We amplified a full-length hepatitis B virus (HBV) genome from the serum of a chronic hepatitis B patient who experienced virological breakthrough with high HBV DNA titer following adefovir (ADV) therapy. The PCR product was cloned and sequencing of the six clones revealed an isolate of C2 subgenotype. Mutation(s) in the polymerase gene responsible for ADV resistance included rtA181T (all clones) and rtN236T (four clones). The rtA181T mutation caused the W172* nonsense mutation in the overlapping S gene. In addition, all the clones harbored another nonsense mutation in the S gene (C69*) and a 207nt in-frame deletion in the preS1 region. These clones were converted to a 1.1mer construct for transient transfection of Huh7 cells. All the clones were deficient in hepatitis B surface antigen production. Three clones had similar levels of DNA replication. Comparison with a wild-type clone of the same genotype revealed a higher intracellular level of replicative DNA for clone c4, which was reduced by putting back the deleted 207nt, but not by co-transfection with an expression construct for the three surface proteins to rescue virion production. The HBcAg expression of the c4 and c4+207nt clones was mainly in the nucleus. Co-transfection with the L/M/S proteins expression construct did not alter the distribution of core. Clone c4 showed a significantly decreased susceptibility to ADV, a mild reduction in susceptibility to lamivudine and tenofovir, but remained sensitive to entecavir. In conclusion, this is an unusual ADV-resistant HBV isolate harboring two nonsense mutations in the S gene and a large in-frame deletion in the preS1 region, but still retains a high replication phenotype, which can provide a platform for recombinant vector construction.

我们从一名慢性乙型肝炎患者的血清中扩增了全长乙型肝炎病毒（HBV）基因组，该患者在阿德福韦（ADV）治疗后经历了高HBV DNA滴度的病毒学突破。克隆了PCR产物，对六个克隆的测序揭示了C2亚基因型的分离物。聚合酶基因中引起ADV抗性的突变包括rtA181T（所有克隆）和rtN236T（四个克隆）。rtA181T突变在重叠的S基因中引起W172 *无意义突变。此外，所有克隆在S基因（C69 *）中都具有另一个无意义的突变，在preS1区中存在207nt的读框缺失。这些克隆被转化为1.1mer构建体，用于Huh7细胞的瞬时转染。所有克隆均缺乏乙型肝炎表面抗原的产生。三个克隆的DNA复制水平相似。与相同基因型的野生型克隆的比较表明，克隆c4的复制DNA的细胞内水平更高，这可以通过放回缺失的207nt来降低，但不能通过与表达构建体共转染以拯救三个表面蛋白而降低病毒体生产。c4和c4 + 207nt克隆的HBcAg表达主要在细胞核中。与L / M / S蛋白表达构建体共转染不会改变核心的分布。克隆c4对ADV的敏感性显着降低，对拉米夫定和替诺福韦的敏感性略有降低，但对恩替卡韦仍然敏感。总之，这是一个不寻常的抗ADV的HBV分离株，在S基因中带有两个无意义的突变，在preS1区中存在较大的框内缺失，