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

Abstract

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.

Ecologically realistic model of infection for exploring the host damage caused by Vibrio aestuarianus

“…F–H). Even though each of these lesions cannot be defined as specific to this disease (Couch, ; Knowles et al ., ; Martenot et al ., ), the presence of all of them in field oysters is generally noticed in V. aestuarianus ‐infected animals sampled during surveillance programs (REPAMO Network, Céline Garcia, Personal Communication) and should constitute a part of a diagnostic method.…”

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

“…However, the detection of low viral DNA amounts (below the quantifiable limit) and viral RNA in some oysters of the J6 batch injected with ASW, suggests that these oysters may have been previously exposed to OsHV-1. This is a recurrent issue and this detection may correspond to either a persistent or recently acquired infection [31,35,39,56,57]. In addition, the hypothesis that viral replication occurred in haemocytes was suggested by the detection of OsHV-1 transcripts and proteins in these cells from OsHV-1 infected animals at 24 and 48hpi.…”

“…Polyclonal antibody targeting a putative apoptosis inhibitor encoded by ORF 87 was selected among three antibodies already used in immunohistochemistry (IHC) to detect OsHV-1 proteins, since satisfactory staining was obtained [57]. Antibody was produced by ProteoGenix (Schiltigheim, France) at 1mg/mL.…”

“…This antibody was previously used by Martenot et al . [57] to describe the localization and the tissue distribution of OsHV-1 proteins during an experimental infection by the virus.…”

“…Antibodies were treated using proteins extracted from non-challenged oysters (considered as non-infected animals) to reduce non-specific epitope interactions [57]. Briefly, antibodies (15μL) were incubated with 110μL of proteins extracted from non-challenged oysters (20mg/mL) and 300μL of Tris-Buffered Saline with additional 0.1% Tween 20 (TBST) under gentle stirring at 4°C overnight.…”