Ultrastructural Localization of Human Papillomavirus Sequences by In Situ Hybridization and Image Analysis

Abstract

Genital infection caused by human papillomavirus (HPV) is of great sanitary importance because of the role it can play in the development of squamous intraepithelial lesions and carcinomas. Because not all lesions have a malignant transformation, it is important that clinical and biological parameters contributing to the evolution of a specific lesion be determined.We used CaSki cells with 400–600 integrated copies of HPV-16 genome for in situ hybridization (ISH) with electron microscopy (EM) to locate HPV sequences. Three biotinylated DNA probes for HPV types 6/11, 16/18 and 31/ 33/51 were used, and hybrid detection was done with an anti-biotin antibody conjugated with 15 nm gold colloids. Electron microphotographs with positive labeling were studied by image analysis to skeletonize them to highlight the heterochromatin fibers and to determinate the spatial localization of colloidal-gold particles. Electron microscopy and ISH technique revealed 2 different distribution patterns: colloidal-gold particles spread over the whole nuclear surface, individually or in small clusters, and large accumulations with many gold particles in the nuclear periphery, next to the membrane. Image analysis revealed there was a spatial association between the gold particles and heterochromatin fibers in all cases.We believe this association is the consequence of viral genome integration. With these results and knowledge that integration of viral DNA is linked to a worse prognosis, we propose the application of this methodology for the future study of HPV infected biopsies to obtain prognostic information in lesions caused by this virus. (The J Histotechnol 21: 295–298, 1998)