Search

Maintaining genetic diversity in cultured shellfish can be challenging due to high variance in individual reproductive success, founder effects, and rapid genetic drift, but is important to retain adaptive potential and avoid inbreeding depression. To support broodstock management and selective breeding in cultured Pacific oysters (Crassostrea (Magallana) gigas), we developed an amplicon panel targeting 592 genomic regions and SNP variants with an average of 50 amplicons per chromosome. Target SNPs were selected based on elevated observed heterozygosity or differentiation in Pacific oyster populations in British Columbia, Canada. The use of the panel for parentage applications was evaluated using multiple generations of oysters from a breeding program on Vancouver Island, Canada (n = 181) and families selected for Ostreid herpesvirus-1 resistance from the Molluscan Broodstock Program in Oregon, USA (n = 136). Population characterization was evaluated using wild, naturalized, farmed, or hatchery oysters sampled throughout the Northern Hemisphere (n = 189). Technical replicates showed high genotype concordance (97.5%; n = 68 replicates). Parentage analysis found suspected pedigree and sample handling errors, demonstrating the panel's value for quality control in breeding programs. Suspected null alleles were identified and found to be largely population dependent, suggesting population-specific variation impacting target amplification. Null alleles were identified using existing data without the need for pedigree information, and once they were removed, assignment rates increased to 93.0 and 86.0% of possible assignments in the two breeding program datasets. A pipeline for analyzing the amplicon sequence data from sequencer output, amplitools, is also provided., Competing Interests: Conflicts of interest B.J.G.S. is affiliated with Sutherland Bioinformatics. The author has no competing financial interests to declare. Some authors affiliated with ThermoFisher Scientific have potential conflicts considering that the AgriSeq Targeted Genotyping by Sequencing solutions and associated Oligo panels that were designed and validated in the study are offered by ThermoFisher Scientific. However, the selection of markers, and data analysis was primarily conducted by other authors. The authors declare that the research was conducted in a scientific manner without any commercial considerations that could be construed as potential conflict of interest and further declare no other conflicts of interest. The other authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Pacific oysters ( Crassostrea or Magallana gigas ) are one of the most economically important aquaculture species globally. Over the past two decades, ostreid herpesvirus (OsHV-1) has become a major pathogen of cultured Pacific oysters, resulting in widespread mortality with a global distribution. Experimental use of OsHV-1 is challenging for many reasons, including both complexity of host-pathogen dynamics and a lack of functioning model systems. The goal of this study was to improve the tools available for working with OsHV-1 in both whole animals and in tissue explants established from oysters maintained in controlled laboratory conditions. Tissue explants were taken from oysters originating from two different sources that have different levels of mortality in experimental OsHV-1 infections and were exposed to OsHV-1. A whole-animal infection experiment was run concurrently as a comparison. Quantitative PCR and electron microscopy were used to confirm that the explants were capable of replicating OsHV-1. Furthermore, the quantitative PCR results suggest that the source of the oysters was significant in determining the outcome of infection in the explants, supporting the validity of the explant model for OsHV-1 infection. This tissue explant approach for studying OsHV-1 allows for the control of confounding factors in the disease outcome that is not possible in whole-animal experiments, providing a new tool for the study of OsHV-1 in Pacific oysters.

To keep pace with rising opportunities for disease emergence and spread, surveillance in aquaculture must enable the early detection of both known and new pathogens. Conventional surveillance systems (designed to provide proof of disease freedom) may not support detection outside of periodic sampling windows, leaving substantial blind spots to pathogens that emerge in other times and places. To address this problem, we organized an expert panel to envision optimal systems for early disease detection, focusing on Ostreid herpesvirus 1 (OsHV-1), a pathogen of panzootic consequence to oyster industries. The panel followed an integrative group process to identify and weight surveillance system traits perceived as critical to the early detection of OsHV-1. Results offer a road map with fourteen factors to consider when building surveillance systems geared to early detection; factor weights can be used by planners and analysts to compare the relative value of different designs or enhancements. The results were also used to build a simple, but replicable, model estimating the system sensitivity (SSe) of observational surveillance and, in turn, the confidence in disease freedom that negative reporting can provide. Findings suggest that optimally designed observational systems can contribute substantially to both early detection and disease freedom confidence. In contrast, active surveillance as a singular system is likely insufficient for early detection. The strongest systems combined active with observational surveillance and engaged joint industry and government involvement: results suggest that effective partnerships can generate highly sensitive systems, whereas ineffective partnerships may seriously erode early detection capability. Given the costs of routine testing, and the value (via averted losses) of early detection, we conclude that observational surveillance is an important and potentially very effective tool for health management and disease prevention on oyster farms, but one that demands careful planning and participation. This evaluation centered on OsHV-1 detection in farmed oyster populations. However, many of the features likely generalize to other pathogens and settings, with the important caveat that the pathogens need to manifest via morbidity or mortality events in the species, life stages and environments under observation., (Published by Elsevier B.V.)

The spread, emergence, and adaptation of pathogens causing marine disease has been problematic to fisheries and aquaculture industries for the last several decades creating the need for strategic management and biosecurity practices. The Pacific oyster (Crassostrea gigas), a highly productive species globally, has been a target of disease and mortality caused by a viral pathogen, the Ostreid herpesvirus 1 (OsHV-1) and its microvariants (OsHV-1 µvars). During routine surveillance to establish health history at a shellfish aquaculture nursery system in San Diego, California, the presence of OsHV-1 in Pacific oyster juveniles was detected. Quantification of OsHV-1 in tissues of oysters revealed OsHV-1 viral loads > 10 6 copies/mg. We characterized and identified the OsHV-1 variant by sequencing of ORFs 4 (C2/C6) and 43 (IA1/IA2), which demonstrated that this variant is a novel OsHV-1 microvariant: OsHV-1 µvar SD. A pilot transmission study indicates that OsHV-1 µvar SD is infectious with high viral loads ~ 7.57 × 10 6 copies/mg detected in dead individuals. The detection of OsHV-1 µvar SD in a large port mirrors previous studies conducted in Australia where aquaculture farms and feral populations near port locations may be at a higher risk of OsHV-1 emergence. Further research is needed to understand the impacts of OsHV-1 µvar SD, such as transmission studies focusing on potential vectors and characterization of virulence as compared to other OsHV-1 µvars. To increase biosecurity of the global aquaculture industry, active and passive surveillance may be necessary to reduce spread of pathogens and make appropriate management decisions., (Copyright © 2021. Published by Elsevier Inc.)

French commercial hatcheries are massively producing Crassostrea gigas selected for their higher resistance to OsHV-1, and soon should also implement selection for increasing resistance to Vibrio aestuarianus. The first objective of this study was to optimize the breeding programs for dual resistance to OsHV-1 and V. aestuarianus to determine the earliest life stage for which oysters are able to develop disease resistance. Wild stocks and selected families were tested using experimental infections by both pathogens at the larval, spat and juvenile stages. Oyster families could be evaluated for OsHV-1 as soon as the larval stage by a bath method, but this only highlighted the most resistant families; those that showed the highest resistance to V. aestuarianus could be determined using the cohabitation method at the juvenile stage. The second objective of this study was to determine if selection to increase/decrease the resistance to OsHV-1 and V. aestuarianus could have an impact on other major pathogens currently detected in hatchery at the larval stage, and in nursery and field at the spat/juveniles stages (V. coralliilyticus, V. crassostreae, V. tasmaniensis, V. neptunius, V. europaeus, V. harveyi, V. chagasi). No relationship was found between mortality caused by V. aestuarianus/OsHV-1 and the mortality caused by the other virulent bacterial strains tested regardless the stages, except between OsHV-1 and V. tasmaniensis at the juvenile stage. Finally, miscellaneous findings were evidenced such as (1) bath for bacterial challenges was not adapted for spat, (2) the main pathogens at the larval stage were OsHV-1 and V. coralliilyticus using bath, while it was V. coralliilyticus, V. europaeus, and V. neptunius at the juvenile stage by injection, and (4) variation in mortality was observed among families/wild controls for all pathogens at larval and juvenile stages, except for V. harveyi for larvae., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Interferon (IFN) system is considered as the first defense line against viral infection, and it has been extensively studied in vertebrates from fish to mammals. In invertebrates, Vagos from arthropod and IFN-like protein ( Cg IFNLP) from Crassostrea gigas appeared to function as IFN-like antiviral cytokines. In the present study, the Cg IFNLP protein in hemocytes was observed to increase after Poly (I:C) stimulation. After Cg IFNLP was knocked down by RNAi, the mRNA expression of IFN-stimulated genes ( Cg ISGs) was significantly inhibited. Both cyclic GMP-AMP synthase ( Cg cGAS) and stimulator of interferon gene ( Cg STING) identified from oyster were able to recognize the double-stranded nucleic acid [Poly (I:C) and dsDNA] and expressed at high level after Poly (I:C) stimulation. The expression of Cg IFNLP and interferon regulatory factors ( Cg IRF1/8) and the nuclear translocation of Cg IRF8 were all suppressed in Cg cGAS-RNAi or Cg STING-RNAi oysters after Poly (I:C) stimulation. The expression level of Cg STING and TANK binding kinase1 ( Cg TBK1) did not decrease in Cg cGAS-RNAi oysters. After Cg STING was knocked down, the high expression of Cg TBK1 induced by Poly (I:C) was prevented significantly. These results indicated that there was a primitive IFN-like antiviral mechanism dependent on the cGAS/STING-TBK1-IRFs regulatory axis in mollusks, which was different from the classic cGAS-STING-TBK1 signal pathway in mammals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qiao, Zong, Liu, Wu, Li, Wang and Song.)

Norovirus contamination of oysters is the lead cause of non-bacterial gastroenteritis and a significant food safety concern for the oyster industry. Here, norovirus reduction from Pacific oysters (Crassostrea gigas), contaminated in the marine environment, was studied in laboratory depuration trials and in two commercial settings. Norovirus concentrations were measured in oyster digestive tissue before, during and post-depuration using the ISO 15216-1 quantitative real-time RT-PCR method. Results of the laboratory-based studies demonstrate that statistically significant reductions of up to 74% of the initial norovirus GII concentration was achieved after 3 days at 17-21 °C and after 4 days at 11-15 °C, compared to 44% reduction at 7-9 °C. In many trials norovirus GII concentrations were reduced to levels below 100 genome copies per gram (gcg -1 ; limit of quantitation; LOQ). Virus reduction was also assessed in commercial depuration systems, routinely used by two Irish oyster producers. Up to 68% reduction was recorded for norovirus GI and up to 90% for norovirus GII reducing the geometric mean virus concentration close to or below the LOQ. In both commercial settings there was a significant difference between the levels of reduction of norovirus GI compared to GII (p < 0.05). Additionally, the ability to reduce the norovirus concentration in oysters to < LOQ differed when contaminated with concentrations below and above 1000 gcg -1 . These results indicate that depuration, carried out at elevated (> 11 °C) water temperatures for at least 3 days, can reduce the concentration of norovirus in oysters and therefore consumer exposure providing a practical risk management tool for the shellfish industry.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Petton, Destoumieux-Garzón, Pernet, Toulza, de Lorgeril, Degremont and Mitta.)

Massive mortality outbreaks affecting Pacific oysters ( Crassostrea gigas ) spat/juveniles are often associated with the detection of a herpesvirus called ostreid herpesvirus type 1 (OsHV-1). In this work, experimental infection trials of C. gigas spat with OsHV-1 were conducted using two contrasted Pacific oyster families for their susceptibility to viral infection. Live oysters were sampled at 12, 26, and 144 h post infection (hpi) to analyze host-pathogen interactions using comparative proteomics. Shotgun proteomics allowed the detection of seven viral proteins in infected oysters, some of them with potential immunomodulatoy functions. Viral proteins were mainly detected in susceptible oysters sampled at 26 hpi, which correlates with the mortality and viral load observed in this oyster family. Concerning the Pacific oyster proteome, more than 3,000 proteins were identified and contrasted proteomic responses were observed between infected A- and P-oysters, sampled at different post-injection times. Gene ontology (GO) and KEGG pathway enrichment analysis performed on significantly modulated proteins uncover the main immune processes (such as RNA interference, interferon-like pathway, antioxidant defense) which contribute to the defense and resistance of Pacific oysters to viral infection. In the more susceptible Pacific oysters, results suggest that OsHV-1 manipulate the molecular machinery of host immune response, in particular the autophagy system. This immunomodulation may lead to weakening and consecutively triggering death of Pacific oysters. The identification of several highly modulated and defense-related Pacific oyster proteins from the most resistant oysters supports the crucial role played by the innate immune system against OsHV-1 and the viral infection. Our results confirm the implication of proteins involved in an interferon-like pathway for efficient antiviral defenses and suggest that proteins involved in RNA interference process prevent viral replication in C. gigas . Overall, this study shows the interest of multi-omic approaches applied on groups of animals with differing sensitivities and provides novel insight into the interaction between Pacific oyster and OsHV-1 with key proteins involved in viral infection resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Leprêtre, Faury, Segarra, Claverol, Degremont, Palos-Ladeiro, Armengaud, Renault and Morga.)

Norovirus in oysters is a significant food safety risk. A recent ISO detection method allows for reliable and repeatable estimates of norovirus concentrations in pooled samples, but there is insufficient data to estimate a distribution of copies per animal from this. The spread of norovirus accumulated across individual oysters is useful for risk assessment models. Six sets of thirty individual Crassostrea gigas oysters were tested for norovirus concentration levels by reverse-transcription quantitative PCR (RT-qPCR): three from a commercial harvest site, and three post-depuration. Five sets had norovirus GII means above the limit of quantification (LOQ), and one below the LOQ, but above the limit of detection. No norovirus GI was detected in pooled tests, and individual oysters were not tested for norovirus GI. Depuration was shown to reduce the mean concentration of GII copies, but not to affect the shape of the distribution around the mean. Deconvoluting the uncertainty of the method, the coefficient of variation was stationary (0.45 ± 0.2). The best fit distribution was either a lognormal distribution or a gamma. Multiplying these distributions by the weight of oyster digestive tissues gave an estimate for the count mean. This was used as the parameter λ in three compound Poisson distributions: Poisson-lognormal, Poisson-gamma, and Poisson-K. No model was found to fit better than the others, with advantages for each. All three could be used in future risk assessments. Preliminary validation of sampling uncertainty using repeated testing data from a previous study suggests that these results have predictive power., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Oyster production is an economic activity of great interest worldwide. Recently, oysters have been suffering significant mortalities from OsHV-1infection, which has resulted in substantial economic loses in several countries around the world. Understanding viral pathogenicity mechanisms is of central importance for the establishment of disease control measures. Thus, the present work aimed to identify and characterize miRNAs from OsHV-1 as well as to predict their target transcripts in the virus and the host. OsHV-1 genome was used for the in silico discovery of pre-miRNAs. Subsequently, viral and host target transcripts of the OsHV-1 miRNAs were predicted according to the base pairing interaction between mature miRNAs and mRNA 3' untranslated regions (UTRs). Six unique pre-miRNAs were found in different regions of the viral genome, ranging in length from 85 to 172 nucleotides. A complex network of self-regulation of viral gene expression mediated by the miRNAs was identified. These sequences also seem to have a broad ability to regulate the expression of host immune-related genes, especially those associated with pathogen recognition. Our results suggest that OsHV-1 encodes miRNAs with important functions in the infection process, inducing self-regulation of viral transcripts, as well as affecting the regulation of Pacific oyster transcripts related to immunity. Understanding the molecular basis of host-pathogen interactions can help mitigate the recurrent events of oyster mass mortalities by OsHV-1 observed worldwide., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The common cockle Cerastoderma edule plays an important ecological role in the marine ecosystem both as an infaunal engineer (reef forming and bioturbation) and a food source for protected bird species in its European range. Cockle beds are found in close proximity to aquaculture and fisheries operations, which can be "hot spots" for infectious agents including viruses and bacteria. Ostreid herpesvirus-1 microVar (OsHV-1 μVar) has spread to many Pacific oyster Crassostrea gigas culture sites globally, where it has been associated with significant mortalities in this cultured bivalve. Knowledge on the impact of the virus on the wider ecosystem, is limited. As the likelihood of released virus dispersing into the wider aquatic ecosystem is high, the plasticity of the virus and the susceptibility of C. edule to act as hosts or carriers is unknown., Methods: In this study, wild C. edule were sampled biweekly at two C. gigas culture sites over a four-month period during the summer when OsHV-1 μVar prevalence is at its highest in oysters. C. edule were screened for the virus molecularly (PCR, qPCR and Sanger sequencing) and visually (in situ hybridisation (ISH)). The cockle's ability to act as a carrier and transmit OsHV-1 μVar to the oyster host at a temperature of 14 ℃, when the virus is considered to be dormant until water temperatures exceed 16 ℃, was also assessed in laboratory transmission trials., Results: The results demonstrated that OsHV-1 μVar was detected in all C. edule size/age cohorts, at both culture sites. In the laboratory, viral transmission was effected from cockles to naïve oysters for the first time, five days post-exposure. The laboratory study also demonstrated that OsHV-1 μVar was active and was successfully transmitted from the C. edule at lower temperatures., Conclusions: This study demonstrates that OsHV-1 μVar has the plasticity to infect the keystone species C. edule and highlights the possible trophic transmission of the virus from cockles to their mobile top predators. This scenario would have important implications, as a greater geographical range expansion of this significant pathogen via migratory bird species may have an impact on other species that reside in bird habitats most of which are special areas of conservation.

Background: Since 2008, the aquaculture production of Crassostrea gigas was heavily affected by mass mortalities associated to Ostreid herpesvirus 1 (OsHV-1) microvariants worldwide. Transcriptomic studies revealed the major antiviral pathways of the oyster immune response while other findings suggested that also small non-coding RNAs (sncRNA) such as microRNAs might act as key regulators of the oyster response against OsHV-1. To explore the explicit connection between small non-coding and protein-coding transcripts, we performed paired whole transcriptome analysis of sncRNA and messenger RNA (mRNA) in six oysters selected for different intensities of OsHV-1 infection., Results: The mRNA profiles of the naturally infected oysters were mostly governed by the transcriptional activity of OsHV-1, with several differentially expressed genes mapping to the interferon, toll, apoptosis, and pro-PO pathways. In contrast, miRNA profiles suggested more complex regulatory mechanisms, with 15 differentially expressed miRNAs (DE-miRNA) pointing to a possible modulation of the host response during OsHV-1 infection. We predicted 68 interactions between DE-miRNAs and oyster 3'-UTRs, but only few of them involved antiviral genes. The sncRNA reads assigned to OsHV-1 rather resembled mRNA degradation products, suggesting the absence of genuine viral miRNAs., Conclusions: We provided data describing the miRNAome during OsHV-1 infection in C. gigas. This information can be used to understand the role of miRNAs in healthy and diseased oysters, to identify new targets for functional studies and, eventually to disentangle cause and effect relationships during viral infections in marine mollusks.

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 herpesvirus 1 (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.

Background: Vibriosis has been implicated in major losses of larvae at shellfish hatcheries. However, the species of Vibrio responsible for disease in aquaculture settings and their associated virulence genes are often variable or undefined. Knowledge of the specific nature of these factors is essential to developing a better understanding of the environmental and biological conditions that lead to larvae mortality events in hatcheries. We tested the virulence of 51 Vibrio strains towards Pacific Oyster (Crassostreae gigas) larvae and sequenced draft genomes of 42 hatchery-associated vibrios to determine groups of orthologous genes associated with virulence and to determine the phylogenetic relationships among pathogens and non-pathogens of C. gigas larvae., Results: V. coralliilyticus strains were the most prevalent pathogenic isolates. A phylogenetic logistic regression model identified over 500 protein-coding genes correlated with pathogenicity. Many of these genes had straightforward links to disease mechanisms, including predicted hemolysins, proteases, and multiple Type 3 Secretion System genes, while others appear to have possible indirect roles in pathogenesis and may be more important for general survival in the host environment. Multiple metabolism and nutrient acquisition genes were also identified to correlate with pathogenicity, highlighting specific features that may enable pathogen survival within C. gigas larvae., Conclusions: These findings have important implications on the range of pathogenic Vibrio spp. found in oyster-rearing environments and the genetic determinants of virulence in these populations.

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., (Copyright © 2020 Tang, Huang, Lin, Wang, Zhang and Li.)

Noroviruses (NoV) are responsible for many shellfish outbreaks. Purification processes may be applied to oysters before marketing to decrease potential fecal pollution. This step is rapidly highly effective in reducing Escherichia coli ; nevertheless, the elimination of virus genomes has been described to be much slower. It is therefore important to identify (i) the purification conditions that optimize virus removal and (ii) the mechanism involved. To this end, the effects of oyster stress, nutrients, and the presence of a potential competitor to NoV adhesion during purification were investigated using naturally contaminated oysters. Concentrations of NoV (genomes) and of the viral indicator F-specific RNA bacteriophage (FRNAPH; genomes and infectious particles) were regularly monitored. No significant differences were observed under the test conditions. The decrease kinetics of both virus genomes were similar, again showing the potential of FRNAPH as an indicator of NoV behavior during purification. The T 90 (time to reduce 90% of the initial titer) values were 47.8 days for the genogroup I NoV genome, 26.7 days for the genogroup II NoV genome, and 43.9 days for the FRNAPH-II genome. Conversely, monitoring of the viral genomes could not be used to determine the behavior of infectious viruses because the T 90 values were more than two times lower for infectious FRNAPH (20.6 days) compared to their genomes (43.9 days). Finally, this study highlighted that viruses are primarily inactivated in oysters rather than released in the water during purification processes. IMPORTANCE This study provides new data about the behavior of viruses in oysters under purification processes and about their elimination mechanism. First, a high correlation has been observed between F-specific RNA bacteriophages of subgroup II (FRNAPH-II) and norovirus (NoV) in oysters impacted by fecal contamination when both are detected using molecular approaches. Second, when using reverse transcription-quantitative PCR and culture to detect FRNAPH-II genomes and infectious FRNAPH in oysters, respectively, it appears that genome detection provides limited information about the presence of infectious particles. The comparison of both genomes and infectious particles highlights that the main mechanism of virus elimination in oysters is inactivation. Finally, this study shows that none of the conditions tested modify virus removal., (Copyright © 2020 American Society for Microbiology.)

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., (© 2020 Stichting International Foundation for Animal Genetics.)

Given the threat that ostreid herpesvirus 1 (OsHV-1) poses to shellfish aquaculture, the need for rapid, user-friendly and cost-effective methods to detect this marine pathogen and minimise its impact is evident. In this work, an electrochemical biosensor for the detection of OsHV-1 based on isothermal recombinase polymerase amplification (RPA) was developed. The system was first tested and optimised on maleimide microtitre plates as a proof-of-concept, before being implemented on miniaturised gold electrodes. Amperometric detection of the isothermally amplified product was achieved through a sandwich hybridisation assay with an immobilised thiolated capture probe and a horseradish peroxidase (HRP)-labelled reporter probe. Calibration curves were constructed using PCR-amplified OsHV-1 DNA, achieving a limit of detection of 207 OsHV-1 target copies. The biosensor was applied to the analysis of 16 oyster samples from an infectivity experiment, and results were compared with those obtained by qPCR analysis, showing a strong degree of correlation (r = 0.988). The simplicity, rapidity, cost-effectiveness and potential for in-situ testing with the developed biosensor provide a valuable tool for the detection of OsHV-1 in aquaculture facilities, improving their management., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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 × 10 7 copies/mg at 18 hours) compared to 18 °C (1.38 × 10 5  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.

More stable than bacteria in environmental samples, enteric viruses are generally related to outbreaks of gastroenteritis caused by the consumption of contaminated oysters. This study evaluated: i) the dynamic processes of enteric viral models bioaccumulation by Crassostrea gigas oysters artificially contaminated; ii) the stability of these viruses in oysters in controlled temperature conditions and iii) the effect of UV light in inactivating these viruses in depurated oysters. Plaque assay (PA) was used to assess the infectivity of both viral models. Cell culture coupled with RT-qPCR (ICC-RT-qPCR) was used to measure infectious adenovirus type 2 (HAdV-2) genomes and qPCR to measure genome copies of murine norovirus (MNV-1). The virus uptake through bioaccumulation behave differently: HAdV-2 reached its peak of uptake faster than MNV-1. Both viruses showed high stability in oysters when maintained under 4 °C, but were completely inactivated in steamed oysters. The HAdV-2 was completely inactivated after 12 h of depuration with UV light and after 24 h without UV light. After 72 h of depuration, MNV-1 was still detected in both tanks, probably due to the stronger interaction of this virus with the oyster's tissues. This study demonstrated the importance of a secure depuration time in ensuring a clean and safe product, and that the steaming process is the safest way to prepare oysters for consumption., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Food provisioning influences disease risk and outcome in animal populations in two ways. On the one hand, unrestricted food supply improves the physiological condition of the host and lowers its susceptibility to infectious disease, reflecting a trade-off between immunity and other fitness-related functions. On the other hand, food scarcity limits the resources available to the pathogen and slows the growth and metabolism of the host on which the pathogen depends to proliferate. Here, we investigated how food availability, growth rate and energetic reserves drive the outcome of a viral disease affecting an ecologically relevant model host, the Pacific oyster, Crassostrea gigas We selected fast- and slow-growing animals, and we exposed them to high and low food rations. We evaluated their energetic reserves, challenged them with a pathogenic virus, monitored daily survival and developed a mortality risk model. Although high food levels and oyster growth were associated with a higher risk of mortality, energy reserves were associated with a lower risk. Food availability acts both as an enabling factor for mortality by increasing oyster growth and as a limiting factor by increasing their energy reserves. This study clarifies how food resources have an impact on susceptibility to disease and indicates how the host's physiological condition could mitigate epidemics. Practically, we suggest that growth should be optimized rather than maximized, considering that trade-offs occur with disease resistance or tolerance., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

This study investigated the synergistic effects of combined chlorine (200, 500, 700, and 1000 ppm) and vitamin B 1 (1000, 2000, and 3000 ppm) on the murine norovirus-1 (MNV-1), a human norovirus (NoV) surrogate, on oyster surface. Vitamin B 1 slightly reduced MNV-1 (0.04-0.3 log-reduction), whereas chlorine significantly reduced MNV-1 (0.4-1.0 log-reduction). The combined chlorine and vitamin B 1 resulted in a 0.52-1.97 log-reduction of MNV-1. The synergistic reduction in the MNV titer was not dependent on the concentrations of chlorine and vitamin B 1 , and it ranged between 0.08 and 1.03 log 10 PFU/mL. The largest synergistic reduction observed was for the combined 700 ppm chlorine and 1000 ppm vitamin B 1 . The pH and mechanical texture of the oysters were not significantly changed by the combined 0-1000 ppm chlorine and 3000 ppm vitamin B 1 . The overall sensory acceptability were significantly (P < 0.05) reduced in oysters treated with 1000 ppm chlorine and 3000 ppm vitamin B 1 than in those treated with 0-700 ppm chlorine and 3000 ppm vitamin B 1 . This study suggests that the combined 700 ppm chlorine and 3000 ppm vitamin B 1 could potentially be used to reduce NoV on oyster surface without causing concomitant changes in the mechanical texture, pH, or sensory qualities of the oysters.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

This study was conducted to evaluate the microbiological quality of a mangrove estuary in the Vitória Bay region, Espírito Santo, Brazil. We analyzed the presence and concentration of enteric viruses and thermotolerant coliforms in water, mussels (Mytella charruana and Mytella guyanensis), and oysters (Crassostrea rhizophorae), collected over a 13-month period. Human adenovirus, rotavirus A (RVA), and norovirus genogroup II were analyzed by quantitative PCR. The highest viral load was found in RVA-positive samples with a concentration of 3.0 × 10 4  genome copies (GC) L -1 in water samples and 1.3 × 10 5  GC g -1 in bivalves. RVA was the most prevalent virus in all matrices. Thermotolerant coliforms were quantified as colony-forming units (CFU) by the membrane filtration method. The concentration of these bacteria in water was in accordance with the Brazilian standard for recreational waters (< 250 CFU 100 mL -1 ) during most of the monitoring period (12 out of 13 months). However, thermotolerant coliform concentrations of 3.0, 3.1, and 2.6 log CFU 100 g -1 were detected in M. charruana, M. guyanensis, and C. rhizophorae, respectively. The presence of human-specific viruses in water and bivalves reflects the strong anthropogenic impact on the mangrove and serves as an early warning of waterborne and foodborne disease outbreaks resulting from the consumption of shellfish and the practice of water recreational activities in the region.

Male-specific coliphages (MSCs) are currently used to assess the virologic quality of shellfish-growing waters and to assess the impact of sewage release or adverse weather events on bivalve shellfish. Since MSC can have either DNA or RNA genomes, and most research has been performed exclusively on RNA MSCs, persistence of M13, a DNA MSC, was evaluated for its persistence as a function of time and temperature within Eastern oysters (Crassostrea virginica). Oysters were individually exposed to seawater containing a total of 10 10 to 10 12 pfu of M13 for 24 h at 15 °C followed by maintenance in tanks with as many as 21 oysters in continuously UV-sterilized water for up to 6 weeks at either 7, 15, or 22 °C. Two trials for each temperature were performed combining three shucked oysters per time point which were assayed by tenfold serial dilution in triplicate. Initial contamination levels averaged 10 6.9 and ranged from 10 6.0 to 10 7.0 of M13. For oysters held for 3 weeks, log 10 reductions were 1.7, 3.8, and 4.2 log 10 at 7, 15, and 22 °C, respectively. Oysters held at 7 and 15 °C for 6 weeks showed average reductions of 3.6 and 5.1 log 10 , respectively, but still retained infectious M13. In total, this work shows that DNA MSC may decline within shellfish in a manner analogous to RNA MSCs.

Shifting environmental conditions are known to be important triggers of oyster diseases. The mechanism(s) behind these synergistic effects (interplay between host, environment and pathogen/s) are often not clear, although there is evidence that shifts in environmental conditions can affect oyster immunity, and pathogen growth and virulence. However, the impact of shifting environmental parameters on the oyster microbiome and how this affects oyster health and susceptibility to infectious pathogens remains understudied. In this review, we summarise the major diseases afflicting oysters with a focus on the role of environmental factors that can catalyse or amplify disease outbreaks. We also consider the potential role of the oyster microbiome in buffering or augmenting oyster disease outbreaks and suggest that a deeper understanding of the oyster microbiome, its links to the environment and its effect on oyster health and disease susceptibility, is required to develop new frameworks for the prevention and management of oyster diseases., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (© 2018 John Wiley & Sons Ltd.)

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.

Ostreid herpesvirus-1 (OsHV-1) has been involved in mass mortality episodes of Pacific oysters Crassostrea gigas throughout the world, causing important economic losses to the aquaculture industry. In the present study, magnetic beads (MBs) coated with an anionic polymer were used to capture viable OsHV-1 from two types of naturally infected matrix: oyster homogenate and seawater. Adsorption of the virus on the MBs and characterisation of the MB-virus conjugates was demonstrated by real-time quantitative PCR (qPCR). To study the infective capacity of the captured virus, MB-virus conjugates were injected in the adductor muscle of naïve spat oysters, using oyster homogenate and seawater without MBs as positive controls, and bare MBs and sterile water as negative controls. Mortalities were induced after injection with MB-virus conjugates and in positive controls, whereas no mortalities were recorded in negative controls. Subsequent OsHV-1 DNA and RNA analysis of the oysters by qPCR and reverse transcription qPCR (RT-qPCR), respectively, confirmed that the virus was the responsible for the mortality event and the ability of the MBs to capture viable viral particles. The capture of viable OsHV-1 using MBs is a rapid and easy isolation method and a promising tool, combined with qPCR, to be applied to OsHV-1 detection in aquaculture facilities., Competing Interests: The authors have declared that no competing interests exist.

Long noncoding RNAs (lncRNAs) may play widespread roles in various biological processes. However, systematic profiles of lncRNAs in the biological responses of Pacific Oyster (Crassostrea gigas) to pathogen infection have not yet been demonstrated. Here, we have conducted an exhaustive comparative transcriptome analysis using a bioinformatics approach to exam the functions of lncRNAs response to Ostreid herpesvirus 1μVar (OsHV-1μVar) challenge. In total, 101 differentially expressed lncRNAs (DE-lncRNA) during OsHV-1μVar infections were identified. Compared with differentially expressed mRNAs (DE-mRNA), DE-lncRNAs are shorter in terms of overall length but longer in terms of exon length. These lncRNAs shared similar characteristics with previously reported invertebrate lncRNAs, such as relatively low GC content, low exon number and low sequence conservation, but low expression level were not observed. 20 DE-lncRNAs are typically co-expressed with their neighboring genes annotated as GO terms (GO: 0044237), indicating that these lncRNAs are involved in binding and cellular process functions in cis mode. The weighted gene co-expression network (WGCNA) analysis resulted in 15 modules. The highlighted blue module was specifically demonstrated a co-expression relationship between 14 DE-lncRNAs and 17 immune-related DE-mRNAs (IR-DE-mRNA). Three hub lncRNAs within this module were co-expressed with one hub IR-DE-mRNA involved in fibrinogen-related protein. It was speculated that lncRNAs is extensively involved in oyster antiviral innate immune system. The present study will facilitate subsequently experimental studies to unravel the function of lncRNAs in marine invertebrate response to pathogen infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Temperature triggers marine diseases by changing host susceptibility and pathogen virulence. Oyster mortalities associated with the Ostreid herpesvirus type 1 (OsHV-1) have occurred seasonally in Europe when the seawater temperature range reaches 16-24 °C. Here we assess how temperature modulates oyster susceptibility to OsHV-1 and pathogen virulence. Oysters were injected with OsHV-1 suspension incubated at 21 °C, 26 °C and 29 °C and were placed in cohabitation with healthy oysters (recipients) at these three temperatures according to a fractional factorial design. Survival was followed for 14 d and recipients were sampled for OsHV-1 DNA quantification and viral gene expression. The oysters were all subsequently placed at 21 °C to evaluate the potential for virus reactivation, before being transferred to oyster farms to evaluate their long-term susceptibility to the disease. Survival of recipients at 29 °C (86%) was higher than at 21 °C (52%) and 26 °C (43%). High temperature (29 °C) decreased the susceptibility of oysters to OsHV-1 without altering virus infectivity and virulence. At 26 °C, the virulence of OsHV-1 was enhanced. Differences in survival persisted when the recipients were all placed at 21 °C, suggesting that OsHV-1 did not reactivate. Additional oyster mortality followed the field transfer, but the overall survival of oysters infected at 29 °C remained higher., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Marine diseases have major impacts on ecosystems and economic consequences for aquaculture and fisheries. Understanding origin, spread and risk factors of disease is crucial for management, but data in the ocean are limited compared to the terrestrial environment. Here we investigated how the marine environment drives the spread of viral disease outbreak affecting The Pacific oyster worldwide by using a spatial epidemiology framework. We collected environmental and oyster health data at 46 sites spread over an area of 300 km 2 along an inshore-offshore gradient during an epizootic event and conducted risk analysis. We found that disease broke out in the intertidal farming area and spread seaward. Mortalities and virus detection were observed in oysters placed 2 km from the farming areas, but oysters of almost all sites were subclinically infected. Increasing food quantity and quality, growth rate and energy reserves of oyster were associated with a lower risk of mortality offshore whereas increasing turbidity, a proxy of the concentration of suspended particulate matter, and terrestrial inputs, inferred from fatty acid composition of oysters, were associated with a higher risk of mortality. Offshore farming and maintenance of good ecological status of coastal waters are options to limit disease risk in oysters.

The Pacific oyster Crassostrea gigas contributes significantly to global aquaculture; however, C. gigas culture has been affected by ostreid herpesvirus-1 (OsHV-1) and variants. The dynamics of how the virus maintains itself at culture sites is unclear and the role of carriers, reservoirs or hosts is unknown. Both wild and cultured mussels Mytilus spp. (Mytilus edulis, Mytilus galloprovincialis and hybrids) are commonly found at C. gigas culture sites. The objective of this study was to investigate if Mytilus spp. can harbour the virus and if viral transmission can occur between mussels and oysters. Mytilus spp. living at oyster trestles, 400-500 m higher up the shore from the trestles and up to 26 km at non-culture sites were screened for OsHV-1 and variants by all the World Organization for Animal Health (OIE) recommended diagnostic methods including polymerase chain reaction (PCR), quantitative PCR (qPCR), histology, in situ hybridization and confirmation using direct sequencing. The particular primers that target OsHV-1 and variants, including OsHV-1 microVar (μVar), were used in the PCR and qPCR. OsHV-1 μVar was detected in wild Mytilus spp. at C. gigas culture sites and more significantly the virus was detected in mussels at non-culture sites. Cohabitation of exposed wild mussels and naïve C. gigas resulted in viral transmission after 14 days, under an elevated temperature regime. These results indicate that mussels can harbour OsHV-1 μVar; however, the impact of OsHV-1 μVar on Mytilus spp. requires further investigation.

Since 2008, juvenile Crassostrea gigas oysters have suffered from massive mortalities in European farming areas. This disease of complex etiology is still incompletely understood. Triggered by an elevated seawater temperature, it has been associated to infections by a herpes virus named OsHV-1 as well as pathogenic vibrios of the Splendidus clade. Ruling out the complexity of the disease, most of our current knowledge has been acquired in controlled experiments. Among the many unsolved questions, it is still ignored what role immunity plays in the capacity oysters have to survive an infectious episode. Here we show that juvenile oysters susceptible to the disease mount an inefficient immune response associated with microbial permissiveness and death. We found that, in contrast to resistant adult oysters having survived an earlier episode of mortality, susceptible juvenile oysters never exposed to infectious episodes died by more than 90% in a field experiment. Susceptible oysters were heavily colonized by OsHV-1 herpes virus as well as bacteria including vibrios potentially pathogenic for oysters, which proliferated in oyster flesh and body fluids during the mortality event. Nonetheless, susceptible oysters were found to sense microbes as indicated by an overexpression of immune receptors and immune signaling pathways. However, they did not express important immune effectors involved in antimicrobial immunity and apoptosis and showed repressed expression of genes involved in ROS and metal homeostasis. This contrasted with resistant oysters, which expressed those important effectors, controlled bacterial and viral colonization and showed 100% survival to the mortality event. Altogether, our results demonstrate that the immune response mounted by susceptible oysters lacks some important immune functions and fails in controlling microbial proliferation. This study opens the way to more holistic studies on the "mass mortality syndrome", which are now required to decipher the sequence of events leading to oyster mortalities and determine the relative weight of pathogens, oyster genetics and oyster-associated microbiota in the disease., (Copyright © 2018. Published by Elsevier Ltd.)

The surveillance activities for abnormal bivalve mortality events in Italy include the diagnosis of ostreid herpesvirus type 1 (OsHV-1) in symptomatic oysters. OsHV-1-positive oysters (Crassostrea gigas) were used as a source for in vivo virus propagation and a virus-rich sample was selected to perform shotgun sequencing based on Illumina technology. Starting from this unpurified supernatant sample from gills and mantle, we generated 3.5 million reads (2×300 bp) and de novo assembled the whole genome of an Italian OsHV-1 microvariant (OsHV-1-PT). The OsHV-1-PT genome encodes 125 putative ORFs, 7 of which had not previously been predicted in other sequenced Malacoherpesviridae. Overall, OsHV-1-PT displays typical microvariant OsHV-1 genome features, while few polymorphisms (0.08 %) determine its uniqueness. As little is known about the genetic determinants of OsHV-1 virulence, comparing complete OsHV-1 genomes supports a better understanding of the virus pathogenicity and provides new insights into virus-host interactions.

Marine herpesviruses are responsible for epizootics in economically, ecologically and culturally significant taxa. The recent emergence of microvariants of Ostreid herpesvirus 1 (OsHV-1) in Pacific oysters Crassostrea gigas has resulted in socioeconomic losses in Europe, New Zealand and Australia however, there is no information on their origin or mode of transmission. These factors need to be understood because they influence the way the disease may be prevented and controlled. Mortality data obtained from experimental populations of C. gigas during natural epizootics of OsHV-1 disease in Australia were analysed qualitatively. In addition we compared actual mortality data with those from a Reed-Frost model of direct transmission and analysed incubation periods using Sartwell's method to test for the type of epizootic, point source or propagating. We concluded that outbreaks were initiated from an unknown environmental source which is unlikely to be farmed oysters in the same estuary. While direct oyster-to-oyster transmission may occur in larger oysters if they are in close proximity (< 40 cm), it did not explain the observed epizootics, point source exposure and indirect transmission being more common and important. A conceptual model is proposed for OsHV-1 index case source and transmission, leading to endemicity with recurrent seasonal outbreaks. The findings suggest that prevention and control of OsHV-1 in C. gigas will require multiple interventions. OsHV-1 in C. gigas, which is a sedentary animal once beyond the larval stage, is an informative model when considering marine host-herpesvirus relationships.

Betanodaviruses are small ssRNA viruses responsible for viral encephalopathy and retinopathy, otherwise known as viral nervous necrosis, in marine fish worldwide. These viruses can be either horizontally or vertically transmitted and have been sporadically detected in invertebrates, which seem to be one of the possible viral sources. Twenty-eight new betanodavirus strains were retrieved in three molluscs species collected from different European countries between 2008 and 2015. The phylogenetic analyses revealed that strains retrieved from bivalve molluscs are closely related to viruses detected in finfish in Southern Europe in the period 2000-2009. Nevertheless, a new betanodavirus strain, markedly different from the other members of the RGNNV genotype, was detected. Such a massive and varied presence of betanodaviruses in bivalve molluscs greatly stresses the risks of transmission previously feared for other invertebrates. Bivalve molluscs reared in the same area as farmed and wild finfish could act as a reservoir of the virus. Furthermore, current European regulations allow relaying activities and the sale of live bivalve molluscs, which could pose a real risk of spreading betanodaviruses across different geographic regions. To our knowledge, this is the first study, which focuses on the detection and genetic characterization of betanodaviruses in bivalve molluscs., (© 2017 John Wiley & Sons Ltd.)

Ostreid herpesvirus (OsHV) can cause mass mortality events in Pacific oyster aquaculture. While various factors impact on the severity of outbreaks, it is clear that genetic resistance of the host is an important determinant of mortality levels. This raises the possibility of selective breeding strategies to improve the genetic resistance of farmed oyster stocks, thereby contributing to disease control. Traditional selective breeding can be augmented by use of genetic markers, either via marker-assisted or genomic selection. The aim of the current study was to investigate the genetic architecture of resistance to OsHV in Pacific oyster, to identify genomic regions containing putative resistance genes, and to inform the use of genomics to enhance efforts to breed for resistance. To achieve this, a population of ∼1,000 juvenile oysters were experimentally challenged with a virulent form of OsHV, with samples taken from mortalities and survivors for genotyping and qPCR measurement of viral load. The samples were genotyped using a recently-developed SNP array, and the genotype data were used to reconstruct the pedigree. Using these pedigree and genotype data, the first high density linkage map was constructed for Pacific oyster, containing 20,353 SNPs mapped to the ten pairs of chromosomes. Genetic parameters for resistance to OsHV were estimated, indicating a significant but low heritability for the binary trait of survival and also for viral load measures (h2 0.12 - 0.25). A genome-wide association study highlighted a region of linkage group 6 containing a significant QTL affecting host resistance. These results are an important step toward identification of genes underlying resistance to OsHV in oyster, and a step toward applying genomic data to enhance selective breeding for disease resistance in oyster aquaculture., (Copyright © 2018 Gutierrez et al.)

The Pacific oyster, Crassostrea gigas , is becoming a valuable model for investigating antiviral defense in the Lophotrochozoa superphylum. In the past five years, improvements to laboratory-based experimental infection protocols using Ostreid herpesvirus I (OsHV-1) from naturally infected C. gigas combined with next-generation sequencing techniques has revealed that oysters have a complex antiviral response involving the activation of all major innate immune pathways. Experimental evidence indicates C. gigas utilizes an interferon-like response to limit OsHV-1 replication and spread. Oysters injected with a viral mimic (polyI:C) develop resistance to OsHV-1. Improved survival following polyI:C injection was found later in life (within-generational immune priming) and in the next generation (multi-generational immune priming). These studies indicate that the oyster's antiviral defense system exhibits a form of innate immune-memory. An important priority is to identify the molecular mechanisms responsible for this phenomenon. This knowledge will motivate the development of practical and cost-effective treatments for improving oyster health in aquaculture., Competing Interests: The authors declare no conflict of interest.

Male-specific bacteriophages have been proposed as human enteric virus indicators for shellfish. In this study, Eastern oysters (Crassostrea virginica) were individually exposed to 5.6 × 10 10 PFU of MS-2 for 48 h at 15 °C followed by collective maintenance in continuously UV-sterilized seawater for 0-6 weeks at either 7, 15, or 24 °C. Initial contamination levels of MS-2 were >6 log PFU. Assessment of weekly declines of viable MS-2 indicated that cooler temperatures dramatically enhanced the persistence of MS-2 within oyster tissues. At 3 weeks, the average log PFU reductions for MS-2 within oysters were 2.28, 2.90, and 4.57 for oysters held at 7, 15, and 24 °C, respectively. Fitting temporal survival data with linear and nonlinear Weibull models indicated that the Weibull model best fit the observed reductions. In total, these data can serve as a guideline for regulatory agencies regarding the influence of water temperature on indicator phage after episodic sewage exposure.

Voltage-dependent anion channel (VDAC) is a key mitochondrial protein. VDAC drives cellular energy metabolism by controlling the influx and efflux of metabolites and ions through the mitochondrial membrane, playing a role in its permeabilization. This protein exerts a pivotal role during the white spot syndrome virus (WSSV) infection in shrimp, through its involvement in a particular metabolism that plays in favor of the virus, the Warburg effect. The Warburg effect corresponds to an atypical metabolic shift toward an aerobic glycolysis that provides energy for rapid cell division and resistance to apoptosis. In the Pacific oyster Crassostrea gigas, the Warburg effect occurs during infection by Ostreid herpesvirus (OsHV-1). At present, the role of VDAC in the Warburg effect, OsHV-1 infection and apoptosis is unknown. Here, we developed a specific antibody directed against C. gigas VDAC. This tool allowed us to quantify the tissue-specific expression of VDAC, to detect VDAC oligomers, and to follow the amount of VDAC in oysters deployed in the field. We showed that oysters sensitive to a mortality event in the field presented an accumulation of VDAC. Finally, we propose to use VDAC quantification as a tool to measure the oyster susceptibility to OsHV-1 depending on its environment.

The Ostreid herpes virus type 1 (OsHV-1) is one of the most devastating pathogen in oyster cultures. Among several factors, as food limitation, oxygen depletion, salinity and temperature variations, episodes of "summer mortality" of the Pacific oyster Crassostrea gigas have also been associated with OsHV-1 infection. Mortalities of C. gigas spat and juveniles have increased significantly in Europe, and contemporary mortality records of this mollusk in México have been associated with the occurrence of OsHV-1. In the present study, the expression of the heat shock protein 70 gene from the Pacific oyster correlates with the abundance of DNA polymerase transcripts from the OsHV-1. This may suggest that the induction on the expression of the Pacific oyster hsp70 may potentially participate in the immune response against the virus. Furthermore, this study reports for the first time a TEM representative image of the OsHV-1 in aqueous solution, which possesses an icosahedral shape with a diameter of 70 nm × 100 nm. Finally, the examined sequence encoding the ORF4 of the OsHV-1 isolate from northwest Mexico showed specific sequence variations when compared with OsHV-1 isolates from distant geographical areas., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Since 2008, mass mortality outbreaks associated with the detection of particular variants of OsHV-1 have been reported in Crassostrea gigas spat and juveniles in several countries. Recent studies have reported information on viral replication during experimental infection. Viral DNA and RNA were also detected in the haemolymph and haemocytes suggesting that the virus could circulate through the circulatory system. However, it is unknown if the virus is free in the haemolymph, passively associated at the surface of haemocytes, or able to infect and replicate inside these cells inducing (or not) virion production. In the present study, we collected haemocytes from the haemolymphatic sinus of the adductor muscle of healthy C. gigas spat and exposed them in vitro to a viral suspension. Results showed that viral RNAs were detectable one hour after contact and the number of virus transcripts increased over time in association with an increase of viral DNA detection. These results suggested that the virus is able to initiate replication rapidly inside haemocytes maintained in vitro. These in vitro trials were also used to carry out a dual transcriptomic study. We analyzed concomitantly the expression of some host immune genes and 15 viral genes. Results showed an up regulation of oyster genes currently studied during OsHV-1 infection. Additionally, transmission electron microscopy examination was carried out and did not allow the detection of viral particles. Moreover, All the results suggested that the in vitro model using haemocytes can be valuable for providing new perspective on virus-oyster interactions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ostreid herpes virus causes serious disease in the Pacific oyster (Crassostrea gigas), but not in the Sydney Rock Oyster (Saccostrea glomerata). To investigate differences in disease progression, we injected oysters with double stranded RNA (dsRNA). dsRNA is known to mimic viral infection, and can evoke immune responses when Toll-like receptors detect the dsRNA, leading to the production of type 1 interferon and inflammation cytokines. The uptake and processing of dsRNA was tracked in gill and mantle tissue of Crassostrea gigas and Saccostrea glomerata after injection of fluorochrome labelled poly (I:C) dsRNA. The two species showed significant differences in tissue uptake and clearance, and differences in immune responses confirmed by real time PCR. These results showed that S. glomerata was more efficient in processing dsRNA than C. gigas, and that the gill tissue is an important site of dsRNA processing and response., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In the last decade, a paradigm shift has emerged in comparative immunology. Invertebrates can no longer be considered to be devoid of specific recognition and immune memory. However, we still lack a comprehensive view of these phenomena and their molecular mechanisms across phyla, especially in terms of duration, specificity, and efficiency in a natural context. In this study, we focused on a Lophotrochozoan/virus interaction, as antiviral priming is mostly overlooked in molluscs. Juvenile Crassostrea gigas oysters experience reoccurring mass mortalities events from Ostreid herpes virus 1 with no existing therapeutic treatment. Our results showed that various nucleic acid injections can prime oysters to trigger an antiviral state ultimately protecting them against a subsequent viral infection. Focusing on poly(I:C) as elicitor, we evidenced that it protected from an environmental infection, by mitigating viral replication. That protection seemed to induce a specific antiviral response as poly(I:C) fails to protect against a pathogenic bacteria. Finally, we showed that this phenomenon was long-lasting, persisting for at least 5 months thus suggesting for the first time the existence of innate immune memory in this invertebrate species. This study strengthens the emerging hypotheses about the broad conservation of innate immune priming and memory mechanisms in Lophotrochozoans.

Infections with Ostreid herpesvirus 1 (OsHV-1) microvariants in young Pacific oysters are associated with massive mortality events and significant economic losses. Previous studies, focusing on few regions of the genome, have revealed the genomic diversity of these genotypes with respect to the reference type. We used a NGS process to sequence the whole genome of the OsHV-1 µVar in infected individuals, collected during mortality events in France and Ireland. The final genome length of OsHV-1 µVar was approximately 205kbp, shorter than the reference genotype and the overall genome organisation resembled herpes simplex viruses. 94.4% similarity was observed with the OsHV-1 reference genotype. Large indels, including five deletions and three insertions were found to induce the loss and the addition of several ORFs, summed with codon substitutions in 64% of genes shared with the reference type. This diversity raises the question of the exact origin and evolution of OsHV-1 µVar., (Copyright © 2017 Elsevier Inc. All rights reserved.)