First detection of Ostreid herpesvirus 1 in wild Crassostrea gigas in Argentina.

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.

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.

Genetic parameters of resistance to Vibrio aestuarianus, and OsHV-1 infections in the Pacific oyster, Crassostrea gigas, at three different life stages.

BACKGROUND: In France, two main diseases threaten Pacific oyster production. Since 2008, Crassostrea gigas spat have suffered massive losses due to the ostreid herpesvirus OsHV-1, and since 2012, significant mortalities in commercial-size adults have been related to infection by the bacterium Vibrio aestuarianus. The genetic basis for resistance to V. aestuarianus and OsHV-1 and the nature of the genetic correlation between these two traits were investigated by using 20 half-sib sire families, each containing two full-sib families. For each disease, controlled infectious challenges were conducted using naïve oysters that were 3 to 26 months old. In addition, siblings were tested under field, pond and raceway conditions to determine whether laboratory trials reflected mortality events that occur in the oyster industry. RESULTS: First, we estimated the genetic basis of resistance to V. aestuarianus in C. gigas. Susceptibility to the infection was low for oysters in spat stage but increased with later life stages. Second, we confirmed a strong genetic basis of resistance to OsHV-1 infection at early stages and demonstrated that it was also strong at later stages. Most families had increased resistance to OsHV-1 infection from the spat to adult stages, while others consistently showed low or high mortality rates related to OsHV-1 infection, regardless of the life stage. Our third main finding was the absence of genetic correlations between resistance to OsHV-1 infection and resistance to V. aestuarianus infection. CONCLUSIONS: Selective breeding to enhance resistance to OsHV-1 infection could be achieved through selective breeding at early stages and would not affect resistance to V. aestuarianus infection. However, our results suggest that the potential to select for improved resistance to V. aestuarianus is lower. Selection for dual resistance to OsHV-1 and V. aestuarianus infection in C. gigas might reduce the impact of these two major diseases by selecting families that have the highest breeding values for resistance to both diseases.

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.

Viruses infecting marine molluscs.

Although a wide range of viruses have been reported in marine molluscs, most of these reports rely on ultrastructural examination and few of these viruses have been fully characterized. The lack of marine mollusc cell lines restricts virus isolation capacities and subsequent characterization works. Our current knowledge is mostly restricted to viruses affecting farmed species such as oysters Crassostrea gigas, abalone Haliotis diversicolor supertexta or the scallop Chlamys farreri. Molecular approaches which are needed to identify virus affiliation have been carried out for a small number of viruses, most of them belonging to the Herpesviridae and birnaviridae families. These last years, the use of New Generation Sequencing approach has allowed increasing the number of sequenced viral genomes and has improved our capacity to investigate the diversity of viruses infecting marine molluscs. This new information has in turn allowed designing more efficient diagnostic tools. Moreover, the development of experimental infection protocols has answered some questions regarding the pathogenesis of these viruses and their interactions with their hosts. Control and management of viral diseases in molluscs mostly involve active surveillance, implementation of effective bio security measures and development of breeding programs. However factors triggering pathogen development and the life cycle and status of the viruses outside their mollusc hosts still need further investigations.

Flat oyster follows the apoptosis pathway to defend against the protozoan parasite Bonamia ostreae.

The in vitro model Ostrea edulis hemocyte - Bonamia ostreae is interesting to investigate host-parasite interactions at the cellular level. Indeed, this unicellular parasite infects the flat oyster Ostrea edulis and multiplies within hemocytes, the central effectors of oyster defenses. Apoptosis is a mechanism used by many organisms to eliminate infected cells. In order to study the potential involvement of this mechanism in the oyster response to B. ostreae, in vitro experiments were carried out by exposing hemocytes from the naturally susceptible oyster O. edulis and a resistant oyster species Crassostrea gigas to live and heat-inactivated parasites. Hemocyte apoptotic response was measured using a combination of flow cytometry and microscopy analyses. Whatever the host species was, the parasite was engulfed in hemocytes and induced an increase of apoptotic parameters including intracytoplasmic calcium concentration, mitochondrial membrane potential or phosphatidyl-serine externalization as well as ultrastructural modifications. However, the parasite appears more able to infect flat oyster than cupped oyster hemocytes and the apoptotic response was more important against live than dead parasites in the natural host than in C. gigas. Our results suggest that O. edulis specifically responds to B. ostreae by inducing apoptosis of hemocytes.

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.

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.

Induction of apoptosis by UV in the flat oyster, Ostrea edulis.

Apoptosis is a fundamental feature in the development of many organisms and tissue systems. It is also a mechanism of host defense against environmental stress factors or pathogens by contributing to the elimination of infected cells. Hemocytes play a key role in defense mechanisms in invertebrates and previous studies have shown that physical or chemical stress can increase apoptosis in hemocytes in mollusks. However this phenomenon has rarely been investigated in bivalves especially in the flat oyster Ostrea edulis. The apoptotic response of hemocytes from flat oysters, O. edulis, was investigated after exposure to UV and dexamethasone, two agents known to induce apoptosis in vertebrates. Flow cytometry and microscopy were combined to demonstrate that apoptosis occurs in flat oyster hemocytes. Investigated parameters like intracytoplasmic calcium activity, mitochondrial membrane potential and phosphatidyl-serine externalization were significantly modulated in cells exposed to UV whereas dexamethasone only induced an increase of DNA fragmentation. Morphological changes were also observed on UV-treated cells using fluorescence microscopy and transmission electron microscopy. Our results confirm the apoptotic effect of UV on hemocytes of O. edulis and suggest that apoptosis is an important mechanism developed by the flat oyster against stress factors.

Mass mortality in bivalves and the intricate case of the Pacific oyster, Crassostrea gigas.

Massive mortality outbreaks in cultured bivalves have been reported worldwide and they have been associated with infection by a range of viral and bacterial pathogens. Due to their economic and social impact, these episodes constitute a particularly sensitive issue in Pacific oyster (Crassostrea gigas) production. Since 2008, mortality outbreaks affecting C. gigas have increased in terms of intensity and geographic distribution. Epidemiologic surveys have lead to the incrimination of pathogens, specifically OsHV-1 and bacteria of the Vibrio genus, in particular Vibrio aestuarianus. Pathogen diversity may partially account for the variability in the outcome of infections. Host factors (age, reproductive status...) including their genetic background that has an impact on host susceptibility toward infection, also play a role herein. Finally, environmental factors have significant effects on the pathogens themselves, on the host and on the host-pathogen interaction. Further knowledge on pathogen diversity, classification, and spread, may contribute toward a better understanding of this issue and potential ways to mitigate the impact of these outbreaks.

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.

Ostreid herpesvirus 1 infection among Pacific oyster (Crassostrea gigas) Spat: relevance of water temperature to virus replication and circulation prior to the onset of mortality.

A number of bivalve species worldwide, including the Pacific oyster, Crassostrea gigas, have been affected by mass mortality events associated with herpesviruses, resulting in significant losses. A particular herpesvirus was purified from naturally infected larval Pacific oysters, and its genome was completely sequenced. This virus has been classified as Ostreid herpesvirus 1 (OsHV-1) within the family Malacoherpesviridae. Since 2008, mass mortality outbreaks among C. gigas in Europe have been related to the detection of a variant of OsHV-1 called µVar. Additional data are necessary to better describe mortality events in relation to environmental-parameter fluctuations and OsHV-1 detection. For this purpose, a single batch of Pacific oyster spat was deployed in 4 different locations in the Marennes-Oleron area (France): an oyster pond ("claire"), a shellfish nursery, and two locations in the field. Mortality rates were recorded based on regular observation, and samples were collected to search for and quantify OsHV-1 DNA by real-time PCR. Although similar massive mortality rates were reported at the 4 sites, mortality was detected earlier in the pond and in the nursery than at both field sites. This difference may be related to earlier increases in water temperature. Mass mortality was observed among oysters a few days after increases in the number of PCR-positive oysters and viral-DNA amounts were recorded. An initial increment in the number of PCR-positive oysters was reported at both field sites during the survey in the absence of significant mortality. During this period, the water temperature was below 16°C.

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.

Genotyping of a microsatellite locus to differentiate clinical Ostreid herpesvirus 1 specimens.

Ostreid herpesvirus 1 (OsHV-1) is a DNA virus belonging to the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 has been associated with mortality outbreaks in different bivalve species including the Pacific cupped oyster, Crassostrea gigas. Since 2008, massive mortality events have been reported among C. gigas in Europe in relation to the detection of a variant of OsHV-1, called µVar. Since 2009, this variant has been mainly detected in France. These results raise questions about the emergence and the virulence of this variant. The search for association between specific virus genetic markers and clinical symptoms is of great interest and the characterization of the genetic variability of OsHV-1 specimens is an area of growing interest. Determination of nucleotide sequences of PCR-amplified virus DNA fragments has already been used to characterize OsHV-1 specimens and virus variants have thus been described. However, the virus DNA sequencing approach is time-consuming in the high-scale format. Identification and genotyping of highly polymorphic microsatellite loci appear as a suitable approach. The main objective of the present study was the development of a genotyping method in order to characterise clinical OsHV-1 specimens by targeting a particular microsatellite locus located in the ORF4 area. Genotyping results were compared to sequences already available. An excellent correlation was found between the detected genotypes and the corresponding sequences showing that the genotyping approach allowed an accuraté discrimination between virus specimens.

Transcriptomic study of 39 ostreid herpesvirus 1 genes during an experimental infection.

Massive mortality outbreaks have been reported in France since 2008 among Pacific oysters, Crassostrea gigas, with the detection of a particular OsHV-1 variant called µVar. Virus infection can be induced in healthy spat in experimental conditions allowing to better understand the disease process, including viral gene expression. Although gene expression of other herpesviruses has been widely studied, we provide the first study following viral gene expression of OsHV-1 over time. In this context, an in vivo transcriptomic study targeting 39 OsHV-1 genes was carried out during an experimental infection of Pacific oyster spat. For the first time, several OsHV-1 mRNAs were detected by real-time PCR at 0 h, 2 h, 4 h, 18 h, 26 h and 42 h post-injection. Several transcripts were detected at 2h post-infection and at 18 h post-infection for all selected ORFs. Quantification of virus gene expression at different times of infection was also carried out using an oyster housekeeping gene, Elongation factor. Developing an OsHV-1-specific reverse transcriptase real time PCR targeting 39 viral gene appears a new tool in terms of diagnosis and can be used to complement viral DNA detection in order to monitor viral replication.

First description of French V. tubiashii strains pathogenic to mollusk: I. Characterization of isolates and detection during mortality events.

Nine dominant bacterial isolates were obtained from different batches of Crassostrea gigas spat experiencing high mortality rates in a French experimental hatchery/nursery in 2007. Using phenotypic analysis combined with multilocus sequence analysis, the isolates were shown to be genetically close to the Vibrio tubiashii type strain. Based on (1) analyses of the recA gene sequences; (2) the results of DNA-DNA hybridization assays between 07/118 T2 (LMG 27884=CECT 8426), which is a representative strain, and the V. tubiashii type strain (69%); and (3) phenotypic traits, the bacteria were classified in a group close to American V. tubiashii strain. Its virulence (70% of mortalities) and the toxicity of the extracellular products of 07/118 T2 was demonstrated (41% of mortalities). Moreover, a QPCR diagnostic tool targeting the gyrB gene was developed to investigate the epidemiological significance of V. tubiashii in French oyster mortality outbreaks recorded by the national surveillance network. Of the 21 batches originating from hatcheries, only two were positive, whereas V. tubiashii DNA could not be detected in any of the batches of moribund animals collected in field/outdoor facilities. These results demonstrate the existence of a group of virulent V. tubiashii in France that episodically infect C. gigas.

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.

Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas.

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

Complete genome sequence of acute viral necrosis virus associated with massive mortality outbreaks in the Chinese scallop, Chlamys farreri.

BACKGROUND: Acute viral necrosis virus (AVNV) is the causative agent of a serious disease resulting in high mortality in cultured Chinese scallops, Chlamys farreri. We have sequenced and analyzed the complete genome of AVNV. RESULTS: The AVNV genome is a linear, double-stranded DNA molecule of 210,993 bp with a nucleotide composition of 38.5% G + C. A total of 123 open reading frames were predicted to encode functional proteins, ranging from 41 to 1,878 amino acid residues. The DNA sequence of AVNV is 97% identical to that of ostreid herpesvirus 1 (OsHV-1), and the amino acid sequences of the encoded proteins of these two viruses are 94-100% identical. The genomic organization of AVNV is similar to that of OsHV-1, and consists of two unique regions (170.4 kb and 3.4 kb, respectively), each flanked by two inverted repeats (7.6 kb and 10.2 kb, respectively), with a third unique region (1.5 kb) situated between the two internal repeats. CONCLUSIONS: Our results indicate that AVNV is a variant of OsHV-1. The AVNV genome sequence provides information useful for understanding the evolution and divergence of OsHV-1 in marine molluscs.

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.