OsHV-1 μVar Research Articles

Assessing selection potential for Pacific oyster (Crassostrea gigas) to a North American OsHV-1 μvar: Comparing two experimental assay methods

Ostreid Herpesvirus - 1 is a global threat to Pacific oyster aquaculture as catastrophic farm losses have occurred where OsHV-1 microvariants (μvars) become endemic. Genetic selection has proven effective for increasing survival to OsHV-1 exposure in multiple Pacific oyster breeding programs, but a novel μvar detected in San Diego Bay California has the potential to severely impact commercial aquaculture along the North American Pacific coast as no OsHV-1 μvar selected population is available commercially. Using a Molluscan Broodstock Program population descending from imports originating in the Miyagi prefecture (Japan), we evaluated the potential for genetic selection to improve survival against the San Diego Bay OsHV-1 μvar using a laboratory trial. Two experimental methods were evaluated, a common garden method where all 15 full-sibling families were pooled, and a petri dish method, where each family was confounded within replicate. No striking differences were found between experimental methods, and both experiments displayed high heritability (0.69 and 0.64) of the tested trait, a broad range of estimated breeding values, and a moderate prediction accuracy for the survival trait. A significant genotype-by-environment interaction occurred, highlighting variability in laboratory-based disease challenges, but the two experiments yielded similar results with minor to moderate re-ranking of mean family survival. Overall, the Miyagi population of Pacific oysters curated by the Molluscan Broodstock Program is a promising population to use for artificial selection to increase survival against San Diego Bay OsHV-1 μvar in attempts to obviate farm losses if the virus expands its currently isolated range.

Cross-talk and mutual shaping between the immune system and the microbiota during an oyster's life.

The Pacific oyster Crassostrea gigas lives in microbe-rich marine coastal systems subjected to rapid environmental changes. It harbours a diversified and fluctuating microbiota that cohabits with immune cells expressing a diversified immune gene repertoire. In the early stages of oyster development, just after fertilization, the microbiota plays a key role in educating the immune system. Exposure to a rich microbial environment at the larval stage leads to an increase in immune competence throughout the life of the oyster, conferring a better protection against pathogenic infections at later juvenile/adult stages. This beneficial effect, which is intergenerational, is associated with epigenetic remodelling. At juvenile stages, the educated immune system participates in the control of the homeostasis. In particular, the microbiota is fine-tuned by oyster antimicrobial peptides acting through specific and synergistic effects. However, this balance is fragile, as illustrated by the Pacific Oyster Mortality Syndrome, a disease causing mass mortalities in oysters worldwide. In this disease, the weakening of oyster immune defences by OsHV-1 µVar virus induces a dysbiosis leading to fatal sepsis. This review illustrates the continuous interaction between the highly diversified oyster immune system and its dynamic microbiota throughout its life, and the importance of this cross-talk for oyster health. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

Rapid detection of Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus (AngHV-1) in Japanese eel (Anguilla japonica) using a qLAMP assay

Two DNA viruses, Japanese eel endothelial cell-infecting virus (JEECV) and Anguillid herpesvirus 1 (AngHV-1), cause serious diseases in eels that can negatively affect the economy of eel aquaculture industries in South Korea. Efficient virus detection is crucial for disease management; however, the practical application of current methodologies is challenging, in part owing to extensive time and specialized equipment requirements. The aim of this study was to develop a rapid real-time quantitative loop-mediated isothermal amplification (qLAMP) technique for the detection of JEECV and AngHV-1. Six different primers were designed to target eight different regions of the large T-antigen gene for JEECV detection, and the DNA polymerase catalytic subunit gene for AngHV-1 detection. Both sets of primers worked optimally at 65 °C. The qLAMP assay exhibited a minimum detection limit of 2.39 × 104 copies/μL for JEECV and 2.37 × 104 copies/μL for AngHV-1. Moreover, the primers demonstrated excellent specificity, showing no reaction with two major viruses, namely ostreid herpesvirus-1 microvariant (OsHV-1 μvar) and red sea bream iridovirus (RSIV). In addition, JEECV primers did not react with AngHV-1, and vice versa. Based on the standard curve and coefficient of determination values, both viruses could be quantified using this method. The qLAMP assay was validated with 104 clinical samples, including 72 infected and 32 healthy eels, and the assay findings were compared with previously reported duplex qPCR results that reported high sensitivity and specificity. The qLAMP results concorded well with the previous results. Evaluation of the threshold time (Tt) of the qLAMP results revealed that the samples could be analyzed in less than 15 min. The clinical diagnostic sensitivity (dSe) and diagnostic specificity (dSp) were assessed based on the confusion matrix; dSe and dSp for JEECV were 100% and 94.1%, respectively, and dSe and dSp for AngHV-1 were 96.8% and 77.8%, respectively. The Kappa values, which were used to represent the level of agreement between qLAMP and duplex qPCR results, for JEECV and AngHV-1 were 0.956 and 0.755, respectively, indicating that the qLAMP assay is a promising detection method for those viruses. Thus, the qLAMP assay developed herein could be applied as a rapid and sensitive tool to diagnose diseases caused by JEECV and AngHV-1 in the eel aquaculture industry.

A core of functional complementary bacteria infects oysters in Pacific Oyster Mortality Syndrome

BackgroundThe Pacific oyster Crassostrea gigas is one of the main cultivated invertebrate species worldwide. Since 2008, oyster juveniles have been confronted with a lethal syndrome known as the Pacific Oyster Mortality Syndrome (POMS). POMS is a polymicrobial disease initiated by a primary infection with the herpesvirus OsHV-1 µVar that creates an oyster immunocompromised state and evolves towards a secondary fatal bacteremia.ResultsIn the present article, we describe the implementation of an unprecedented combination of metabarcoding and metatranscriptomic approaches to show that the sequence of events in POMS pathogenesis is conserved across infectious environments. We also identified a core bacterial consortium which, together with OsHV-1 µVar, forms the POMS pathobiota. This bacterial consortium is characterized by high transcriptional activities and complementary metabolic functions to exploit host’s resources. A significant metabolic specificity was highlighted at the bacterial genus level, suggesting low competition for nutrients between members of the core bacteria.ConclusionsLack of metabolic competition between the core bacteria might favor complementary colonization of host tissues and contribute to the conservation of the POMS pathobiota across distinct infectious environments.

Diversity and molecular epidemiology of Ostreid herpesvirus 1 in farmed Crassostrea gigas in Australia: Geographic clusters and implications for “microvariants” in global mortality events

Since 2010, mass mortality events known as Pacific oyster mortality syndrome (POMS) have occurred in Crassostrea gigas in Australia associated with Ostreid herpesvirus 1. The virus was thought to be an OsHV-1 µVar or “microvariant”, i.e. one of the dominant variants associated with POMS in Europe, but there are few data to characterize the genotype in Australia. Consequently, the genetic identity and diversity of the virus was determined to understand the epidemiology of the disease in Australia. Samples were analysed from diseased C. gigas over five summer seasons between 2011 and 2016 in POMS-affected estuaries: Georges River in New South Wales (NSW), Hawkesbury River (NSW) and Pitt Water in Tasmania. Sequencing was attempted for six genomic regions. Numerous variants were identified among these regions (n = 100 isolates) while twelve variants were identified from concatenated nucleotide sequences (n = 61 isolates). Nucleotide diversity of the seven genotypes of C region among Australian isolates (Pi 0.99 × 10−3) was the lowest globally. All Australian isolates grouped in a cluster distinct from other OsHV-1 isolates worldwide. This is the first report that Australian outbreaks of POMS were associated with OsHV-1 distinct from OsHV-1 reference genotype, µVar and other microvariants from other countries. The findings illustrate that microvariants are not the only variants of OsHV-1 associated with mass mortality events in C. gigas. In addition, there was mutually exclusive spatial clustering of viral genomic and amino acid sequence variants between estuaries, and a possible association between genotype/amino acid sequence and the prevalence and severity of POMS, as this differed between these estuaries. The sequencing findings supported prior epidemiological evidence for environmental reservoirs of OsHV-1 for POMS outbreaks in Australia.

Understanding the Dynamic of POMS Infection and the Role of Microbiota Composition in the Survival of Pacific Oysters, Crassostrea gigas.

For over a decade, Pacific oyster mortality syndrome (POMS), a polymicrobial disease, induced recurring episodes of massive mortality affecting Crassostrea gigas oysters worldwide. Recent studies evidenced a combined infection of the ostreid herpesvirus (OsHV-1 μVar) and opportunistic bacteria in affected oysters. However, the role of the oyster microbiota in POMS is not fully understood. While some bacteria can protect hosts from infection, even minor changes to the microbial communities may also facilitate infection and worsen disease severity. Using a laboratory-based experimental infection model, we challenged juveniles from 10 biparental oyster families with previously established contrasted genetically based ability to survive POMS in the field. Combining molecular analyses and 16S rRNA gene sequencing with histopathological observations, we described the temporal kinetics of POMS and characterized the changes in microbiota during infection. By associating the microbiota composition with oyster mortality rate, viral load, and viral gene expression, we were able to identify both potentially harmful and beneficial bacterial amplicon sequence variants (ASVs). We also observed a delay in viral infection resulting in a later onset of mortality in oysters compared to previous observations and a lack of evidence of fatal dysbiosis in infected oysters. Overall, these results provide new insights into how the oyster microbiome may influence POMS disease outcomes and open new perspectives on the use of microbiome composition as a complementary screening tool to determine shellfish health and potentially predict oyster vulnerability to POMS. IMPORTANCE For more than a decade, Pacific oyster mortality syndrome (POMS) has severely impacted the Crassostrea gigas aquaculture industry, at times killing up to 100% of young farmed Pacific oysters, a key commercial species that is cultivated globally. These disease outbreaks have caused major financial losses for the oyster aquaculture industry. Selective breeding has improved disease resistance in oysters, but some levels of mortality persist, and additional knowledge of the disease progression and pathogenicity is needed to develop complementary mitigation strategies. In this holistic study, we identified some potentially harmful and beneficial bacteria that can influence the outcome of the disease. These results will contribute to advance disease management and aquaculture practices by improving our understanding of the mechanisms behind genetic resistance to POMS and assisting in predicting oyster vulnerability to POMS.

Immunomodulatory and Antiviral Effects of Macroalgae Sulphated Polysaccharides: Case Studies Extend Knowledge on Their Importance in Enhancing Shellfish Health, and the Control of a Global Viral Pathogen Ostreid Herpesvirus-1 microVar

Macroalgae are the primary source of non-animal sulphated polysaccharides (SPs) in the marine environment with fucoidans derived from brown algae (Phaeophyta) and carrageenans from red algae (Rhodophyta). Much research has been carried out on SP effects on Asian shrimp species (genera Penaeus and Metapenaeus) but their effect on commercially important bivalve mollusc species is limited and in Pacific oyster Crassostrea gigas is unknown. Knowledge of their impact on bivalve pathogens and Palaemon shrimp is unknown. The objectives of this study were to assess the effects of Fucus vesiculosus (Phaeophyta), Mastocarpus stellatus (Rhodophyta) and algal derivatives (fucoidan and κ-carrageenan) on C. gigas performance, and on ostreid herpesvirus-1 microvar (OsHV-1 μVar) and bacteria Vibrio spp. development. Both pathogens have been associated with significant oyster mortalities and economic losses globally. The effects of sulphated galactan from Gracilaria fisheri (Rhodophyta) on European common prawn Palaemon serratus, an important fishery species, was also assessed. Findings indicate a rapid and prolonged increase in total blood cell count, lysozyme (enzyme that destroys pathogens), and a difference in the ratio of blood cell types in treated individuals compared to their control counterparts. A significantly lower OsHV-1 μVar prevalence was observed in treated oysters and κ-carrageenan was found to suppress viral replication (loads), while OsHV-1 μVar was not detected in the fucoidan treated oysters from Day 8 of the 26-day trial. No antibacterial effect was observed however, the oysters did not succumb to vibriosis. These findings contribute further knowledge to macroalgae sulphated polysaccharide biotherapeutic properties, their twofold effect on animal health and viral suppression.

Changes in benthic macrofauna in oyster parks during an OsHV-1 μVar oyster spat mortality outbreak

In intertidal areas, oyster farming creates a crosshatching pattern between oyster tables and aisles. Tables provide a refuge from the current and solar irradiance and the oysters facilitate the accumulation of OM, thereby structuring the spatial organization of the associated macrozoobenthic community at mesoscale. The aim of this study was to describe the quality of the oyster table environment at small scale and the response of the macrozoobenthic community to OsHV-1 μvar oyster mortality. The species assemblage was dominated by Golfingia vulgaris, Tubificoides benedii, Capitella capitata and Scoloplos armiger. The table habitat appeared to be in a bad ecological state throughout the 2-month survey (May and June 2017), whereas in the aisle, eutrophication occurred lately and was clearly related to be due to the massive stranding of dead seaweed at the end of the survey (in early July). So, this disturbance of the species assemblage seemed to occur in two phases: 1) after oyster spat mortality and 2) after seaweed stranding resulted in a bad ecological status, as revealed by macrofaunal indicators. Large quantities of OsHV-1 DNA were also found in some species, including small crabs and amphipods, one week after the mortality crisis, but there is no apparent virus reservoir found in the benthic species.

The Pacific Oyster Mortality Syndrome, a Polymicrobial and Multifactorial Disease: State of Knowledge and Future Directions.

The Pacific oyster (Crassostreae gigas) has been introduced from Asia to numerous countries around the world during the 20th century. C. gigas is the main oyster species farmed worldwide and represents more than 98% of oyster production. The severity of disease outbreaks that affect C. gigas, which primarily impact juvenile oysters, has increased dramatically since 2008. The most prevalent disease, Pacific oyster mortality syndrome (POMS), has become panzootic and represents a threat to the oyster industry. Recently, major steps towards understanding POMS have been achieved through integrative molecular approaches. These studies demonstrated that infection by Ostreid herpesvirus type 1 µVar (OsHV-1 µvar) is the first critical step in the infectious process and leads to an immunocompromised state by altering hemocyte physiology. This is followed by dysbiosis of the microbiota, which leads to a secondary colonization by opportunistic bacterial pathogens, which in turn results in oyster death. Host and environmental factors (e.g. oyster genetics and age, temperature, food availability, and microbiota) have been shown to influence POMS permissiveness. However, we still do not understand the mechanisms by which these different factors control disease expression. The present review discusses current knowledge of this polymicrobial and multifactorial disease process and explores the research avenues that must be investigated to fully elucidate the complexity of POMS. These discoveries will help in decision-making and will facilitate the development of tools and applied innovations for the sustainable and integrated management of oyster aquaculture.

First detection of OsHV-1 in the cephalopod Octopus vulgaris. Is the octopus a dead-end for OsHV-1?

The ostreid herpes virus (OsHV-1), associated with massive mortalities in the bivalve Crassostrea gigas, was detected for the first time in the cephalopod Octopus vulgaris. Wild adult animals from a natural breeding area in Spain showed an overall prevalence of detection of 87.5% between 2010 and 2015 suggesting an environmental source of viral material uptake. Overall positive PCR detections were significantly higher in adult animals (p = 0.031) compared to newly hatched paralarvae (62%). Prevalence in embryos reached 65%. Sequencing of positive amplicons revealed a match with the variant OsHV-1 µVar showing the genomic features that distinguish this variant in the ORF4. Gill tissues from adult animals were also processed for in situ hybridization and revealed positive labelling. Experimental exposure trials in octopus paralarvae were carried out by cohabitation with virus injected oysters and by immersion in viral suspension observing a significant decrease in paralarval survival in both experiments. An increase in the number of OsHV-1 positive animals was detected in dead paralarvae after cohabitation with virus injected oysters. No signs of viral replication were observed based on lack of viral gene expression or visualization of viral structures by transmission electron microscopy. The octopus response against OsHV-1 was evaluated by gene expression of previously reported transcripts involved in immune response in C. gigas suggesting that immune defences in octopus are also activated after exposure to OsHV-1.

The role of invasive tunicates as reservoirs of molluscan pathogens

Ascidian tunicates frequently display rapid expansion when introduced beyond their native range and are considered successful invaders. This invasive potential may be exacerbated by a warming climate, allowing for the occupation of environmental niches previously held by native species. Research into tunicate invasion ecology is prevalent, but less is known about their role in pathogen maintenance. This study investigated the impact of invasive tunicates on the maintenance of pathogens that affect commercial bivalves, including the cultured species Ostrea edulis (European flat oyster) and Crassostrea gigas (Pacific oyster), and the fished species Cerastoderma edule (Common cockle). Focal pathogens included ostreid herpesvirus OsHV-1 μVar, Vibrio aestuarianus, Bonamia ostreae and Minchinia spp. The range of pathogens in their molluscan hosts was determined and the tunicates Botrylloides violaceus, Didemnum vexillum and Styela clava were then screened for these same pathogens, using both field samples from oyster culture sites and marinas and a series of laboratory cohabitation trials. Sample sites reflected areas close to and further away from known pathogen sources. PCR, Sanger sequencing and histology confirmed the presence of B. ostreae and Minchinia mercenariae-like in S. clava, and V. aestuarianus was confirmed by qPCR in B. violaceus and D. vexillum. Furthermore, histology confirmed Minchinia mercenariae-like sporonts in S. clava suggesting that the tunicate can facilitate replication of this species. S. clava also maintained B. ostreae in tanks with no oysters present. The results indicate that tunicates can act as reservoirs of infection in areas where disease occurs and potentially transport diseases to uninfected sites.

CgMyD88s Serves as an Innate Immune System Plug During Ostreid Herpesvirus 1 Infection in the Pacific Oyster (Crassostrea gigas).

Ostreid herpesvirus-1 microvariant (OsHV-1 μVar) is considered a major infectious microbe that can reduce the survival of natural or cultured oysters in summer. Because they lack an adaptive immune system, oysters are dependent on their innate immune systems to fight pathogens. The duplication and functional divergence of innate immune genes in the oyster have been studied, but the contribution of molecular mechanisms underlying innate immunity remains to be defined. Here, we identified the interacting proteins associated with Crassostrea gigas Toll-like receptors (CgTLR) using a yeast two-hybrid (Y2H) screening system. A total of eight proteins were identified that could interact with CgTLR. Three of these appeared at least four times in the screening and were related to MyD88. Two genes encoding these MyD88-like proteins, CgMyD88-1 and CgMyD88-2, possessed typical death and TIR domains. The third gene encoding an MyD88-like protein possessed only a TIR domain, and we named it CgMyD88s. CgMyD88s interacted only with CgTLR, but not CgMyD88-1 or CgMyD88-2. Both CgMyD88-1 and CgMyD88-2 mRNAs were upregulated after OsHV-1 μVar infection, whereas the expression of CgMyD88s decreased. When overexpressed in HEK293T cells, CgMyD88-1 and CgMyD88-2 activated an NF-κB reporter, whereas CgMyD88s impaired activation induced by CgMyD88-1 or CgMyD88-2. Intriguingly, the silencing of CgMyD88s using double-stranded RNA (dsRNA)-mediated RNA interference increased the expression of CgMyD88-1 and CgMyD88-2. Taken together, our results revealed that CgMyD88-1, CgMyD88-2, and CgMyD88s may all participate in the TLR-mediated innate immune pathway and that CgMyD88s served as a plug to avoid oysters from excessive inflammatory response during OsHV-1 μVar infections.

Contribution of Viral Genomic Diversity to Oyster Susceptibility in the Pacific Oyster Mortality Syndrome.

Juvenile Pacific oysters (Crassostrea gigas) are subjected to recurrent episodes of mass mortalities that constitute a threat for the oyster industry. This mortality syndrome named “Pacific Oyster Mortality Syndrome” (POMS) is a polymicrobial disease whose pathogenesis is initiated by a primary infection by a variant of an Ostreid herpes virus named OsHV-1 μVar. The characterization of the OsHV-1 genome during different disease outbreaks occurring in different geographic areas has revealed the existence of a genomic diversity for OsHV-1 μVar. However, the biological significance of this diversity is still poorly understood. To go further in understanding the consequences of OsHV-1 diversity on POMS, we challenged five biparental families of oysters to two different infectious environments on the French coasts (Atlantic and Mediterranean). We observed that the susceptibility to POMS can be different among families within the same environment but also for the same family between the two environments. Viral diversity analysis revealed that Atlantic and Mediterranean POMS are caused by two distinct viral populations. Moreover, we observed that different oyster families are infected by distinct viral populations within a same infectious environment. Altogether these results suggest that the co-evolutionary processes at play between OsHV-1 μVar and oyster populations have selected a viral diversity that could facilitate the infection process and the transmission in oyster populations. These new data must be taken into account in the development of novel selective breeding programs better adapted to the oyster culture environment.

Effects of Environmental Factors and Husbandry Practices on Summer Mortality Events in the Cultivated Pacific Oyster Crassostrea gigas in the North of Ireland

The Pacific oyster Crassostrea gigas (Thunberg, 1793) is an important part of the aquaculture industry in Europe; however, since 2008, oyster mortality events (>20%) have become a regular summer occurrence. This study monitored and recorded C. gigas mortality events during a 3-y period at a commercial oyster site in Lough Foyle, North of Ireland, and related these to environmental conditions, oyster physiology, and husbandry practices. Early summer temperature rises above 16°C were consistently correlated with large mortality events, but warmer temperatures in late summer did not cause mortalities, including in 1 y when there were no early summer mortalities. Mortalities were also correlated with a sharp early summer decrease in salinity from 22.2 to 16.9. In the laboratory, the combined effects of various temperatures and salinities on oyster stress levels were investigated using total hemocyte count and neutral red retention techniques. The cell membrane integrity and hemocyte density in C. gigas are sensitive to temperatures of 20°C or greater and at salinities of 20 or lower. This suggests that high temperature and low salinity combinations can negatively impact oyster stress levels and potentially increase oyster vulnerability to infections, including oyster herpes OsHV-1 µvar. Comparing survivorship in experimental bags simulating commercial growing conditions, these results equivocally support previous studies in which higher shore position was correlated with higher survivorship. Bags of oysters at lower density in lower shore positions were equally successful and may be more protected from climate-related stressors. Focusing on a single stressor is insufficient to predict future oyster mortalities, but balancing effects and relieving stress on oyster stocks through husbandry could limit the impacts of summer mortalities.

Microbiota Composition and Evenness Predict Survival Rate of Oysters Confronted to Pacific Oyster Mortality Syndrome.

Pacific Oyster Mortality Syndrome (POMS) affects Crassostrea gigas oysters worldwide and causes important economic losses. Disease dynamic was recently deciphered and revealed a multiple and progressive infection caused by the Ostreid herpesvirus OsHV-1 μVar, triggering an immunosuppression followed by microbiota destabilization and bacteraemia by opportunistic bacterial pathogens. However, it remains unknown if microbiota might participate to protect oysters against POMS, and if microbiota characteristics might be predictive of oyster mortalities. To tackle this issue, we transferred full-sib progenies of resistant and susceptible oyster families from hatchery to the field during a period in favor of POMS. After 5 days of transplantation, oysters from each family were either sampled for individual microbiota analyses using 16S rRNA gene-metabarcoding or transferred into facilities to record their survival using controlled condition. As expected, all oysters from susceptible families died, and all oysters from the resistant family survived. Quantification of OsHV-1 and bacteria showed that 5 days of transplantation were long enough to contaminate oysters by POMS, but not for entering the pathogenesis process. Thus, it was possible to compare microbiota characteristics between resistant and susceptible oysters families at the early steps of infection. Strikingly, we found that microbiota evenness and abundances of Cyanobacteria (Subsection III, family I), Mycoplasmataceae, Rhodobacteraceae, and Rhodospirillaceae were significantly different between resistant and susceptible oyster families. We concluded that these microbiota characteristics might predict oyster mortalities.

IKKε-like plays an important role in the innate immune signaling of the Pacific oyster (Crassostrea gigas)

IκB-related kinase ε (IKKε) plays a crucial role in the activation of nuclear factor κB (NF-κB) by phosphorylating inhibitor of NF-κB (IκB) and in the regulation of interferon (IFN) gene expression by phosphorylating IFN regulatory factors (IRFs). In this study, we cloned an IKKε homologue cDNA (designated as CgIKKε-like) from the Pacific oyster, Crassostrea gigas. The full 2896-bp cDNA sequence comprised a 2163-bp open reading frame (ORF) encoding 720 amino acids. CgIKKε-like is ubiquitously expressed, and its mRNA levels in hemocytes after poly I:C, V. alginolyticus, or OsHV-1 μVar challenge were analyzed by real-time PCR. Compared to that in the control, CgIKKε-like mRNA expression levels were significantly increased at 3 h and peaked at 6 h after OsHV-1 μVar challenge; no obvious changes were observed in expression levels until 24 h after either V. alginolyticus or poly I:C challenge, reaching a maximum at 24 h (p < 0.01) and then rapidly decreasing. CgIKKε-like transfection into human cell lines induced NF-κB and ISRE activation, while transfection with CgIKKε-like deletion mutants abolished NF-κB and ISRE reporter gene activation. Additionally, CgIKKε-like could interact with CgTBK1 and could form homodimers strongly, which may be critical for the immune signaling transduction. Last but not least, we found that CgIKKε-like may increase CgIκBs phosphorylation and could interact with CgIRF8. Together, these results suggest that CgIKKε-like could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB and ISRE reporter genes. Thus, CgIKKε-like could play an important role in the oyster immune system.

Mass mortality in Korean bay scallop (Argopecten irradians) associated with Ostreid Herpesvirus-1 μVar.

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 5-10days 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.

Variability in the Composition of Pacific Oyster Microbiomes Across Oyster Families Exhibiting Different Levels of Susceptibility to OsHV-1 μvar Disease.

Oyster diseases are a major impediment to the profitability and growth of the oyster aquaculture industry. In recent years, geographically widespread outbreaks of disease caused by ostreid herpesvirus-1 microvariant (OsHV-1 μvar) have led to mass mortalities among Crassostrea gigas, the Pacific Oyster. Attempts to minimize the impact of this disease have been largely focused on breeding programs, and although these have shown some success in producing oyster families with reduced mortality, the mechanism(s) behind this protection is poorly understood. One possible factor is modification of the C. gigas microbiome. To explore how breeding for resistance to OsHV-1 μvar affects the oyster microbiome, we used 16S rRNA amplicon sequencing to characterize the bacterial communities associated with 35 C. gigas families, incorporating oysters with different levels of susceptibility to OsHV-1 μvar disease. The microbiomes of disease-susceptible families were significantly different to the microbiomes of disease-resistant families. OTUs assigned to the Photobacterium, Vibrio, Aliivibrio, Streptococcus, and Roseovarius genera were associated with low disease resistance. In partial support of this finding, qPCR identified a statistically significant increase of Vibrio-specific 16S rRNA gene copies in the low disease resistance families, possibly indicative of a reduced host immune response to these pathogens. In addition to these results, examination of the core microbiome revealed that each family possessed a small core community, with OTUs assigned to the Winogradskyella genus and the Bradyrhizobiaceae family consistent members across most disease-resistant families. This study examines patterns in the microbiome of oyster families exhibiting differing levels of OsHV-1 μvar disease resistance and reveals some key bacterial taxa that may provide a protective or detrimental role in OsHV-1 μvar disease outbreaks.

Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters

Infectious diseases are mostly explored using reductionist approaches despite repeated evidence showing them to be strongly influenced by numerous interacting host and environmental factors. Many diseases with a complex aetiology therefore remain misunderstood. By developing a holistic approach to tackle the complexity of interactions, we decipher the complex intra-host interactions underlying Pacific oyster mortality syndrome affecting juveniles of Crassostrea gigas, the main oyster species exploited worldwide. Using experimental infections reproducing the natural route of infection and combining thorough molecular analyses of oyster families with contrasted susceptibilities, we demonstrate that the disease is caused by multiple infection with an initial and necessary step of infection of oyster haemocytes by the Ostreid herpesvirus OsHV-1 µVar. Viral replication leads to the host entering an immune-compromised state, evolving towards subsequent bacteraemia by opportunistic bacteria. We propose the application of our integrative approach to decipher other multifactorial diseases that affect non-model species worldwide.

Role of the intertidal predatory shore crab Carcinus maenas in transmission dynamics of ostreid herpesvirus-1 microvariant.

Ostreid herpesvirus-1 microVar (OsHV-1 µVar) has been responsible for significant mortalities globally in the Pacific oyster Crassostrea gigas. While the impact of this virus on the Pacific oyster has been significant, this pathogen may have wider ecosystem consequences. It has not been definitively determined how the virus is sustaining itself in the marine environment and whether other species are susceptible. The shore crab Carcinus maenas is a mobile predator and scavenger of C. gigas, commonly found at Pacific oyster culture sites. The aim of this study was to investigate the role of the crab in viral maintenance and transmission to the Pacific oyster. A field trial took place over 1 summer at different shore heights at 2 Irish Pacific oyster culture sites that are endemic for OsHV-1 µVar. Infection of OsHV-1 µVar in tissues of C. maenas at both shore heights of both sites was detected by polymerase chain reaction (PCR), quantitative PCR (qPCR), in situ hybridization and direct Sanger sequencing. In addition, a laboratory trial demonstrated that transmission of the virus could occur to naïve C. gigas within 4 d, from C. maenas previously exposed to the virus in the wild. These findings provide some insight into the possibility that the virus can be transmitted through marine food webs. The results also suggest viral plasticity in the hosts required by the virus and potential impacts on a range of crustacean species with wider ecosystem impacts if transmission to other species occurs.