Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection.
ABSTRACT During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from approximately 35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (approximately 1 mg) containing, in total, approximately 3 x 10(7) liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, approximately 30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.
Article: Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections.[show abstract] [hide abstract]
ABSTRACT: During long-term studies of the natural history of woodchuck hepatitis virus infection, five cases of histologically confirmed, primary hepatocellular carcinoma were observed in a total of 92 woodchucks which had recovered, by analysis of viral serologic markers (WHsAg-, anti-WHc+, anti-WHs+), from experimental acute woodchuck hepatitis virus infections 20 to 30 months prior to the detection of hepatocellular carcinoma. No hepatocellular carcinoma was observed in 167 uninfected controls at least 3 years of age and held in the same laboratory environment. Southern blot hybridization analysis of liver tissue taken from four of these recovered woodchucks revealed the presence of low levels (0.1 to 0.3 copies per cell) of integrated woodchuck hepatitis virus DNA in hepatocellular carcinoma (four of four animals) and nonneoplastic tissue (three of four animals). Similarly, hepatocellular carcinoma tissue obtained from two wild-caught, naturally infected and serologically recovered woodchucks also contained low levels of integrated woodchuck hepatitis virus DNA. Liver tissues from another 27 of these 92 recovered woodchucks (without hepatocellular carcinoma) were examined for woodchuck hepatitis virus nucleic acids 13 to 31 months following experimental woodchuck hepatitis virus infection. Nonreplicating woodchuck hepatitis virus DNA was present in the liver of eight (30%) and in the peripheral blood lymphocytes from eight (30%) of these 27 animals. These results were in marked contrast to the analysis of woodchuck hepatitis virus DNA in the liver tissue of chronic woodchuck hepatitis virus carriers (20 experimentally infected and nine naturally infected). In these animals, high levels of replicating woodchuck hepatitis virus DNA (up to 2,000 copies per cell) were observed in all hepatocellular carcinoma and nonneoplastic liver tissue. Integrated woodchuck hepatitis virus DNA was found in eight of 60 individual hepatocellular carcinomas detected in 29 chronic carriers, 15 to 40 months postinfection. Integrated woodchuck hepatitis virus DNA was present in the nonneoplastic tissue from four of these 29 chronic woodchuck hepatitis virus carriers.Hepatology 04/1989; 9(3):461-70. · 11.66 Impact Factor