Propidium monoazide PCR, a method to determine OsHV-1 undamaged capsids and to estimate virus Lethal Dose 50.

Ostreid herpes virus 1 (OsHV-1) has been classified within the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 is the etiological agent of a contagious viral disease of Pacific oysters, C. gigas, affecting also other bivalve species. Mortality rates reported associated with the viral infection vary considerably between sites and countries and depend on the age of affected stocks. A variant called µVar has been reported since 2008 in Europe and other variants in Australia and in New Zealand last decade. These variants are considered as the main causative agents of mass mortality events affecting C. gigas. Presently there is no established cell line that allows for the detection of infectious OsHV-1. In this context, a technique of propidium monoazide (PMA) PCR was developed in order to quantify "undamaged" capsids. This methodology is of interest to explore the virus infectivity. Being able to quantify viral particles getting an undamaged capsid (not only an amount of viral DNA) in tissue homogenates prepared from infected oysters or in seawater samples can assist in the definition of a Lethal Dose (LD) 50 and gain information in the experiments conducted to reproduce the viral infection. The main objectives of the present study were (i) the development/optimization of a PMA PCR technique for OsHV-1 detection using the best quantity of PMA and verifying its effectiveness through heat treatment, (ii) the definition of the percentage of undamaged capsids in four different tissue homogenates prepared from infected Pacific oysters and (iii) the approach of a LD50 during experimental viral infection assays on the basis of a number of undamaged capsids. Although the developped PMA PCR technique was unable to determine OsHV-1 infectivity in viral supensions, it could greatly improve interpretation of virus positive results obtained by qPCR. This technique is not intended to replace the quantification of viral DNA by qPCR, but it does make it possible to give a form of biological meaning to the detection of this DNA.

Evaluation of tangential flow filtration coupled to long-read sequencing for ostreid herpesvirus type 1 genome assembly.

Whole-genome sequencing is widely used to better understand the transmission dynamics, the evolution and the emergence of new variants of viral pathogens. This can bring crucial information to stakeholders for disease management. Unfortunately, aquatic virus genomes are usually difficult to characterize because most of these viruses cannot be easily propagated in vitro. Developing methodologies for routine genome sequencing of aquatic viruses is timely given the ongoing threat of disease emergence. This is particularly true for pathogenic viruses infecting species of commercial interest that are widely exchanged between production basins or countries. For example, the ostreid herpesvirus type 1 (OsHV-1) is a Herpesvirus widely associated with mass mortality events of juvenile Pacific oyster Crassostrea gigas. Genomes of Herpesviruses are large and complex with long direct and inverted terminal repeats. In addition, OsHV-1 is unculturable. It therefore accumulates several features that make its genome sequencing and assembly challenging. To overcome these difficulties, we developed a tangential flow filtration (TFF) method to enrich OsHV-1 infective particles from infected host tissues. This virus purification allowed us to extract high molecular weight and high-quality viral DNA that was subjected to Illumina short-read and Nanopore long-read sequencing. Dedicated bioinformatic pipelines were developed to assemble complete OsHV-1 genomes with reads from both sequencing technologies. Nanopore sequencing allowed characterization of new structural variations and major viral isomers while having 99,98 % of nucleotide identity with the Illumina assembled genome. Our study shows that TFF-based purification method, coupled with Nanopore sequencing, is a promising approach to enable in field sequencing of unculturable aquatic DNA virus.

Monitoring Autophagy at Cellular and Molecular Level in Crassostrea gigas During an Experimental Ostreid Herpesvirus 1 (OsHV-1) Infection.

Mortality outbreaks of young Pacific oysters, Crassostrea gigas, have seriously affected the oyster-farming economy in several countries around the world. Although the causes of these mortality outbreaks appear complex, a viral agent has been identified as the main factor: a herpesvirus called ostreid herpesvirus 1 (OsHV-1). Autophagy is an important degradation pathway involved in the response to several pathologies including viral diseases. In C. gigas, recent studies indicate that this pathway is conserved and functional in at least haemocytes and the mantle. Furthermore, an experimental infection in combination with compounds known to inhibit or induce autophagy in mammals revealed that autophagy is involved in the response to OsHV-1 infection. In light of these results, the aim of this study was to determine the role of autophagy in the response of the Pacific oyster to infection by virus OsHV-1. For this purpose, an experimental infection in combination with a modulator of autophagy was performed on Pacific oysters known to have intermediate susceptibility to OsHV-1 infection. In haemolymph and the mantle, the autophagy response was monitored by flow cytometry, western blotting, and real-time PCR. At the same time, viral infection was evaluated by quantifying viral DNA and RNA amounts by real-time PCR. Although the results showed activation of autophagy in haemolymph and the mantle 14 hours post infection (after viral replication was initiated), they were also indicative of different regulatory mechanisms of autophagy in the two tissues, thus supporting an important function of autophagy in the response to virus OsHV-1.

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.

First comparison of French and Australian OsHV-1 µvars by bath exposure.

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

Identification of the autophagy pathway in a mollusk bivalve, Crassostrea gigas.

The Pacific oyster, Crassostrea gigas, is a mollusk bivalve commercially important as a food source. Pacific oysters are subjected to stress and diseases during culture. The autophagy pathway is involved in numerous cellular processes, including responses to starvation, cell death, and microorganism elimination. Autophagy also exists in C. gigas, and plays a role in the immune response against infections. Although this process is well-documented and conserved in most animals, it is still poorly understood in mollusks. To date, no study has provided a complete overview of the molecular mechanism of autophagy in mollusk bivalves. In this study, human and yeast ATG protein sequences and public databases (Uniprot and NCBI) were used to identify protein members of the C. gigas autophagy pathway. A total of 35 autophagy related proteins were found in the Pacific oyster. RACE-PCR was performed on several genes. Using molecular (real-time PCR) and protein-based (western blot and immunohistochemistry) approaches, the expression and localization of ATG12, ATG9, BECN1, MAP1LC3, MTOR, and SQSTM1, was investigated in different tissues of the Pacific oyster. Comparison with human and yeast counterparts demonstrated a high homology with the human autophagy pathway. The results also demonstrated that the key autophagy genes and their protein products were expressed in all the analyzed tissues of C. gigas. This study allows the characterization of the complete C. gigas autophagy pathway for the first time. Abbreviations: ATG: autophagy related; Atg1/ULK: unc-51 like autophagy activating kinase; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG12: autophagy related 12; BECN1: beclin 1; BSA: bovine serum albumin; cDNA: complementary deoxyribonucleic acid; DNA: deoxyribonucleic acid; GABARAP: GABA type A receptor-associated protein; IHC: immunohistochemistry; MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NCBI: national center for biotechnology information; ORF: open reading frame; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PtdIns3K: class III phosphatidylinositol 3-kinase; RACE-PCR: rapid amplification of cDNA-ends by polymerase chain reaction; RNA: ribonucleic acid; SQSTM1: sequestosome 1; Uniprot: universal protein resource; WIPI: WD repeat domain, phosphoinositide interacting.

Comparative Proteomics of Ostreid Herpesvirus 1 and Pacific Oyster Interactions With Two Families Exhibiting Contrasted Susceptibility to Viral Infection.

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.

Exploring First Interactions Between Ostreid Herpesvirus 1 (OsHV-1) and Its Host, Crassostrea gigas: Effects of Specific Antiviral Antibodies and Dextran Sulfate.

Viral entry mechanisms of herpesviruses constitute a highly complex process which implicates several viral glycoproteins and different receptors on the host cell surfaces. This initial infection stage was currently undescribed for Ostreid herpes virus 1 (OsHV-1), a herpesvirus infecting bivalves including the Pacific oyster, Crassostrea gigas. To identify OsHV-1 glyproteins implicated in the attachment of the virus to oyster cells, three viral putative membrane proteins, encoded by ORF 25, 41, and 72, were selected and polyclonal antibodies against these targets were used to explore first interactions between the virus and host cells. In addition, effects of dextran sulfate, a negative charged sulfated polysaccharide, were investigated on OsHV-1 infection. Effects of antiviral antibodies and dextran sulfate were evaluated by combining viral DNA and RNA detection in spat (in vivo trials) and in oyster hemolymph (in vitro trials). Results showed that viral protein encoded by ORF 25 appeared to be involved in interaction between OsHV-1 and host cells even if other proteins are likely implicated, such as proteins encoded by ORF 72 and ORF 41. Dextran sulfate at 30 µg/mL significantly reduced the spat mortality rate in the experimental conditions. Taken together, these results contribute to better understanding the pathogenesis of the viral infection, especially during the first stage of OsHV-1 infection, and open the way toward new approaches to control OsHV-1 infection in confined facilities.

A study of autophagy in hemocytes of the Pacific oyster, Crassostrea gigas.

Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, Crassostrea gigas. However, the autophagy pathway has never been explored in the hemocytes of C. gigas, which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of C. gigas and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level. Abbreviations: MAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NH4Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy.

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.

Contribution of in Vivo Experimental Challenges to Understanding Flat Oyster Ostrea edulis Resistance to Bonamia ostreae.

Bonamiosis due to the parasite Bonamia ostreae has been associated with massive mortality outbreaks in European flat oyster stocks in Europe. As eradication and treatment are not possible, the control of the disease mainly relies on transfer restriction. Moreover, selection has been applied to produce resistant flat oyster families, which present better survival and lower prevalence than non-selected oysters. In order to better understand the mechanisms involved in resistance to bonamiosis, cellular and molecular responses of 2 oyster groups (selected oysters and wild-type oysters) were analyzed in the context of experimental injection and cohabitation infections. Cellular responses including non-specific esterases detection, ROS production and phagocytosis activity were analyzed by flow cytometry. Four genes homologous to those shown to be involved in immunity were selected (Inhibitor of apotosis OeIAP, Fas ligand OeFas-ligand, Oe-SOD, and OeEc-SOD) and monitored by quantitative reverse-transcription PCR (qRT-PCR). Infected oysters showed higher phagocytosis activity than controls. Infected selected oyster show a lower phagocytosis activity which might be a protection against the parasite infection. The expression of OeIAP and OeFas-ligand gene was significantly increased in selected oysters at 5 days post-injection. OeIAP gene expression appeared to be significantly increased in wild-type oysters at 8 days post-injection. Our results suggest that resistance to bonamiosis partly relies on the ability of the oysters to modulate apoptosis.

Haemocytes from Crassostrea gigas and OsHV-1: A promising in vitro system to study host/virus interactions.

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.

Long dsRNAs promote an anti-viral response in Pacific oyster hampering ostreid herpesvirus 1 replication.

Double-stranded RNA (dsRNA)-mediated genetic interference (RNAi) is a widely used reverse genetic tool for determining the loss-of-function phenotype of a gene. Here, the possible induction of an immune response by long dsRNA was tested in a marine bivalve (Crassostrea gigas), as well as the specific role of the subunit 2 of the nuclear factor κB inhibitor (IκB2). This gene is a candidate of particular interest for functional investigations in the context of oyster mass mortality events, as Cg-IκB2 mRNA levels exhibited significant variation depending on the amount of ostreid herpesvirus 1 (OsHV-1) DNA detected. In the present study, dsRNAs targeting Cg-IκB2 and green fluorescent protein genes were injected in vivo into oysters before being challenged by OsHV-1. Survival appeared close to 100% in both dsRNA-injected conditions associated with a low detection of viral DNA and a low expression of a panel of 39 OsHV-1 genes as compared with infected control. Long dsRNA molecules, both Cg-IκB2- and GFP-dsRNA, may have induced an anti-viral state controlling the OsHV-1 replication and precluding the understanding of the specific role of Cg-IκB2 Immune-related genes including Cg-IκB1, Cg-Rel1, Cg-IFI44, Cg-PKR and Cg-IAP appeared activated in the dsRNA-injected condition, potentially hampering viral replication and thus conferring a better resistance to OsHV-1 infection. We revealed that long dsRNA-mediated genetic interference triggered an anti-viral state in the oyster, emphasizing the need for new reverse genetics tools for assessing immune gene function and avoiding off-target effects in bivalves.

In situ localization and tissue distribution of ostreid herpesvirus 1 proteins in infected Pacific oyster, Crassostrea gigas.

Immunohistochemistry (IHC) assays were conducted on paraffin sections from experimentally infected spat and unchallenged spat produced in hatchery to determine the tissue distribution of three viral proteins within the Pacific oyster, Crassostrea gigas. Polyclonal antibodies were produced from recombinant proteins corresponding to two putative membrane proteins and one putative apoptosis inhibitor encoded by ORF 25, 72, and 87, respectively. Results were then compared to those obtained by in situ hybridization performed on the same individuals, and showed a substantial agreement according to Landis and Koch numeric scale. Positive signals were mainly observed in connective tissue of gills, mantle, adductor muscle, heart, digestive gland, labial palps, and gonads of infected spat. Positive signals were also reported in digestive epithelia. However, few positive signals were also observed in healthy appearing oysters (unchallenged spat) and could be due to virus persistence after a primary infection. Cellular localization of staining seemed to be linked to the function of the viral protein targeted. A nucleus staining was preferentially observed with antibodies targeting the putative apoptosis inhibitor protein whereas a cytoplasmic localization was obtained using antibodies recognizing putative membrane proteins. The detection of viral proteins was often associated with histopathological changes previously reported during OsHV-1 infection by histology and transmission electron microscopy. Within the 6h after viral suspension injection, positive signals were almost at the maximal level with the three antibodies and all studied organs appeared infected at 28h post viral injection. Connective tissue appeared to be a privileged site for OsHV-1 replication even if positive signals were observed in the epithelium cells of different organs which may be interpreted as a hypothetical portal of entry or release for the virus. IHC constitutes a suited method for analyzing the early infection stages of OsHV-1 infection and a useful tool to investigate interactions between OsHV-1 and its host at a protein level.

Detection and distribution of ostreid herpesvirus 1 in experimentally infected Pacific oyster spat.

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

Pesticides and Ostreid Herpesvirus 1 Infection in the Pacific Oyster, Crassostrea gigas.

Since 2008, mass mortality outbreaks have been reported in all French regions producing Pacific oysters, and in several Member States of the European Union. These mass mortality events of Pacific oysters are related to OsHV-1 infection. They occur during spring and summer periods leaving suspect the quality of the marine environment and the role of seasonal use of pesticides associated with the arrival of freshwater in oyster rearing areas. Pesticides have been also detected in French coastal waters, especially in areas of oyster production. Using PMA real-time PCR we showed that a mixture of 14 pesticides has no effect on the integrity of virus capsids from viral suspension in the conditions tested. A contact of oysters with this pesticide mixture was related to higher mortality rates among experimentally infected animals in comparison with control ones (no previous pesticide exposure before experimental infection). We therefore suggest that pesticides at realistic concentration can exert adverse effects on Pacific oysters and causes an increased susceptibility to the viral infection in experimental conditions.

Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas.

An in situ hybridization protocol for detecting mRNAs of ostreid herpesvirus type 1 (OsHV-1) which infects Pacific oysters, Crassostrea gigas, was developed. Three RNA probes were synthesized by cloning three partial OsHV-1 genes into plasmids using three specific primer pairs, and performing a transcription in the presence of digoxigenin dUTP. The RNA probes were able to detect the virus mRNAs in paraffin sections of experimentally infected oysters 26 h post-injection. The in situ hybridization showed that the OsHV-1 mRNAs were mainly present in connective tissues in gills, mantle, adductor muscle, digestive gland and gonads. DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also performed and results were compared with those obtained using RNA probes.

Ostreid herpesvirus type 1 replication and host response in adult Pacific oysters, Crassostrea gigas.

Since 2008, massive mortality outbreaks associated with OsHV-1 detection have been reported in Crassostrea gigas spat and juveniles in several countries. Nevertheless, adult oysters do not demonstrate mortality in the field related to OsHV-1 detection and were thus assumed to be more resistant to viral infection. Determining how virus and adult oyster interact is a major goal in understanding why mortality events are not reported among adult Pacific oysters. Dual transcriptomics of virus-host interactions were explored by real-time PCR in adult oysters after a virus injection. Thirty-nine viral genes and five host genes including MyD88, IFI44, IkB2, IAP and Gly were measured at 0.5, 10, 26, 72 and 144 hours post infection (hpi). No viral RNA among the 39 genes was detected at 144 hpi suggesting the adult oysters are able to inhibit viral replication. Moreover, the IAP gene (oyster gene) shows significant up-regulation in infected adults compared to control adults. This result suggests that over-expression of IAP could be a reaction to OsHV-1 infection, which may induce the apoptotic process. Apoptosis could be a main mechanism involved in disease resistance in adults. Antiviral activity of haemolymph against herpes simplex virus (HSV-1) was not significantly different between infected adults versus control.

First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated.

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

BACKGROUND: Massive mortality outbreaks affecting Pacific oyster (Crassostrea gigas) spat in various countries have been associated with the detection of a herpesvirus called ostreid herpesvirus type 1 (OsHV-1). However, few studies have been performed to understand and follow viral gene expression, as it has been done in vertebrate herpesviruses. In this work, experimental infection trials of C. gigas spat with OsHV-1 were conducted in order to test the susceptibility of several bi-parental oyster families to this virus and to analyze host-pathogen interactions using in vivo transcriptomic approaches. RESULTS: The divergent response of these oyster families in terms of mortality confirmed that susceptibility to OsHV-1 infection has a significant genetic component. Two families with contrasted survival rates were selected. A total of 39 viral genes and five host genes were monitored by real-time PCR. Initial results provided information on (i) the virus cycle of OsHV-1 based on the kinetics of viral DNA replication and transcription and (ii) host defense mechanisms against the virus. CONCLUSIONS: In the two selected families, the detected amounts of viral DNA and RNA were significantly different. This result suggests that Pacific oysters are genetically diverse in terms of their susceptibility to OsHV-1 infection. This contrasted susceptibility was associated with dissimilar host gene expression profiles. Moreover, the present study showed a positive correlation between viral DNA amounts and the level of expression of selected oyster genes.