Treatment of chronic hepatitis B for the prevention of development of hepatocellular carcinoma and analyses of hepatitis B virus insertional mutagenesis as a first hit during multistep liver carcinogenesis.

1.核苷类似物拉米夫定自2000年起在日本获准用于治疗慢性乙型肝炎。据报道，长期服用该药会导致病毒DNA聚合酶中酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸（YMDD）基序点突变的耐药病毒的出现和临床病程的加剧。我们为这些突变病毒开发了一种灵敏的检测方法，并分析了它们的动态和出现机制。我们发现，在一些先前未接受拉米夫定治疗的HBV携带者中，已经存在少数具有YMDD基序突变的HBV变体，并且YMDD突变早在拉米夫定给药后6个月内出现。然而，尽管持续使用拉米夫定，这些突变体有时也会自发消失。我们还对YMDD突变体的早期检测进行了敏感的定量分析，并建议在拉米夫定治疗期间预测病毒突破的阈值为10^<2.7>拷贝/ml。越来越多的证据表明，HBV整合和由此产生的插入突变在肝细胞癌（HCC）的细胞生长或维持中起着重要作用。我们分析了无HCC的慢性肝炎组织中的HBV整合，以探索HBV在HCC发展前几十年引起的早期遗传变化。我们发现具有相同HBV整合的几组肝细胞在慢性肝炎组织中扩展，并确定了受HBV插入影响的几个基因。这些基因包括一个已知的肿瘤抑制基因，三个对器官发育至关重要的果蝇基因的人类同源基因，一个假定的致癌基因和一个最近发现的趋化因子。这些数据提供了HBV在慢性感染早期在肝细胞中整合的确凿证据，并揭示了HBV相关病毒标记方法在识别与细胞生长和维持相关的基因方面的有效性。

1.Lamivudine, a nucleoside analog, has been licensed for chronic hepatitis B in Japan since 2000. It is reported that prolonged administration of this drug results in emergence of resistant viruses with point mutations at tyrosine- methionine- aspartate- aspartate (YMDD) motif of viral DNA polymerase and exacerbation of clinical course. We developed a sensitive assay for these mutant viruses and analyzed their dynamics and mechanisms of emergence. We revealed that variant HBV with mutations at YMDD motif already exists as minority in some HBV carriers without previous lamivudine treatment and YMDD mutants appear as early as within 6 months after lamivudine administration. However, these mutants sometimes disappear spontaneously despite continuous lamivudine administration. We also performed sensitive quantitative assay for early detection of YMDD mutants and a threshold of 10^<2.7> copies/ml was suggested for predicting viral breakthrough during lamivudine treatment.2.Growing evidence demonstrates that HBV integration and resulting insertional mutagenesis plays an important role in cell growth or maintenance in hepatocellular carcinoma (HCC). We analyzed HBV integration in chronic hepatitis tissues without HCC to explore early genetic change due to HBV preceding the development of HCC by a few decades. We found that a few groups of liver cells with a same HBV integration expand in chronic hepatitis tissues and identified several genes affected by HBV insertion. These genes included one known tumor suppressor gene, three human homologs of drosophila genes that are critical for organ development, one putative oncogene and one recently found chemokine. These data provided firm evidence of HBV integration in hepatocytes at an early stage of chronic infection and revealed usefulness of the HBV-related virus tagging approach to identify genes associated with cell growth and maintenance.