Assessment of genotype and molecular evolution of hepatitis C virus in formalin-fixed paraffin-embedded liver tissue from patients with chronic hepatitis C virus infection

Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liv er tissue have satisfactorily been solved by RT-PCR amplification of the 5' non-coding region (5'NCR). However, detection of this highly conserved regi on does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from par affin-embedded liver specimens by RT-PGR, and whether the amplified materia l is suitable for further molecular analyses. RT-PCR amplification of 5'NCR , HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, par affin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 19 85, 8 from subjects with normal liver histology and 4 from sequential biops ies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1 996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5'NCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documen tation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5'NCR in the 13 cases analyzed. HVR -1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive f or the 5'NCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analy zed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence af ter transplantation. These observations indicate that paraffin-embedded liv er tissue, even when stored for more than 20 years, is appropriate for adva nced studies on the molecular biology of HCV.