共价闭合环状DNA（cccDNA）是乙肝病毒（HBV）慢性感染的分子基础。HBV研究中多采用基因组串联重复、或部分重复的线性DNA复制子起始病毒复制周期，被认为可能具有与天然cccDNA模板不同的病毒学特征和宿主反应性。本项研究中，我们拟通过Cre/loxP介导的位点特异性重组策略诱导HBV 重组cccDNA（rcccDNA）。由于HBV基因组高度压缩，我们在单拷贝基因组中插入一段外源内含子序列，以容纳DNA重组残留的loxP位点；外源内含子在病毒RNA转录过程中被有效拼接，并不干扰正常的HBV复制和表达。我们将应用尾静脉高压注射rcccDNA前体质粒，或构建腺病毒载体携带rcccDNA前体序列感染免疫功能正常的Cre转基因小鼠，发展基于rcccDNA的病毒持续感染和慢性炎症模型，对HBV特征性的慢性病理机制进行深入研究；同时，建立以(r)cccDNA为靶标的抗病毒药物体内、体外评价系统。

Covalently closed circular (ccc) DNA is an essential component of the HBV replication cycle and is regarded as a primary molecular mechanism for HBV persistence. Investigators in the field have used linear HBV DNA replicons with overlength genome for transfection to ensure the transcription of HBV mRNAs. However, the linear HBV replicon with foreign-sequence inserted may not fully surrogate the authentic cccDNA template in HBV virology, and is likely to cause distinct host immune responses. We hereby propose to induce a recombinant (r) cccDNA through Cre/loxP-mediated DNA recombination. A loxP-chimeric intron is specially engineered into the viral genome to enable functionally seamless DNA recombination. This chimeric intron could be spliced from RNA transcripts without interrupting the HBV life cycle. In the study, we plan to construct a precursor plasmid (prcccDNA) or the adenoviral vector encoding the rcccDNA sequence (prcccDNA). Hydrodynamic injection of prcccDNA or intravenous infusion of rAd/rcccDNA to Alb-Cre transgenic mice would result in accumulation of nuclear rcccDNA in the host liver. Our study will focus on how to develop rcccDNA-based HBV persistence and immunopathogenesis in the immunocompetent mouse model. Virological and immunological assays will be performed for understanding the molecular mechanism of HBV persistence. Our study will also make efforts to establish a model system in vitro and in vivo for evaluating antiviral treatments directly targeting HBV (r)cccDNA.