title: Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas.
abstract: An in situ hybridization protocol for detecting mRNAs of ostreid herpesvirus type 1 (OsHV-1) which infects Pacific oysters, Crassostrea gigas, was developed. Three RNA probes were synthesized by cloning three partial OsHV-1 genes into plasmids using three specific primer pairs, and performing a transcription in the presence of digoxigenin dUTP. The RNA probes were able to detect the virus mRNAs in paraffin sections of experimentally infected oysters 26 h post-injection. The in situ hybridization showed that the OsHV-1 mRNAs were mainly present in connective tissues in gills, mantle, adductor muscle, digestive gland and gonads. DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also performed and results were compared with those obtained using RNA probes.
authors.affiliation: Australian Animal Health Laboratory, CSIRO Animal Food Health Sciences, Geelong, Victoria 3220, Australia. Electronic address: Serge.Corbeil@csiro.au.
authors.forename: Serge
authors.initials: S
authors.lastname: Corbeil
authors.affiliation: Ifremer, Unité Santé Génétique et Microbiologie des Mollusques (SG2M), Laboratoire de Génétique et Pathologie des Mollusques Marins (LGPMM), 17390 La Tremblade, France. Electronic address: nfaury@ifremer.fr.
authors.forename: Nicole
authors.initials: N
authors.lastname: Faury
authors.affiliation: Ifremer, Unité Santé Génétique et Microbiologie des Mollusques (SG2M), Laboratoire de Génétique et Pathologie des Mollusques Marins (LGPMM), 17390 La Tremblade, France. Electronic address: asegarra@ifremer.fr.
authors.forename: Amélie
authors.initials: A
authors.lastname: Segarra
authors.affiliation: Ifremer, Unité Santé Génétique et Microbiologie des Mollusques (SG2M), Laboratoire de Génétique et Pathologie des Mollusques Marins (LGPMM), 17390 La Tremblade, France. Electronic address: trenault@ifremer.fr.
authors.forename: Tristan
authors.initials: T
authors.lastname: Renault
mesh_terms: D000825:Animal Structures / Q000821:virology; D000818:Animals; D049896:Crassostrea / Q000821:virology*; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D017403:In Situ Hybridization / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014730:Veterinary Medicine / Q000379:methods; D014773:Virology / Q000379:methods
chemical_list: D015345:Oligonucleotide Probes; D012333:RNA, Messenger
keywords: Crassostrea gigas; In situ hybridization; Ostreid herpesvirus type 1; Pacific oyster; Viral infection; mRNA