Demonstration of Hepatitis C Virus RNA withIn SituHybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

Abstract

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.

Artificial Nucleic Acid Probes and Their Applications in Clinical Microbiology

“…In addition, and unlike natural NA probes, when these probes are to be introduced into live cells, they are highly resistant to enzymatic degradation and can be further modified as needed to increase cellular uptake (Amann & Fuchs, 2008). Unlike microarray technology, there has been an increased interest in developing FISH assays for the use in clinical microbiology particularly targeted to pathogen identification whereas the majority of research work with LNAs has been directed at the detection of human miRNA and mRNA (Doné & Beltcheva, 2014;Nielsen, 2012;Singh et al, 2014) or viral targets (Cerqueira et al, 2008;Robertson, Verhoeven, Thach, & Chang, 2010;Shiogama et al, 2013).…”

Going full circle: Validation of P-body dispersion in hepatitis C virus-infected patients

“…A major hurdle for such in vivo studies is the low abundance of HCV antigen and RNA in infected cells. With respect to the detection of HCV RNA in infected tissues, various techniques have been developed such as in situ hybridization or in situ reversetranscription polymerase chain reaction ( [17] and references cited therein). Detection of viral antigens was first achieved by conventional immunohistochemistry or fluorescence microscopy ( [15] and references cited therein).…”