Abstract
Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.
Highlights
During the last decade, mass mortality outbreaks of young Pacific oysters, Crassostrea gigas, have been regularly reported each year since 2008 in France and throughout the world, in association with the detection of particular genotypic variants of ostreid herpesvirus type 1 (OsHV-1)
Mortality monitoring and DNA quantification in oyster mantle and haemocytes Oyster mortality was first observed at 48hpi and increased gradually from 48hpi to 96hpi for the two batches, regardless of the animal age (Fig 1)
No viral DNA was present in the mantle of NSI 01/15 oysters injected with ASW whereas OsHV-1 DNA was detected below the quantification limit of the real time PCR in the mantle and the haemolymph of J6 oysters injected with artificial seawater
Summary
Mass mortality outbreaks of young Pacific oysters, Crassostrea gigas, have been regularly reported each year since 2008 in France and throughout the world, in association with the detection of particular genotypic variants of ostreid herpesvirus type 1 (OsHV-1). This virus is one of the major pathogens affecting C. gigas and constitutes the type species within the genus Ostreavirus and the Malacoherpesviridae family [1,2,3]. A homolog to the anti-apoptotic protein, B-cell lymphoma 2 (Bcl-2), involved in the intrinsic apoptosis pathway, was reported from Pacific oyster haemocytes exposed to OsHV-1 during in vitro assays [33] and in the Mediterranean mussels, Mytilus galloprovincialis [44]
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