Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

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 Resale or republication not permitted without written consent of the publisherDis Aquat Org 44: [87][88][89][90][91][92][93][94][95][96] 2001 tion methods including histology, in situ hybridization or PCR (Lightner et al. 1993, Pantoja & Lightner 2000.HPV infection in cultured shrimp has been linked to chronic mortalities during the early larval and/or postlarval stages (Lightner et al. 1993, Spann et al. 1997, and it may result in stunted growth during the juvenile stages (Flegel et al. 1992, Limsuwan 1999. The effect of HPV on adult shrimp is unknown but it may compromise their survival if the infection is severe and if the shrimp is in a highly demanding metabolic state (i.e., during gonad maturation) (D.V.L. unpubl).Currently, HPV is considered as a member of the Parvoviridae ; however, its position within the family still remains uncertain. Apparently HPV shares more similarities with the autonomous parvoviruses than with the arthropod-infecting parv...

Detection of Panulirus argus Virus 1 (PaV1) in the Caribbean spiny lobster using fluorescence in situ hybridization (FISH)

“…ISH has been subsequently applied to diagnosis of several crustacean viruses, such as Baculovirus penaei (BP) (Bruce et al 1993(Bruce et al , 1994, white spot syndrome virus (WSSV) (Durand et al 1996, Lo et al 1997, Nunan & Lightner 1997, Chang et al 1998, hepatopancreatic parvovirus (HPV) (Pantoja & Lightner 2001, Phromjai et al 2002 and gill-associated virus (GAV) (Spann et al 2003). ISH has also been applied to the diagnosis of several other pathogens of marine organisms (Stokes & Burreson 1995, Chang et al 1996, Lo et al 1997, Pantoja & Lightner 2001, Carnegie et al 2003, Small et al 2006.…”

Enhanced detection of Rickettsia species in Ixodes pacificus using highly sensitive fluorescence in situ hybridization coupled with Tyramide Signal Amplification

“…Even though this resin has been successfully used in immunocytochemical studies (De Jonge et al, 2005; Takechi et al, 1999), in this study no fluorescent bacteria could be obviously detected using eubacterial EUB338 probes conjugated to FITC or Cy3 or Alexa 568 (data not shown). Unicryl is a single-component resin that has been shown to be an effective embedding medium for in situ hybridization studies (Pantoja and Lightner, 2001). This resin has the added advantage that the same embedded tissue sample can be processed for both FISH and ultrastructural analysis (Pantoja and Lightner, 2001).…”

Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells

“…For Unicryl resin-embedded tissue sections, the hybridization protocol was modified from a protocol that was developed for HPV (Pantoja & Lightner 2001). Specifically, semi-thin sections were first re-hydrated at RT for 10 min each with HPLC water and 1× TNE (50 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 7.4).…”

“…human parechovirus type 1, dengue virus, hepatitis C virus, foot-andmouth disease virus (FMDV), and severe acute respiratory syndrome-associated coronavirus (SARS-CoV), has been characterized using transmission electron microscopy (TEM), in situ hybridization (ISH), and immunoelectron microscopy (IEM; Grief et al 1997, Gosert et al 2003, Krogerus et al 2003, Goldsmith et al 2004, Monaghan et al 2004. Additionally, our laboratory has previously developed an ISH protocol using a specific cDNA probe to follow the intracellular translocation of hepatopancreatic parvovirus (HPV) in penaeid shrimp (Pantoja & Lightner 2001).…”

“…The slides were then mounted with Permount (Fisher Scientific) and examined under a light microscope. The hybridization using only hybridization buffer without the TSV-specific gene probes was also performed on appropriate slides (negative control).For Unicryl resin-embedded tissue sections, the hybridization protocol was modified from a protocol that was developed for HPV (Pantoja & Lightner 2001). Specifically, semi-thin sections were first re-hydrated at RT for 10 min each with HPLC water and 1× TNE (50 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 7.4).…”