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 b iopsies to localize both positive and negative RNA strands of hepatiti s 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, a nd labeled using digoxigenin-ll-UTP. Positive HCV-RNA strands were det ected in all 20 of the patients analyzed, whereas negative strands wer e detected in only nine patients, as confirmed using computerized imag e analysis. Both probes labeled the cytoplasm of hepatocytes with a pe rinuclear intensification. Few of the mononuclear cells infiltrating t he portal connective space contained positive HCV-RNA strands only, St acks of dilated endoplasmic reticulum cisternae were observed by elect ron microscopy and their relationship with the infection was discussed , This study confirmed that non-radioactive in situ hybridization repr esents a useful tool to analyze the localization and replication of he patitis C virus in liver tissue. (C) Journal of Hepatology.