Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas

• An in situ hybridization protocol for detecting OsHV-1 mRNAs in paraffin sections of experimentally infected Pacific oysters was developed.
• Three RNA probes were synthesized targeting viral transcripts from ORF7, ORF25 and ORF87.
• OsHV-1 mRNAs were mainly detected in connective tissues of gills, mantle, adductor muscle, digestive gland and gonads of infected oysters.
• DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also carried out.

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.