Replication of Naturally Occurring Woodchuck Hepatitis Virus Deletion Mutants in Primary Hepatocyte Cultures and after Transmission to Naive Woodchucks

Abstract

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans.We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNAenriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.Mutations within the core gene of hepatitis B virus (HBV) were often found in chronically HBV-infected patients (3,5,8,21; for a review, see reference 14). HBV core internal deletion (CID) is a common type of mutation (1,2,12,13,17,20,26,27,28). In some patients, HBV CID mutants emerged in association with severe liver diseases. For example, HBV CID mutants occurred in renal allograft recipients who suffered from endstage liver diseases (12,13,18). CID mutations lead to the expression of truncated, unstable core proteins (24, 29). Thus, HBV CID mutants are defective in replication and require trans-complementation by wild-type virus (22,29). This fact explains why HBV CID mutants always co-occurred with wildtype virus in patients examined so far. However, our knowledge about the replication of HBV CID mutants in the host is limited. It is also not known whether the presence of HBV CID mutants influences ...

Helper-Dependent Adenoviral Vector-Mediated Delivery of Woodchuck-Specific Genes for Alpha Interferon (IFN-α) and IFN-γ: IFN-α but Not IFN-γ Reduces Woodchuck Hepatitis Virus Replication in Chronic Infection In Vivo

“…Primary woodchuck hepatocytes were prepared as described in detail previously (21). Briefly, the liver cells were harvested by perfusion of the liver with a collagenase medium.…”

“…WHV replication intermediates in liver biopsies were analyzed. Total liver DNA from snap-frozen liver samples was extracted with the QIAamp tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and as described previously in detail (5,21). WHV replication intermediates were analyzed by Southern blot hybridization with a full-length WHV8 genome as a probe.…”

Woodchuck Gamma Interferon Upregulates Major Histocompatibility Complex Class I Transcription but Is Unable To Deplete Woodchuck Hepatitis Virus Replication Intermediates and RNAs in Persistently Infected Woodchuck Primary Hepatocytes

“…Liver perfusion was carried out according to the protocol described previously (23). Anesthetized woodchucks received an intravenous injection of 2 ml of heparin (10 4 U/ml).…”

Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients

“…Most attempts for the quantitation of cccDNA have been made with liver biopsies from ducks or woodchucks. [11][12][13][14][15][16] Quantitation of cccDNA in human peripheral blood mononuclear cells and liver biopsies has been performed. [17][18][19][20] In these studies, primers spanning across the gap in the minus strand and corresponding to the variable region on the plus strand were used to amplify across noninterrupted cccDNA.…”