%0 Journal Article
%T Demonstration of Hepatitis C Virus RNA with In Situ Hybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver
%A Kazuya Shiogama
%A Ken-ichi Inada
%A Michinori Kohara
%A Hidemi Teramoto
%A Yasuyoshi Mizutani
%A Takanori Onouchi
%A Yutaka Tsutsumi
%J International Journal of Hepatology
%D 2013
%I Hindawi Publishing Corporation
%R 10.1155/2013/249535
%X Background. In situ hybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens. 1. Introduction Hepatitis C virus (HCV) is a single-stranded RNA virus, a member of the Flaviviridae family. Since the first identification of the HCV genome by Choo et al. [1], HCV study has progressed mainly in the field of HCV functional analysis and therapeutic implications. Because of low viral levels in the serum, the diagnosis of HCV infection has been made with a branched chain DNA signal amplification assay and reverse-transcription polymerase chain reaction (RT-PCR) [2¨C4]. The sensitivity of HCV detection in the serum thus became reproducible and clinically relevant. Pathologists are often requested to detect viral pathogens within the routinely prepared biopsy tissue samples, and therefore simple and reliable histochemical techniques are needed. Techniques for visualizing HCV localization within diseased hepatocytes have also been developed. Reports have described the detection of HCV in human liver tissue by using immunohistochemistry and in situ hybridization (ISH) [4¨C14], as well as by in situ RT-PCR [4, 6, 15¨C20]. At the moment, HCV detection with immunohistochemistry and ISH is not yet reproducible enough, we believe. In situ RT-PCR may demonstrate highly sensitive signals, but with concomitant increase of false positivity. Reliable histochemical
%U http://www.hindawi.com/journals/ijh/2013/249535/