Characterization of woodchuck hepatitis virus DNA and RNA in the hepatocellular carcinomas of woodchucks

Abstract

Integration and transcription of woodchuck hepatitis virus DNA were studied by Southern and Northern blot analysis in 26 hepatocellular carcinomas and in adjacent nontumor tissue of woodchucks (Marmota monax). All liver tissue chronically infected with woodchuck hepatitis virus contained various amounts of episomal and replicative forms of woodchuck hepatitis virus DNA: episomal and replicative forms of woodchuck hepatitis virus DNA without integration were found in six tumors, episomal and integrated woodchuck hepatitis virus DNA was observed in 18 tumors and exclusively integrated woodchuck hepatitis virus DNA was found in two tumors. In most tumors and in all of the liver tissues chronically infected with woodchuck hepatitis virus, two major woodchuck hepatitis virus RNA species (3.7 and 2.1 kilobases) were detected. In tumors of two other animals (HW76 and HW89) with integrated woodchuck hepatitis virus DNA, only single major transcripts of 3.5 and 2.5 kilobases, respectively, were detected. Hybridization with subcloned woodchuck hepatitis virus DNA probes showed that both aberrant transcripts lacked the C gene and a part of the pre-S1 gene; characterization of corresponding integrated woodchuck hepatitis virus DNA sequences revealed that the C gene was deleted in one tumor, although not in the other. In agreement with the nucleic acid data, we found expression of core protein by Western blotting only in chronically infected liver tissue of these animals, but not in the corresponding tumors. Deletion of the C gene in mRNA may be due to deletion of this gene in the integrated sequences or due to transcriptional regulation.

The Laboratory Woodchuck (Marmota monax)

“…Molecular cloning and analyses of integrated WHY DNA and associated flanking sequences of cellular DNA initially demonstrated that integrations occurred at multiple sites within the woodchuck genome (Fuchs et al, 1989;Kaneko et al, 1986;Korba et al, 1989;Mitamura et al, 1982;Ogston et al, 1982;Rogler et al, 1987). Integrations of WHV DNA have been reported in apparently nontumorous liver of woodchucks (Fuchs et al, 1989;Korba et al, 1989;Mitamura et al, 1982;Ogston et al, 1982;Rogler et al, 1987). One explanation for multiple integrations was that several hepadnavirus integrations could have accumulated in a single hepatocyte and been propagated clonally by the transformed hepatocyte.…”

Application of the woodchuck animal model for the treatment of hepatitis B virus-induced liver cancer

“…Since neutrophils accumulate in woodchuck liver[ 79 ], these cells may be responsible for the intrahepatic recruitment of mononuclear inflammatory cells via neutrophil-derived metalloproteinases, as observed in a transgenic mouse model of acute hepatitis B and in patients with chronic hepatitis B[ 84 , 85 ]. Liver disease then appears to progress to HCC due to the reduced immune-mediated clearance of WHV-infected hepatocytes by both non-cytolytic and cytolytic mechanisms[ 30 , 76 ], continuing chronic microinflammation [ 43 , 86 - 88 ], and viral integration events[ 67 , 72 , 89 - 91 ]. However, as described in more detail below, these deficiencies in humoral and cellular immune responses present in chronic WHV carrier woodchucks can be altered by different means leading to a functional cure (defined as a loss of viral DNA and surface antigen in serum, with or without seroconversion to virus-neutralizing antibodies[ 10 ]) that delays or even prevents HCC onset.…”