Super-infection and virus spread during chronic hepadnaviral infection

描述（由申请人提供）：B肝炎病毒（HBV）是一种原型嗜肝DNA病毒，全球有4亿慢性携带者。慢性HBV感染是肝细胞癌（HCC）的主要危险因素，占所有HCC的50%以上。在感染早期，HBV几乎遍布整个肝脏，但在慢性感染期间， 肝细胞（整个肝细胞群体的20-90%）出现，而不管进行中的病毒血症。这些发现是在HBV感染的人类和黑猩猩以及感染与HBV密切相关的土拨鼠肝炎病毒（WHV）的土拨鼠中发现的。这些观察结果，沿着耐药HBV突变体出现的动力学分析，为HBV领域长期悬而未决的争论奠定了基础，即在慢性感染中，嗜肝DNA病毒的细胞间传播至少是非常低效的（如果发生的话），并且超级感染是不太可能发生的事件。有人提出，慢性嗜肝DNA病毒感染 可以在没有扩散的情况下通过感染的肝细胞的分裂来维持。对HBV相关鸭肝炎B病毒（DHBV）显示重叠感染排除，并建议对HBV和WHV。然而，WHV或HBV慢性感染中不存在病毒细胞间传播和超感染尚未得到证实或排除。因此，在目的1中，我们提出直接确定嗜肝DNA病毒的细胞间传播和已感染细胞的超感染是否在慢性感染期间发生，这将表明传播可能是维持慢性感染的重要因素，因此也可能是迄今未被认识到的HCC风险因素。此外，HCC也经常被报道为在HBV携带者和WHV携带者土拨鼠中明显无病毒，这表明HCC是由改变的肝细胞产生的，这些肝细胞已经失去了支持高效嗜肝DNA病毒复制的能力，因此对于克隆扩增具有选择性优势，因为它们不再被免疫系统靶向。这将我们引导到目标2，其将确定（i）嗜肝DNA病毒诱导的HCC是否仍然对嗜肝DNA病毒感染敏感，以及（ii）控制HCC的表观无病毒状态的因素。我们建议使用一种非常有价值的替代模型来研究HBV感染，特别是HCC的发展- WHV携带者土拨鼠。我们将用不同的天然感染性WHV毒株WHVNY对具有早期HCC的WHV携带者进行超级感染，该毒株具有独特的缺失，因此可以容易地与用于初次感染的毒株WHV 7区分开。我们将（i）直接确定正常肝细胞和HCC对WHVNY超级感染的易感性;（ii）解决免疫系统是否控制细胞再次感染的能力。总的来说，这项研究将大大提高我们对慢性嗜肝DNA病毒感染与HCC发展相关机制的理解。它有可能确定病毒粒子和病毒传播的感染性作为发病机制和HCC风险的重要因素，并可能促进结合HBV受体和阻断病毒传播的进入抑制剂的使用，作为对抗慢性HBV感染和降低HCC风险的新疗法。

DESCRIPTION (provided by applicant): Hepatitis B virus (HBV) is a prototype hepadnavirus with 400 million chronic carriers worldwide. Chronic HBV infection is a main risk factor of hepatocellular carcinoma (HCC), causing >50% of all HCCs. Early in infection, HBV spreads virtually throughout the entire liver, but during chronic infection, areas of apparently virus-free hepatocytes (20-90% of the entire hepatocyte population) appear regardless of the ongoing viremia. These findings were made in HBV-infected humans and chimpanzees and in woodchucks infected with the woodchuck hepatitis virus (WHV), which is closely related to HBV. These observations, along with the analysis of kinetics of the emergence of drug-resistant HBV mutants, laid the foundation for a long-standing unresolved argument in the HBV field that in chronic infection, cell-to-cell spread of a hepadnavirus is at least very inefficient (if it occursat all), and super-infection is an unlikely event. It was proposed that chronic hepadnavirus infection can be maintained exclusively via division of the infected hepatocytes in the absence of the spread. Super-infection exclusion was shown for HBV-related duck hepatitis B virus (DHBV), and was suggested for HBV and WHV. However, the absence of the viral cell-to-cell spread and super-infection in chronic infections with either WHV or HBV has not been confirmed or dismissed. Therefore, in Aim 1, we propose to determine directly whether the cell-to-cell spread of hepadnavirus and super-infection of already infected cells occur during chronic infection, which would indicate that spread could be an essential factor for maintenance of chronic infection and thus could also be a so far unrecognized risk factor of HCC. In addition, HCCs are also frequently reported as being apparently virus-free in HBV carrier humans and WHV carrier woodchucks, suggesting that HCCs arise from altered hepatocytes that have lost the ability to support efficient hepadnavirus replication, and thus have a selective advantage for a clonal expansion since they are no longer targeted by the immune system. This has led us to Aim 2 that will determine (i) if hepadnavirus-induced HCC is still susceptible to infection with a hepadnavirus, and (ii) what controls the apparent virus-free status of HCC. We propose to use an invaluable surrogate model to study HBV infection and especially HCC development - WHV carrier woodchucks. We will super-infect WHV carriers with early HCCs with a different natural infectious WHV strain, WHVNY, which has a unique deletion and thus can be easily discriminated from the strain WHV7 used for the primary infection. We will (i) directly determine the susceptibility of normal hepatocytes and HCCs to WHVNY super-infection; and (ii) address if the immune system controls the ability of cells to get re-infected. Overall, the proposed study will greatly improve our understanding of the mechanism of chronic hepadnavirus infection in relation to HCC development. It has the potential to identify infectivity of virions and virus spred as important factors of pathogenesis and HCC risk, and may facilitate the use of entry inhibitors that bind the HBV receptor and block virus spread as new therapeutics for battling chronic HBV infection and reducing HCC risk.