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

J Virol Methods. 2015 Jan:211:43-50. doi: 10.1016/j.jviromet.2014.10.007. Epub 2014 Oct 25.

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.

Keywords: Crassostrea gigas; In situ hybridization; Ostreid herpesvirus type 1; Pacific oyster; Viral infection; mRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animal Structures / virology
  • Animals
  • Crassostrea / virology*
  • Herpesviridae / genetics
  • Herpesviridae / isolation & purification*
  • In Situ Hybridization / methods*
  • Molecular Diagnostic Techniques / methods*
  • Oligonucleotide Probes / genetics
  • RNA, Messenger / analysis*
  • RNA, Messenger / genetics
  • Veterinary Medicine / methods
  • Virology / methods

Substances

  • Oligonucleotide Probes
  • RNA, Messenger