Demonstration and distribution of HCV RNA sequences by in situ hybridization and HCV-related proteins by immunohistochemistry in the liver tissue of patients with chronic HCV infection

Nonisotopic in situ cytohybridization of HCV RNA was attempted in liver specimens from 12 chronically hepatitis C virus (HCV) infected patients. Oligonucleotides deduced from 5'-noncoding and core regions of the HCV genome were labeled with digoxigenin and used on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline phosphatase-conjugated anti-digoxigenin and alkaline phosphatase substrate. These findings were correlated with the results of tissue immunohistochemistry for HCV antigens identified with specific mouse monoclonal antibodies developed against c22-3 antigen (Ag), a core-encoded protein, and c100-3 Ag, a NS4-encoded protein, and histologic assessment of each liver. HCV RNA detected in the above assay was predominantly cytoplasmic; it was detected in all 12 patients and in none of the controls. Tissue HCV RNA was associated with the presence of cytoplasmic (c100-3 Ag) and membrane (c22-3 Ag) expression of viral proteins in all 9 patients with histological evidence of chronic progressive liver disease as judged by the presence of piecemeal necrosis, and lobular and portal tract inflammation. Despite the presence of abundant HCV RNA, none of 3 patients without histological evidence of chronic liver disease showed intrahepatocyte expression of viral proteins. These findings support the view that tissue HCV antigens are markers of progressive damage and demonstrate that active liver disease does not occur without such markers. It is proposed that synthesis of viral proteins and membrane accumulation of c22-3 Ag may be involved in the pathogenesis of hepatocyte injury in chronic hepatitis C infection.