Lu M, Hilken G, Yang D, Kemper T, Roggendorf M
Institut für Virologie, Universitätsklinikum Essen, 45122 Essen, Germany.
J Virol. 2001 Apr;75(8):3811-8. doi: 10.1128/JVI.75.8.3811-3818.2001.
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.
与人类中的乙型肝炎病毒突变体一样，核心内部缺失（CID）的土拨鼠肝炎病毒（WHV）突变体自然存在于慢性感染WHV的土拨鼠中。我们研究了WHV缺失突变体在原代土拨鼠肝细胞培养物中的复制情况，以及将其传播给未感染的土拨鼠后在体内的复制情况。通过对14只野生捕获的、慢性感染WHV的土拨鼠进行筛查，发现两只土拨鼠WH69和WH70携带WHV CID突变体。与之前的结果一致，来自这两只动物的WHV CID突变体在WHV核心基因中部有不同长度（90至135 bp）的缺失。在土拨鼠WH69中，WHV CID突变体在血清、正常肝组织以及在较小程度上在肝肿瘤组织中的病毒群体中占主要部分。在WH69的原代肝细胞中，野生型WHV和CID突变体的复制至少维持了7天。尽管WHV CID突变体在细胞内WHV复制中间体的部分中占主导，但突变的共价闭合环状DNA（cccDNA）似乎只是富含cccDNA部分的一小部分。对其他土拨鼠肝脏组织中富含cccDNA部分的分析证实，突变的cccDNA仅占总cccDNA库的一小部分。给四只未感染的土拨鼠接种含有WHV CID突变体的土拨鼠WH69或WH70的血清。所有四只土拨鼠在接种后3至4周（p.i.）出现病毒血症。它们产生了抗WHV核心抗原（WHcAg）抗体、对WHcAg的淋巴细胞增殖反应以及抗WHV表面抗原。在这些土拨鼠的血清中仅发现野生型WHV，未发现CID突变体。在接种后第7周处死的一只土拨鼠的肝脏组织中也未鉴定出WHV CID突变体。其余三只土拨鼠清除了WHV。因此，接种物中WHV CID突变体的存在并未显著改变急性自限性WHV感染的病程。我们的结果表明，WHV CID突变体的复制可能需要一些特定的选择条件。对WHV CID突变体的进一步研究将使我们对嗜肝DNA病毒的复制有更深入的了解。
