Hepatitis C Virus Quasispecies in the Resistance to Antiviral Therapy

描述（由申请人提供）：丙型肝炎病毒（HCV）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 +利巴韦林的最佳抗病毒治疗，治愈了约50%的HCV基因型1患者和约80%的HCV基因型2和3患者。关于我们对治疗耐药性的理解的困难之一与当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抗病毒活性而闻名，它们通过产生非特异性抗病毒状态而不是直接与病毒相互作用。因此，HCV基因组上没有明确的靶点。这些药物的耐药性研究通过常规方法产生了非常有争议的数据，这些方法通常侧重于HCV的短结构域，而不是整个病毒基因组。利用一种新型的长RT-PCR和克隆技术，以及来自丙型肝炎抗病毒长期治疗肝硬化试验（HALT-C）的具有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，病毒对抗病毒治疗的耐药性机制将在多个层面上得到详尽的检验。

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.