics. More particularly, resource availability can have positive effects at the individual level, fostering physiological condition and growth, but negative effects at the population level, increasing magnitude of epidemics.

Competition between organisms interfere in host and pathogen dynamics in ways that are difficult to predict. By one side, competitors can reduce the food supply and cause nutritional stress. Such stress could further modulate the susceptibility to infection by altering immune response or metabolic rate of the host. Alternatively, competitors may trap pathogens before they reach the focal host, and therefore reduce, enhance, or have no effect on infection according to the competitor's susceptibility to the infection. To better understand how competition influences host and pathogen interactions, we experimentally assessed the relative importance of competition for pathogens and resources on the severity of a viral disease infecting the Pacific oyster Crassostrea gigas. We designed an open‐flow system where food enriched seawater flowed to filter‐feeding competitors (or empty controls) before being delivered to recipient oysters. We tested a range of competing species that exhibit both low (ascidians, European oysters, mussels) and high (Pacific oysters) susceptibility to the virus. We assessed the physiological condition of the recipient oysters during acclimation, we added virus‐contaminated seawater upstream of the distribution system, and we monitored host and pathogen dynamics. We found that the presence of competitors, regardless of susceptibility to the virus, indirectly reduced the infection rate of hosts by decreasing their food ingestion and growth rates. Although competitors can reduce viral particles from the seawater, this had no effect on the host population. Our data suggest that the effect of competition for food overwhelmed that of competition for pathogens, thus emphasizing the importance of considering resource availability in host and pathogen dynamics. More particularly, resource availability can have positive effects at the individual level, fostering physiological condition and growth, but negative effects at the population level, increasing magnitude of epidemics. [ABSTRACT FROM AUTHOR]

The interaction between viral membrane associate proteins and host cellular surface molecules should facilitate the attachment and entry of OsHV-1 into host cells. Thus, blocking the putative membrane proteins ORF25 and ORF72 of OsHV-1 with antibodies that have previously been reported to subdue OsHV-1 replication in host cells, especially ORF25. In this study, prey proteins in host hemocytes were screened by pull-down assay with recombinant baits ORF25 and ORF72, respectively. Gene Ontology (GO) analysis of these prey proteins revealed that most of them were mainly associated with binding, structural molecule activity and transport activity in the molecular function category. The protein–protein interaction (PPI) network of the prey proteins was constructed by STRING and clustered via K-means. For both ORF25 and ORF72, three clusters of these prey proteins were distinguished that were mainly associated with cytoskeleton assembly, energy metabolism and nucleic acid processing. ORF25 tended to function in synergy with actins, while ORF72 functioned mainly with tubulins. The above results suggest that these two putative membrane proteins, ORF25 and ORF72, might serve a role in the transport of viral particles with the aid of a cytoskeleton inside cells.

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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.

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 κB inhibitor (IκB2). This gene is a candidate of particular interest for functional investigations in the context of oyster mass mortality events, as Cg-IκB2 mRNA levels exhibited significant variation depending on the amount of ostreid herpesvirus 1 (OsHV-1) DNA detected. In the present study, dsRNAs targeting Cg-IκB2 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-IκB2- 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-IκB2. Immune-related genes including Cg-IκB1, 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. [ABSTRACT FROM AUTHOR]

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 (10 8 and 10 9 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. [ABSTRACT FROM AUTHOR]

Mass mortalities of juvenile Pacific oysters ( Crassostrea gigas ) in aquaculture operations have been linked to the ostreid herpes virus 1 (OsHV-1) or its variants. This oyster has heritable resilience to the virus which can provide a basis for selective breeding for higher OsHV-1 resistance, however, controlled challenge methods for improving selection response towards increased OsHV-1 resistance are required. The objective of this study was to compare the survival of sibling juvenile oysters exposed to either a laboratory virus challenge or a heat shock challenge (as proxy for the virus challenge) and the survival of the same families on two oyster farms where they were exposed to natural field conditions including the presence of OsHV-1. A strong correlation between the family rankings was observed for the two farm sites. Estimated narrow sense heritabilities (h 2 ) on the underlying liability scale were 0.38 (on-farm survival), 0.45 (laboratory virus challenge survival) and 0.15 (heat shock challenge survival). Only on-farm survival and laboratory virus challenge had a high genetic correlation. The method for measuring heat shock resistance in juvenile oysters produced high variability in survival among families but low estimates of heritability. Overall, the results of this study indicate that the laboratory virus challenge is a simple and relatively effective tool for selective breeding specifically towards OsHV-1 resistance or as part of a controlled multi-trait program. Statement of relevance The OsHV-1 virus is a serious problem for commercial aquaculture of Pacific oysters in numerous countries. This paper tests two ways of evaluating oysters for resistance to OsHV-1 that can be incorporated into selective breeding programs. [ABSTRACT FROM AUTHOR]

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.

Ostreid herpesvirus 1 (OsHV-1) infections have been reported in several bivalve species. Mortality of Pacific oyster Crassostrea gigas spat has increased considerably in Europe since 2008 linked to the spread of a variant of OsHV-1 called μvar. In the present study we demonstrated that O. edulis juveniles can be infected by OsHV-1 μvar when administered as an intramuscular injection. Mortality in the oysters injected with OsHV-1 μvar was first detected 4 days after injection and reached 25% mortality at day 10. Moreover, the high viral load observed and the detection of viral transcripts by in situ hybridization in several tissues of dying oysters suggested that OsHV-1 μvar was the cause of mortality in the O. edulis juveniles. This is therefore the first study to provide evidence about the pathogenicity of OsHV-1 μvar in a species that does not belong to the Crassostrea genus. Additionally, we present a novel method to detect OsHV-1 transcripts in infected individuals’ using in situ hybridization. [ABSTRACT FROM AUTHOR]

Cytogenetic abnormalities associated with viral infections, including from viruses of the Herpesvirales order, have been reported in vertebrate species. Ostreid herpesvirus 1 (Os HV-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 Os HV-1 in gills of C. gigas, the Portuguese oyster C. angulata and their F1 hybrids cultured in Ria Formosa (Portugal). Os HV-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 Os HV-1 reference type. Moreover, in situ hybridization confirmed the presence of Os HV-1 in gill tissue. Oysters where Os HV-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 Os HV-1 infection, but the virus is unlikely to be the cause of somatic aneuploidy. [ABSTRACT FROM AUTHOR]

Between May 2011 and February 2014, 205 samples of Crassostrea gigas, farmed on the northwest coast of Italy, were collected and analyzed for OsHV-1, Vibrio parahaemolyticus, V. cholerae, V. alginolyticus, V. aestuarianus, bacteria belonging to V. splendidus clade, E. coli, Salmonella spp., environmental parameters and algae. Statistical analysis was carried out to correlate all the data of the environmental parameters, algae and Vibrio with OsHV-1 in the presence and absence of die-off. Of the samples tested, 46.8 % (96/205) were positive for the detection of OsHV-1 DNA, 0.97 % (2/205) of the oysters were positive for V. harveyi DNA detection and 8.8 % (18/205) were positive for V. aestuarianus. Statistical analysis showed a positive correlation between the presence of V. aestuarianus, OsHV-1 and a die-off occurred in November 2013. Sequencing analysis of the PCR products revealed that the genotype of OsHV-1 is highly related to the variant µVar. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The microvariant genotype of Ostreid herpesvirus 1 (OsHV-1 μVar) has severely disrupted the production of Pacific oysters Crassostrea gigas in Europe, New Zealand, and Australia since its first detection in France in 2008. The disease occurs in the warmer months, recurs annually, and requires new management strategies. Larvae and spat are the most susceptible life history stages, which poses a threat to hatchery production. This is the first study to examine strategies to enable survival of spat in treated seawater in an OsHV-1 μVar endemic area. In 2013 and 2014, seven controlled experiments were conducted in which potentially infected estuarine water was pumped through an upweller nursery system housing spat. Controls died of OsHV-1 infection in 6 of 7 experiments. Filtration of seawater to 5 μm was protective, whereas filtration to 30 μm or 55 μm was not. UV irradiation was not required for protection after 5 μm filtration. Chilling water by 2–3 °C delayed the onset and reduced the mortality rate. Aging seawater for 48 h prior to use prevented mortality, but the mechanism, either sedimentation of particles or inactivation of OsHV-1, was not determined. Some oysters in treatments in which mortality was prevented contained low quantities of OsHV-1 DNA, suggesting that infections may have occurred. The results support a hypothesis that OsHV-1 is carried on particles rather than being uniformly distributed in water; the removal of the putative particulate vector of OsHV-1 from seawater using aging/sedimentation of water or filtration to 5 μm enabled C. gigas spat to survive despite the presence of OsHV-1 μVar in the water supply. [ABSTRACT FROM AUTHOR]

Viral infection caused by Ostreid herpesvirus 1 (OsHV-1) is one of the proximate causes of mass mortalities of cultivated bivalves around the world. The emergence and spread of different variants of OsHV-1 accompanied by different epidemiological characteristics have been reported frequently in different countries around the world. In this paper, we present a study of the detection of OsHV-1 DNA and their variations from 1599 samples over 18 species collected in 27 aquaculture sites and two food markets during 2001–2013 in China. All of the samples were examined by a nested PCR assay targeting the C2/C6 fragment of OsHV-1 followed by sequencing. Our results showed 338 individuals (21.1%) of seven species sampled from 14 (14/27 = 51.9%) sites and the two food markets were positive for viral DNA. Sequencing of 289 PCR products revealed 24 virus types. No shared virus type was found among different countries with 47 types (23 in Japan, 16 in France, 2 in South Korea and 1 in each country of Australia, USA, Ireland, New Zealand, Mexico and China) identified in previous studies. As previously reported, two main phylogenetic groups were identified by phylogenetic analysis based on the 71 virus types; within which 6 separate clades were identified. Our results also demonstrated that two clades were associated with abnormal mortalities of the scallop, Chlamys farrier and the calm, Scapharca broughtonii in China. These findings indicated that cultivated bivalves may face potential threats from OsHV-1 types found in our study. [ABSTRACT FROM AUTHOR]

Background: Massive 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. Results: The 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. Conclusions: In 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 12month) 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 7months. 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. [Copyright &y& Elsevier]

The article focuses on the mortality of fishes caused by the Oyster herpesvirus 1 (OsHV-1) infections in the larvae and the juveniles of several bivalve species, including Pacific oyster or Crassostrea gigas. The serious disease of a new variant in OsHV-1 called ostreid herpesvirus 1 in the oyster aquaculture industry is mentioned. The need to investigate the dynamics of the ostrid herpesvirus and the need for further molecular characterization to know the original source is cited.

Abstract: Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1α (EF-1α), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in normal growing oyster larvae and in OsHV-1 infected larvae. These high quality internal controls will be a valuable resource in future studies of oyster larval mortality. [Copyright &y& Elsevier]