Anin situ hybridization, molecular biological and immunohistochemical study of hepatitis delta virus in woodchucks

The presence of hepatitis delta virus genomic RNA and hepatitis delta antigen was investigated in woodchuck liver and extrahepatic tissues by in situ hybridization using synthetic radiolabeled probes, Northern-blot analysis and immunohistochemical staining for hepatitis delta antigen. Hepatitis D virus RNA and hepatitis delta antigen were detected in the nuclei of infected hepatocytes but in none of the other tissues examined. Northern-blot analysis of total cell RNA confirmed these findings and revealed a series of hepatitis D virus transcripts, including full-length genomic RNA and dimers and trimers of hepatitis D virus RNA that may represent replicative intermediates. Use of single-stranded probes showed genome-size monomers and dimers to be both of genomic and antigenomic polarity, although dimers were found to be predominantly antigenomic. These findings document the strict hepatotropism of hepatitis D virus and support the rolling-circle model of genome replication for this unique, defective RNA virus.