Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis

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.

Fig 1. Cumulative mortality of Pacific oysters (NSI 01/15 and J6) experimentally infected by OsHV-1 or injected with sterilized artificial sea water (ASW) during one week (168h).

Fig 2. Viral DNA quantification in haemolymph and in the mantle of oysters (five animals per sampling time) infected by OsHV-1 at 2, 24, and 48 hours post injection (hpi).

Fig 3. OsHV-1 transcript detection in haemocytes from oysters injected with virus or artificial seawater (ASW) at 2, 24, and 48 hours post injection (hpi).

Fig 4. Double immunofluorescence assay using antibodies against a viral putative apoptosis inhibitor (FITC) and actin (rhodamin) in haemocytes from OsHV-1 infected and control oysters at 2, 24, and 48hpi.

Bar = 10μM.

Fig 5

Transmission electron micrograph of haemocytes or fibroblast-like cells from OsHV-1 infected oysters at 24h (A) and 48h (B) post viral injection in the adductor muscle. Arrows indicate viral nucleo-capsids or empty viral capsids.

Fig 6

Percentages of early apoptotic (A), late apoptotic/secondary necrotic (B), and primary necrotic/dead cells (C) in haemocytes from OsHV-1 infected (virus) and control oysters (ASW) at 2, 24, and 48hpi. **P<0.01, ***P<0.001. Bar = 5μM. Each apoptosis stage was illustrated by fluorescence microscopy figures (dapi = cell nucleus).

Fig 7. Detection of cells with fragmented DNA in haemocytes from OsHV-1 infected and control oysters by TUNEL assay.

Percentages of cells with fragmented DNA in haemocytes from OsHV-1 infected (virus) and control oysters (ASW) at 2, 24, and 48hpi.

Fig 8. Detection of cells with fragmented DNA in haemocytes by epifluorescence microscope (FITC) from OsHV-1 infected and control oysters (TUNEL assay).

Negative control consisted in slides without the Terminal deoxynucleotidyl transferase (Tdt) and positive control corresponded to cells treated with the DNase.

Fig 9. Relative expression of five apoptosic genes in haemoctes from OsHV-1 infected (virus) and control oysters (ASW): B-cell lymphoma 2, caspase 3, tumor necrosis factor 2, tumor necrosis factor receptor, baculoviral IAP repeat-containing protein 2.

Fig 10. OsHV-1 detection in haemocytes from experimentally infected oysters in relation with inhibition of apoptosis.