Super-infection and virus spread during chronic hepadnaviral infection

描述(申请人提供)：乙肝病毒(乙肝病毒)是一种原型肝炎病毒，全球有4亿慢性携带者。慢性乙肝病毒感染是肝细胞癌的主要危险因素，占所有肝细胞癌的50%。在感染早期，乙肝病毒几乎扩散到整个肝脏，但在慢性感染期间，明显没有病毒的区域 不管持续的病毒血症如何，肝细胞(占整个肝细胞总数的20%-90%)都会出现。这些发现是在感染了乙肝病毒的人和黑猩猩以及感染了与乙肝病毒密切相关的土拨鼠肝炎病毒(WHV)的土拨鼠身上发现的。这些观察结果，以及对耐药突变株出现的动力学分析，为乙肝领域一个长期悬而未决的论点奠定了基础，即在慢性感染中，肝病毒在细胞之间的传播至少是非常低效的(如果它全部发生的话)，而超级感染是不太可能发生的事件。有人认为慢性肝炎病毒感染 在没有扩散的情况下，可以完全通过分裂感染的肝细胞来维持。对乙肝病毒相关的鸭乙型肝炎病毒(DHBV)表现为超感染排斥，建议对乙肝病毒和世界卫生病毒(WHV)进行超感染排斥。然而，WHV或乙肝病毒慢性感染中没有病毒细胞间传播和双重感染的说法尚未得到证实或排除。因此，在目标1中，我们建议直接确定在慢性感染过程中是否发生了肝病毒的细胞间传播和已感染细胞的重叠感染，这表明传播可能是维持慢性感染的一个必要因素，因此也可能是迄今为止尚未被认识的肝癌的危险因素。此外，在乙肝病毒携带者人类和WHV携带者土拨鼠中，肝细胞癌也经常被报道为明显无病毒，这表明肝细胞癌源于改变的肝细胞，这些肝细胞已经失去了支持高效复制Hepadna病毒的能力，因此具有克隆扩张的选择性优势，因为它们不再是免疫系统的靶标。这导致了我们的目标2，它将确定(I)肝病毒诱导的肝细胞癌是否仍然容易受到肝病毒的感染，以及(Ii)是什么控制了肝细胞癌表面上的无病毒状态。我们建议使用一个有价值的替代模型来研究乙肝病毒感染，特别是肝细胞癌的发展--WHV携带者土拨鼠。我们将用一种不同的天然感染性WHV株WHVNY来超级感染WHV携带者的早期肝癌，该株具有独特的缺失，因此很容易与用于初次感染的WHV7株区分开来。我们将(I)直接确定正常肝细胞和肝癌细胞对WHVNY重叠感染的易感性；以及(Ii)解决免疫系统是否控制细胞再次感染的能力。总体而言，拟议的研究将极大地提高我们对慢性肝炎病毒感染与肝细胞癌发生相关机制的理解。它有可能确定病毒粒子和病毒的传染性是发病和肝细胞癌风险的重要因素，并可能促进结合乙肝病毒受体和阻止病毒传播的进入抑制剂的使用，作为对抗慢性乙肝感染和降低肝细胞癌风险的新疗法。

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.