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 cccDNA-enriched 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.

FIG. 1

Identification of WHV CID mutant genomes in serum samples of chronically WHV-infected woodchucks WH69 and WH70. (A) PCR with DNA-extracted serum samples from woodchucks WH69 and WH70. WH8 was a virus stock containing only wild-type WHV. PCR fragments corresponding to the wild-type WHV core gene and deletion mutants are marked wild type and CID, respectively. (B) PCR amplification of WHV wild type and CID mutants in defined ratios of 1:10, 1:1, and 10:1. Templates of WHV wild type and CID mutants were mixed in defined ratios and adjusted to different copy numbers.

FIG. 2

Heterogeneity of WHV CID mutants in woodchuck WH69. (A) Analysis of the WHV population in WH69 by the cloning of the PCR product of the WHV core region. The numbering of the nucleotide positions is according to Galibert et al. (9). The positions of CIDs are indicated by broken lines and the nucleotide positions. The numbers on the broken lines indicate the respective length of deletions. Nr, number. (B) Analysis of the WHV population in WH69 by nested PCR with diluted preparations of extracted serum DNA. Samples at dilutions of 1:3 × 10⁶ and 1:10⁷ were subjected to nested PCR. The numbers of WHV wild type (wt), CID mutants, and mixtures of both types (wt+CID) are indicated. Diluted samples resulting in negative PCR are indicated as blank.

FIG. 3

(A) Detection of WHV replication intermediates in normal woodchuck liver tissues (lane 1) and woodchuck HCC tissues from WH69 (lanes 2 through 4). (B) The immunohistological staining of HCC section with anti-WHcAg antibody. Magnification, ×1,000. (C) WHV core-specific PCR with DNA extracted from normal liver tissues (lane 1) and three different parts of HCC tissues from WH69 (lanes 2 through 4).

FIG. 3

(A) Detection of WHV replication intermediates in normal woodchuck liver tissues (lane 1) and woodchuck HCC tissues from WH69 (lanes 2 through 4). (B) The immunohistological staining of HCC section with anti-WHcAg antibody. Magnification, ×1,000. (C) WHV core-specific PCR with DNA extracted from normal liver tissues (lane 1) and three different parts of HCC tissues from WH69 (lanes 2 through 4).

FIG. 4

Detection of WHV CID mutants in HCC tissues from seven chronically WHV-infected woodchucks. WHV core-specific PCR with DNA extracted from HCC tissues from seven woodchucks is shown.

FIG. 5

Detection of WHV CID mutants in a fraction of WHV replication intermediates and cccDNA in woodchuck primary hepatocytes. (A) WHV replication intermediates detected by Southern blot hybridization. WHV core-specific PCRs were performed with total DNA (B), with encapsidated WHV DNA (C), and with cccDNA-enriched fractions (D) extracted from woodchuck primary hepatocytes.

FIG. 6

Detection of WHV CID mutants in total DNA and cccDNA-enriched fractions from normal liver tissues from woodchucks DW3 and DW15-8. WHV replication intermediates (A) and cccDNAs (B) were detected by Southern blot hybridization. (C) The presence of WHV CID mutants in the extracted DNA fractions was analyzed by WHV core-specific PCR.

FIG. 7

Infection of naive woodchucks WH10491 (A) and WH10840 (C) with sera from WH69. OD 490, optical density at 490 nm. The course of WHV infection was monitored by determining anti-WHcAg (anti-WHc titer) and anti-WHsAg (anti-WHs OD 490) antibody levels in woodchuck sera. For the detection of WHV CID, WHV core-specific PCR was performed with DNA extracted from woodchuck sera. The specific lymphoproliferative response of PBMCs to rWHcAg was indicated by a plus if the stimulation index was higher than 3 (19). n.d., not determined. (B) WHV core-specific PCR with DNA extracted from WHV inoculum (WH69) and from liver tissues from woodchuck WH10491 is shown.