DAO 44:87-96 (2001)  -  doi:10.3354/dao044087

ABSTRACT: A post-embedding in situ hybridization procedure was developed to detect hepatopancreatic parvovirus (HPV) of penaeid shrimp at the ultrastructural level. The procedure was optimized using sections of resin-embedded hepatopancreas from HPV-infected juvenile Penaeus monodon and postlarval P. chinensis. The hepatopancreata were fixed using various fixatives, dehydrated, and embedded in the hydrophilic resin Unicryl. A 592 bp HPV-specific DNA probe, labeled with DIG-11-dUTP, was tested both on semi-thin and ultra-thin sections and examined by light and electron microscopy, respectively. Hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles and subsequent silver enhancement. Hybridization signal intensities were similar with all fixatives tested, but ultrastructure was best preserved with either 2 or 6% glutaraldehyde. Post-fixation with 1% osmium tetroxide improved ultrastructure but markedly decreased hybridization signal and induced non-specific deposition of gold and silver. Under optimized conditions, this technique was used to successfully follow the development of HPV from absorption and transport through the cytoplansm to nuclear penetration, replication and release by cytolysis. The probe signal was consistently observed among necrotic cell debris within the lumen of hepatopancreatic tubules, within the microvillous border of tubule epithelial cells, within the cytoplasm, and within diagnostic HPV intranuclear inclusion bodies. The nucleolus and karyoplasm of patently infected cells (i.e., showing HPV intranuclear inclusion bodies) were almost devoid of signal. Electron-lucent structures, known as intranuclear bodies, commonly found within the virogenic stroma, showed only weak labeling. This is the first use of in situ hybridization to detect HPV nucleic acids with the electron microscope. The technique should be useful for studying the pathogenesis of HPV.

KEY WORDS: Parvoviridae · HPV · Ultrastructural in situ hybridization · Transmission electron microscopy · Penaeus monodon · Penaeus chinensis