Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon by in-situ hybridization at transmission electron microscopic level

Kanokporn Chayaburakul; Gun Anantasomboon; Benjamart Pratoomthai; Boonsirm Withyachumnarnkul

Authors

Kanokporn Chayaburakul Anatomy Unit, Faculty of Science, Rangsit University, Pathumthani 12000, Thailand
Gun Anantasomboon Anatomy Unit, Faculty of Science, Rangsit University, Pathumthani 12000, Thailand
Benjamart Pratoomthai Anatomy Unit, Faculty of Science, Rangsit University, Pathumthani 12000, Thailand
Boonsirm Withyachumnarnkul Centex Shrimp and Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

Keywords:

IHHNV, in-situ hybridization for TEM, Penaeus monodon, virus tracking

Abstract

In-situ hybridization procedure was developed to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV) of infected juvenile Penaeus monodon at the ultrastructural level. The tissues were fixed, dehydrated, and embedded in Unicryl^TM hydrophilic resin. A 515 bp IHHNV-specific DNA probe, labeled with DIG-11-dUTP, was tested on both semi-thin and ultra-thin sections, and the sections were examined under light and transmission-electron microscopes, respectively. The hybridized probe was detected by means of an anti-DIG antibody conjugated to 10 nm gold particles, followed by silver enhancement. This technique allows IHHNV to be tracked in the infected cell, from entry into the cytoplasm, nuclear penetration, through replication in the nucleus, and release from the cell by exocytosis. This report is the first description of in-situ hybridization to detect IHHNV at the ultrastructural level. The result showed positive in situ hybridization of IHHNV-infected shrimp tissue at TEM in nucleolus and cytoplasm.

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