Hepatitis C Virus Quasispecies in the Resistance to Antiviral Therapy

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染是公共卫生的主要问题之一。目前，聚乙二醇化干扰素-α联合利巴韦林的最佳抗病毒治疗可治愈约50%的1型丙型肝炎病毒感染者和80%的2型和3型丙型肝炎病毒感染者。我们对治疗耐药性的理解的困难之一与目前抗病毒药物的性质有关。长期以来，干扰素和利巴韦林都以其广谱的抗病毒活性而闻名，它们通过产生一种非特异性的抗病毒状态，而不是直接与病毒相互作用。因此，丙型肝炎病毒基因组上没有明确的靶点被记录下来。用这些药物进行的耐药性研究通过常规方法产生了非常有争议的数据，这些方法往往侧重于丙型肝炎病毒的短域，而不是整个病毒基因组。利用一种新的长RT-PCR和克隆技术，以及来自丙型肝炎抗病毒长期治疗抗肝硬化试验(HALT-C)的良好特征的血清样本，我们提出了一个病毒测序项目，通过该项目，将在多个水平上详尽地检测病毒对抗病毒治疗的耐药机制。 假设：丙型肝炎病毒对抗病毒治疗的耐药性与病毒准种在单个变种或种群水平上的区域相关突变有关。 目的1：探讨感染1a型丙型肝炎病毒的空应答者在丙型肝炎病毒分离株和准种水平上的遗传特征。将从病毒学应答(SVR)为零或持续的患者中生成基线上的全长丙型肝炎病毒准种图谱，然后进行比较分析，以确定与治疗耐药性相关的潜在遗传特征。 目的：探讨丙型肝炎病毒2型在抗病毒治疗中是否存在明显的准种结构。全长的丙型肝炎病毒准种图谱将从20个具有丙型肝炎病毒基因2a的SVR中产生，随后将与来自丙型肝炎病毒基因1a的那些进行比较分析。 目的3：研究抗病毒治疗后复发患者中与丙型肝炎病毒复发相关的变异模式。复发表明丙型肝炎病毒从抗病毒治疗下形成的假定的种群瓶颈中存活下来。丙型肝炎病毒如何应对这种“活体”人口瓶颈？ 这一问题将通过对丙型肝炎病毒准种全长图谱的顺序比较分析来解决。这些研究的数据将立即应用于未来丙型肝炎抗病毒治疗的合理设计，其中聚乙二醇化干扰素和利巴韦林仍是核心成分。

DESCRIPTION (provided by applicant):Hepatitis C virus (HCV) infection is one of the major concerns in public health. Currently, optimal antiviral therapy with pegylated interferon-alpha plus ribavirin, cures ~50% of patients infected with HCV genotype 1 and ~80% of patients infected with HCV genotypes 2 and 3. One of the difficulties regarding our understanding on treatment resistance is related to the nature of current antiviral agents. Both interferon and ribavirin have long been known for their broad-spectrum antiviral activity by creating a non-specific antiviral status rather than the direct interaction with viruses. Consequently, no explicit targets on HCV genome have been documented. Studies on drug resistance with these agents have generated very controversial data through conventional approaches that frequently focus on short domains of the HCV rather than the entire viral genome. Using a novel long RT-PCR and cloning technology and well characterized serum samples from the Hepatitis C Antiviral Long-term Treatment against Cirrhosis trial (HALT-C), we propose a viral sequencing project through which viral mechanisms for the resistance to antiviral therapy will be exhaustively examined at multiple levels. HYPOTHESIS: HCV resistance to antiviral therapy is associated with region-dependent mutations of viral quasispecies at either single variants or the population level. Aim 1: To explore genetic signatures at both HCV isolate and quasispecies levels in null responders infected with HCV genotype 1a. The full-length HCV quasispecies profiles at the baseline will be generated from patients with either null or sustained virological responses (SVR), followed by comparative analyses to identify potential genetic signatures that are associated with the treatment resistance. Aim 2: To demonstrate if there are distinct quasispecies structures of HCV genotype 2 in terms of the high response rate to the antiviral therapy. The full-length HCV quasispecies profiles will be generated from twenty SVRs with HCV genotype 2a, followed by comparative analyses with those derived from HCV genotype 1a. Aim 3: To characterize mutational patterns associated with HCV re-emergence in patients with relapse after initial response to antiviral therapy. The relapse indicates the survival of HCV from a putative population bottleneck formed under antiviral therapy. How does HCV respond to such "in vivo" population bottlenecks? This issue will be addressed through a sequential comparative analysis of full-length HCV quasispecies profiles. Data from these studies will have immediate applications for rational design of future HCV antiviral therapy in which PegIFN-( and ribavirin are still the core components.