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，聚乙二醇干扰素-α加病毒唑的最佳抗病毒治疗可治愈约50%感染HCV基因型1的患者和约80%感染HCV基因型2和3的患者。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。干扰素和利巴韦林长期以来都以其广谱抗病毒活性而闻名，其通过产生非特异性抗病毒状态而不是与病毒直接相互作用。因此，尚无关于HCV基因组的明确靶点的记载。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，这些方法经常关注HCV的短结构域而不是整个病毒基因组。使用一种新的长RT-PCR和克隆技术，以及丙型肝炎抗病毒长期治疗肝硬化试验（HALT-C）的血清样本，我们提出了一个病毒测序项目，通过该项目，将在多个水平上彻底检查病毒对抗病毒治疗的耐药机制。 假设：丙型肝炎病毒抗病毒治疗耐药与病毒准种的区域依赖性突变有关，无论是在单一变异还是在群体水平。 目的1：探讨HCV基因型1a感染的无效应答者中HCV分离株和准种水平的遗传特征。基线时的全长HCV准种谱将从无效或持续病毒学应答（SVR）的患者中生成，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2：证实HCV基因2型是否存在不同的准种结构，以对抗病毒治疗的高应答率。将从20个HCV基因型2a的SVR中生成全长HCV准种谱，然后与来自HCV基因型1a的那些进行比较分析。 目的3：描述抗病毒治疗初始应答后复发患者中与HCV再出现相关的突变模式。复发表明HCV从抗病毒治疗下形成的假定群体瓶颈中存活。HCV如何应对这种“体内”群体瓶颈？ 这个问题将通过全长HCV准种谱的连续比较分析来解决。这些研究的数据将直接应用于未来HCV抗病毒治疗的合理设计，其中聚乙二醇干扰素和利巴韦林仍然是核心成分。

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.