CULTIVATION OF HEPATITIS-C VIRUS IN PRIMARY HEPATOCYTE CULTURE FROM PATIENTS WITH CHRONIC HEPATITIS-C RESULTS IN RELEASE OF HIGH-TITER INFECTIOUS VIRUS

To investigate the viral replication cycle and genomic heterogeneity o f hepatitis C virus (HCV), we established an HCV cultivation system by using a primary hepatocyte culture from patients with chronic hepatit is C. Liver tissue was obtained by needle biopsy or surgery, then hepa tocytes were isolated by collagenase digestion. After several weeks, w e determined the HCV RNA titre of the cultured cells and supernatant b y a competitive polymerase chain reaction (PCR) method. A significant amount of HCV RNA was observed in the cells and supernatant during cul tivation. Negative-strand RNA, regarded as a marker of viral replicati on, could be detected by a strand-specific reverse transcription PCR m ethod and the HCV core protein could be detected by immunofluorescence microscopy. Many HCV particles released into the supernatant were inf ectious. In addition, we compared the nucleotide sequences in the E2/N S1 region of pre- and post-cultivation hepatocytes for 8 weeks. At the beginning of the culture period, three major HCV types containing two subtypes were isolated. Following cultivation, the same types were is olated from the cultured hepatocytes in the same ratio as prior to cul tivation. We could detect the same clones in this patient's serum, but in vivo We observed genetic variability over a 6 month interval. One clone detected throughout the 6 month period mutated extensively in th e hypervariable region. These results indicated that HCV can replicate in cultured hepatocytes, and that infectious virions are released int o the supernatant, This cultivation system should facilitate the study of HCV genomic heterogeneity, infection and replication.