Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells

In situ hybridization was performed using cRNA probes on human liver biopsies to localize both positive and negative RNA strands of hepatitis C virus. From the 5′ non-coding region of the viral genome, 210 bp, were amplified by reverse transcriptase-polymerase chain reaction and cloned in a plasmid....

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Bibliographic Details
Published in:Journal of hepatology 1995-11, Vol.23 (5), p.509-518
Main Authors: Gastaldi, Marguerite, Massacrier, Annick, Planells, Richard, Robaglia-Schlupp, Andrée, Portal-Bartolomei, Isabelle, Bourlière, Marc, Quilici, François, Fiteni, Joëlle, Mazzella, Evelyne, Cau, Pierre
Format: Article
Language:English
Subjects:
Biological and medical sciences
Biopsy
Chronic hepatitis C
Digoxigenin
Digoxigenin-labeled cRNA probes
Electron microscopy
Hepacivirus - isolation & purification
Hepatitis C virus
Human liver
Human viral diseases
Humans
Image Processing, Computer-Assisted
In Situ Hybridization
Infectious diseases
Liver - pathology
Liver - virology
Medical sciences
Microscopy, Electron
Positive and negative strands detection
RNA, Antisense - isolation & purification
RNA, Complementary
RNA, Viral - isolation & purification
Viral diseases
Viral hepatitis
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Summary:In situ hybridization was performed using cRNA probes on human liver biopsies to localize both positive and negative RNA strands of hepatitis C virus. From the 5′ non-coding region of the viral genome, 210 bp, were amplified by reverse transcriptase-polymerase chain reaction and cloned in a plasmid. Probes were produced by in vitro transcription, and labeled using digoxigenin-11-UTP. Positive HCV-RNA strands were detected in all 20 of the patients analyzed, whereas negative strands were detected in only nine patients, as confirmed using computerized image analysis. Both probes labeled the cytoplasm of hepatocytes with a perinuclear intensification. Few of the mononuclear cells infiltrating the portal connective space contained positive HCV-RNA strands only. Stacks of dilated endoplasmic reticulum cisternae were observed by electron microscopy and their relationship with the infection was discussed. This study confirmed that non-radioactive in situ hybridization represents a useful tool to analyze the localization and replication of hepatitis C virus in liver tissue.
ISSN:0168-8278
1600-0641
DOI:10.1016/0168-8278(95)80055-7