Ostreid Herpesvirus Research Articles

Synthesis of the “PLAN DE SAUVEGARDE” using selected all-triploid oysters to reduce the shortage of spat in France due to OsHV-1–associated mortality in Crassostrea gigas

Due to massive mortality of Crassostrea gigas spat in France since 2008, a “plan de sauvegarde” was set up from 2011 to 2014 (hereafter referred to as PS1 to PS4), in order to reduce the shortage of spat. This plan involved the participation of commercial hatcheries, the French Research Institute for Exploitation of the Sea (Ifremer), and the Direction des Pêches Maritimes et de l'Aquaculture (DPMA) of the French Ministry of Agriculture. It was based on selecting diploid lines of C. gigas for their higher resistance to the oyster herpesvirus OsHV-1 (2nR group), and one of these lines was subsequently tetraploidized (4nR group). Both the 2nR and 4nR groups were produced by Ifremer, and then transferred to commercial hatcheries that produced the selected triploids (3nR groups). We report here the mortality rates of the 3nR group for each of the four “plan de sauvegarde” campaigns and compare these with the mortalities of the classic production of commercial hatcheries (both 2n and 3n), benchmarks of selected (2nR group) and unselected (2n-control group) oysters produced by Ifremer, and wild-caught spat, representing a total of 104 diploid and triploids batches. For PS1, the 3nR group had a mean mortality of 67% and did not show any advantage over the 2n- and 3n-commercial groups, suggesting a lack of genetic progress in the 2nR and 4nR groups. For PS2, OsHV-1 resistance was increased in both the 2nR and 4nR groups and, consequently, the 3nR group exhibited a mean mortality of 52%, which was significantly lower than the mortality of the 2n- (87%) and 3n-(76%) commercial groups in 2012. Unfortunately, the mortality of the 3nR group reached 62% and 71% in PS3 and PS4, respectively, although it was expected to be lower than that in PS2. OsHV-1 DNA was quantified in the live oysters at deployment (1356 oysters) and at the endpoint (1171 oysters), as well as in moribund oysters sampled during peak mortality (539 oysters). The results strongly supported the involvement of this pathogen during the main mortality outbreak in May/June. Meanwhile, Vibrio aestuarianus was also suspected to cause unexpected mortality of PS3 oysters in August and September, and it was detected in moribund PS4 oysters during both the mortality events, in May and July. Despite genetic improvement for OsHV-1 resistance, this translated into variable commercial genetic gain. This could be explained by the limited genetic backgrounds of the 2nR and 4nR groups, the reemergence of V. aestuarianus in France since 2012, the changing levels of genetic improvement in both the 3nR group and the commercial groups, as well as the limited broodstock genetic variation where small numbers of males were used. Results on growth and yield are discussed.

Effect of emersion on the mortality of Pacific oysters (Crassostrea gigas) infected with Ostreid herpesvirus-1 (OsHV-1)

Microvariant genotypes of Ostreid herpesvirus-1 (OsHV-1) have been responsible for mass mortalities in farmed Pacific oyster (Crassostrea gigas) populations in Europe, New Zealand and Australia since its first detection in France in 2008. Previous studies conducted in the Georges River estuary, New South Wales, Australia demonstrated a significant protective effect of increased emersion time for adult oysters, when oysters were grown 300 mm above the standard intertidal growing height used by local farmers. The aim of this study was to investigate the protective effect of tidal emersion on infection by OsHV-1 in a controlled laboratory environment. Adult C. gigas (24 months old 70–90 mm) and C. gigas spat (5 months old, 20–40 mm) were infected with OsHV-1 by intramuscular injection and housed with either twice daily emersion or constant immersion. Oysters were monitored daily for gaping of the valves, as an indicator of clinical disease. Contrary to observations in prior field studies, adult oysters subjected to emersion in this study had significantly higher mortalities (67.2%) compared to the constantly immersed adults (11.3%). No significant difference was observed between treatments for the spat. Real-time quantitative PCR confirmed that all mortality was associated with a high concentration of OsHV-1 DNA. Constant immersion appeared to have a protective effect on infected adult oysters, as many gaping oysters recovered. These results suggest that the beneficial effect of high growing height on adult oysters in the field is due to avoidance of infection with OsHV-1, rather than an effect on oyster physiology. Furthermore, it suggests that if infected oysters can be immersed in such a way that predators and secondary infections are avoided, then many may survive the infection.

RNA-seq of HaHV-1-infected abalones reveals a common transcriptional signature of Malacoherpesviruses

Haliotid herpesvirus-1 (HaHV-1) is the viral agent causative of abalone viral ganglioneuritis, a disease that has severely affected gastropod aquaculture. Although limited, the sequence similarity between HaHV-1 and Ostreid herpesvirus-1 supported the assignment of both viruses to Malacoherpesviridae, a Herpesvirales family distantly related with other viruses. In this study, we reported the first transcriptional data of HaHV-1, obtained from an experimental infection of Haliotis diversicolor supertexta. We also sequenced the genome draft of the Chinese HaHV-1 variant isolated in 2003 (HaHV-1-CN2003) by PacBio technology. Analysis of 13 million reads obtained from 3 RNA samples at 60 hours post injection (hpi) allowed the prediction of 51 new ORFs for a total of 117 viral genes and the identification of 207 variations from the reference genome, consisting in 135 Single Nucleotide Polymorphisms (SNPs) and 72 Insertions or Deletions (InDels). The pairing of genomic and transcriptomic data supported the identification of 60 additional SNPs, representing viral transcriptional variability and preferentially grouped in hotspots. The expression analysis of HaHV-1 ORFs revealed one putative secreted protein, two putative capsid proteins and a possible viral capsid protease as the most expressed genes and demonstrated highly synchronized viral expression patterns of the 3 infected animals at 60 hpi. Quantitative reverse transcription data of 37 viral genes supported the burst of viral transcription at 30 and 60 hpi during the 72 hours of the infection experiment, and allowed the distinction between early and late viral genes.

Dual Analysis of Virus-Host Interactions: The Case of Ostreid herpesvirus 1 and the Cupped Oyster Crassostrea gigas

Dual analyses of the interactions between Ostreid herpesvirus 1 (OsHV-1) and the bivalve Crassostrea gigas during infection can unveil events critical to the onset and progression of this viral disease and can provide novel strategies for mitigating and preventing oyster mortality. Among the currently used “omics” technologies, dual transcriptomics (dual RNA-seq) coupled with the analysis of viral DNA in the host tissues has greatly advanced the knowledge of genes and pathways mostly contributing to host defense responses, expression profiles of annotated and unknown OsHV-1 open reading frames (ORFs), and viral genome variability. In addition to dual RNA-seq, proteomics and metabolomics analyses have the potential to add complementary information, needed to understand how a malacoherpesvirus can redirect and exploit the vital processes of its host. This review explores our current knowledge of “omics” technologies in the study of host-pathogen interactions and highlights relevant applications of these fields of expertise to the complex case of C gigas infections by OsHV-1, which currently threaten the mollusk production sector worldwide.

Low pH reduced survival of the oyster Crassostrea gigas exposed to the Ostreid herpesvirus 1 by altering the metabolic response of the host

Environmental change in the marine realm has been accompanied by emerging diseases as new pathogens evolve to take advantage of hosts weakened by environmental stress. Here we investigated how an exposure to reduced seawater pH influenced the response of the oyster Crassostrea gigas to an infection by the Ostreid herpesvirus type I (OsHV-1). Oysters were acclimated at pH 8.1 or pH 7.8 and then exposed to OsHV-1. Their survival was monitored and oyster tissues were sampled for biochemical analyses. The survival of oysters exposed to OsHV-1 at pH 7.8 was lower (33.5%) than that of their counterparts at pH 8.1 (44.8%) whereas levels of OsHV-1 DNA were similar. Energetic reserves, fatty acid composition and prostaglandin levels in oyster did not vary consistently with pH, infection or their interactions. However, there was a reduction in the activities of superoxide dismutase (SOD) and nitric oxide synthase (iNOS) in oysters at low pH, which is associated with the observed difference in survival.

Long-range PCR and high-throughput sequencing of Ostreid herpesvirus 1 indicate high genetic diversity and complex evolution process

Ostreid herpesvirus 1 (OsHV-1) is an important pathogen associated with mass mortalities of cultivated marine mollusks worldwide. Since no cell line allows OsHV-1 replication in vitro, it is difficult to isolate enough high-purity viral DNA for High-Throughput Sequencing (HTS). We developed an efficient approach for the enrichment of OsHV-1 DNA for HTS with long-range PCR. Twenty-three primer pairs were designed to cover 99.3% of the reference genome, and their performances were examined on ten OsHV-1 infected samples. Amplicon mixtures from six successfully amplified samples were sequenced with Illumina platform, and one of them (ZK0118) was also sequenced with the PacBio platform. PacBio reads were assembled into 2 scaffolds compared to 9−68 scaffolds assembled from the Illumina reads. Genomic comparison confirmed high genetic diversity among OsHV-1 variants. Phylogenetic analysis revealed that OsHV-1 evolution was mainly impacted by its host species rather than spatial segregation.

Omics approaches to investigate host–pathogen interactions in mass mortality outbreaks of Crassostrea gigas

AbstractMass mortality events are routinely reported in bivalve cultivation practices worldwide with significant economic consequences for the global aquaculture industry. Major contributors of these bivalve losses are infections caused by viruses and bacteria whose molecular action mechanisms are still not fully understood. This major bottleneck for shellfish cultivation presents an opportunity for fundamental research that can generate practical tools for reliable health assessments. To this end, newly developed omics approaches are likely to facilitate knowledge acquisition, especially if applied in conjunction with established and emergent immunological techniques. In recent years, various omics technologies (e.g. genomics, transcriptomics, proteomics and metabolomics) have been used to study the Pacific oyster mortality events associated with detection of ostreid herpesvirus type 1 (OsHV‐1) and Vibrio bacteria. These new approaches, especially when used in combination have significantly improved our understanding of this syndrome and associated diseases. In this paper, we review the current omics technologies that have been employed to investigate the interactions between Pacific oyster Crassostrea gigas and pathogens (e.g. OsHV‐1 and Vibrio sp.), infection mechanisms, host susceptibility and resistance. Herein, we also highlight some of the current challenges and future directions of omics approaches in shellfish health research, including disease detection, diagnosis and treatment.

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.

Transgenerational plasticity and antiviral immunity in the Pacific oyster (Crassostrea gigas) against Ostreid herpesvirus 1 (OsHV-1)

The oyster's immune system is capable of adapting upon exposure to a pathogen-associated molecular pattern (PAMP) to have an enhanced secondary response against the same type of pathogen. This has been demonstrated using poly(I:C) to elicit an antiviral response in the Pacific oyster (Crassostrea gigas) against Ostreid herpesvirus (OsHV-1). Improved survival following exposure to poly(I:C) has been found in later life stages (within-generational immune priming) and in the next generation (transgenerational immune priming). The mechanism that the oyster uses to transfer immunity to the next generation is unknown. Here we show that oyster larvae have higher survival to OsHV-1 when their mothers, but not their fathers, are exposed to poly(I:C) prior to spawning. RNA-seq provided no evidence to suggest that parental exposure to poly(I:C) reconfigures antiviral gene expression in unchallenged larvae. We conclude that the improved survival of larvae might occur via maternal provisioning of antiviral compounds in the eggs.

Ostreid Herpesvirus-1 Infects Specific Hemocytes in Ark Clam, Scapharca broughtonii.

High levels of ostreid herpesvirus 1 (OsHV-1) were detected in hemocytes of OsHV-1 infected mollusks. Mollusk hemocytes are comprised of different cell types with morphological and functional heterogeneity. Granular cells are considered the main immunocompetent hemocytes. This study aimed to ascertain if OsHV-1 infects specific types of hemocytes in ark clams. Types of hemocytes were first characterized through microexamination and flow cytometry. In addition to a large group of red cells, there were three types of recognizable granular cells in ark clams. Type II granular cells were mostly found with OsHV-1 infection by transmission electron microscope (TEM) examination, and represented the hemocyte type that was susceptible to OsHV-1 infection. The subcellular location of OsHV-1 particles in apoptotic type II granular cells was further analyzed. Some OsHV-1 particles were free inside the apoptotic cells, which may contribute to OsHV-1 transmission among cells in the host, some particles were also found enclosed inside apoptotic bodies. Apoptosis is an important part of the host defense system, but might also be hijacked by OsHV-1 as a strategy to escape host immune attack. Following this investigation, a primary culture of type II granular cells with OsHV-1 infection would facilitate the research on the interaction between OsHV-1 and mollusk hosts.

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

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

Herpes virus OsHV-1 and the protistPerkinsus marinusmodify the expression of the Down syndrome cell adhesion molecule gene in gill and mantle ofCrassostreaspp.

The Down syndrome cell adhesion molecule (Dscam) gene in invertebrates encodes numerous isoforms derived by alternative splicing as a pathogen-specific response. The role of Dscam in immune system responses has been established in fruit fly, mosquito, shrimp, crab, and crayfish, among other arthropods. In molluscs, this gene has not yet been clearly identified, and more importantly, its function has not been elucidated. The present study examined Dscam expression in Crassostrea gigas (CgDscam) and Crassostrea corteziensis (CcDscam) infected with Ostreid herpesvirus type 1 (OsHV-1) and Perkinsus marinus, respectively, through in situ hybridization and quantitative PCR. The consensus coding sequences for CgDscam and CcDscam showed the same organization of domains identified in other taxonomic groups. Furthermore, conserved regions from the signal peptide to Ig1 and from FNIII6 to the cytoplasmic tail were identified. The Dscam transcript was detected in gills and mantles of both species. The hybridization signals were remarkable in infected tissues. QPCR analysis showed higher Dscam expression levels in the infected gills of both species, but in the mantle, no difference in CgDscam expression was observed between control and infected tissues. Moreover, CcDscam expression was inhibited in infected tissue. These results suggest that Dscam could be directly involved in bivalve immune responses.

A novel divergent group of Ostreid herpesvirus 1 μVar variants associated with a mortality event in Pacific oyster spat in Normandy (France) in 2016.

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.

Dual transcriptomic analysis of Ostreid herpesvirus 1 infected Scapharca broughtonii with an emphasis on viral anti-apoptosis activities and host oxidative bursts

The ark shell, Scapharca (Anadara) broughtonii, is an economically important marine shellfish species in Northwestern Pacific. Mass mortalities of ark shell adults related to Ostreid herpesvirus-1 (OsHV-1) infection have occurred frequently since 2012. However, due to the lack of transcriptomic resource of ark shells, the molecular mechanisms underpinning the virus-host interaction remains largely undetermined. In the present study, we resolved the dual transcriptome changes of OsHV-1 infected ark shell with Illumina sequencing. A total of 44 M sequence reads were generated, of which 67,119 reads were mapped to the OsHV-1 genome. De novo assembly of host reads resulted in 276,997 unigenes. 74,529 (26.90%), 47,653 (17.20%) and 19, 611 (7.07%) unigenes were annotated into GO, KOG and KEGG database, respectively. According to RSEM expression values, we identified 2998 differentially expressed genes (DEGs) between control and challenged groups, which included 2065 up-regulated unigenes and 933 down-regulated unigenes. Further analysis of functional pathways indicated that OsHV-1 could inhibit host cell apoptosis mainly by the up-regulation of inhibitor of apoptosis protein (IAP), and thus facilitating its successful replication. While host hemoglobins could induce oxidative burst by suppressing its peroxidase activity, and thus defense against OsHV-1 infection. Although we reported a narrow expression of the OsHV-1 genome compared to Crassostrea gigas infection, we highlighted several common viral genes highly expressed in the two hosts, suggesting an important functional role. This study offers insights into the pathogenesis mechanisms of OsHV-1 infection in bivalve mollusks of the Arcidae family.

The complex interactions of Ostreid herpesvirus 1, Vibrio bacteria, environment and host factors in mass mortality outbreaks of Crassostrea gigas

AbstractFrom the first detection in the Eastern oysters (Crassostrea virginica) in 1972, herpes‐like viruses/herpesviruses have been identified in at least 20 bivalve species and have caused massive mortalities in six cultured species of bivalves. Among them, the Pacific oyster (Crassostrea gigas) is most threatened by massive mortalities associated with detection of Ostreid herpesvirus 1 (OsHV‐1) and its variants. These mortality events have been reported in more than 12 countries, and OsHV‐1 itself has been reported in 15 countries worldwide. Traditionally, the genotype OsHV‐1 (or OsHV‐1 Var) is the main contributor to bivalve mortalities, but the emergence of variants (e.g. OsHV‐1 μVar and related ones, acute viral necrosis virus—AVNV, OsHV‐1‐SB) is now responsible for new mortality outbreaks in Pacific oysters and other bivalve species. In addition to OsHV‐1, Vibrio bacteria and other abiotic and host factors also significantly contribute to mortality outbreaks. To this end, the current contribution aims to synthesize the body of literature on our current knowledge of these mass mortality events to provide insights into the role of each risk factor and complex interactions in the disease process. Specifically, this review encompasses our current knowledge and critical gaps. Important suggestions for future investigation are also presented.

Differential lncRNA expression profiles reveal the potential roles of lncRNAs in antiviral immune response of Crassostrea gigas

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.

Characterisation of the Pacific Oyster Microbiome During a Summer Mortality Event.

The Pacific oyster, Crassostrea gigas, is a key commercial species that is cultivated globally. In recent years, disease outbreaks have heavily impacted C. gigas stocks worldwide, with many losses incurred during summer. A number of infectious agents have been associated with these summer mortality events, including viruses (particularly Ostreid herpesvirus 1, OsHV-1) and bacteria; however, cases where no known aetiological agent can be identified are common. In this study, we examined the microbiome of disease-affected and disease-unaffected C. gigas during a 2013-2014 summer mortality event in Port Stephens (Australia) where known oyster pathogens including OsHV-1 were not detected. The adductor muscle microbiomes of 70 C. gigas samples across 12 study sites in the Port Stephens estuary were characterised using 16S rRNA (V1-V3 region) amplicon sequencing, with the aim of comparing the influence of spatial location and disease state on the oyster microbiome. Spatial location was found to be a significant determinant of the disease-affected oyster microbiome. Furthermore, microbiome comparisons between disease states identified a significant increase in rare operational taxonomic units (OTUs) belonging to Vibrio harveyi and an unidentified member of the Vibrio genus in the disease-affected microbiome. This is indicative of a potential role of Vibrio species in oyster disease and supportive of previous culture-based examination of this mortality event.

Temperature modulate disease susceptibility of the Pacific oyster Crassostrea gigas and virulence of the Ostreid herpesvirus type 1

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.

Validation of housekeeping genes for quantitative mRNA expression analysis in OsHV-1 infected ark clam, Scapharca broughtonii.

Ostreid herpesvirus-1 (OsHV-1) presents interspecies transmission among bivalves. Recently, events of mass mortalities of ark clams (Scapharca broughtonii) infected with OsHV-1 have been recorded. To accurately assess the gene responding patterns of ark clams post OsHV-1 infection, constant stable housekeeping genes (HKGs) are needed as internal control to normalize raw mRNA expression data. In this study, ten candidate HKGs were selected, including 18S rRNA (18S), beta-actin (ACT), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NADH dehydrogenase subunit (NADH), Elongation factor-1a (EF-1a), Elongation factor-1β (EF-1β), Elongation factor-1γ (EF-1γ), Ribosomal protein L7 (RL7), Ribosomal protein L15 (RL15) and Ribosomal protein S18 (S18). The expression levels of ten candidate HKGs were analyzed by real-time PCR under given experimental conditions, including various tissues, OsHV-1 challenge, temperature stress and OsHV-1 challenge at different temperature. Their expression stability values were further calculated using two different statistical models (geNorm and NormFinder). The results showed that different tissues presented distinct best pair genes combinations for gene expression analysis under OsHV-1 challenge. RL15 was comparatively more stable than other HKGs under various experimental conditions, while commonly used 18s and ACT seemed to be more greatly influenced by most given experimental conditions in ark clams. This study emphasized the necessity of prior validation of HKGs and would facilitate future gene expression analysis in ark clams or other shellfishes.

Counting the dead to determine the source and transmission of the marine herpesvirus OsHV-1 in Crassostrea gigas

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.