HCV Genotype 6: A Model for HCV Genetics and Molecular Epidemiology Studies

描述(申请人提供)：在六个基因类型中，基因6代表最古老、最多样化和基因最复杂的病毒。该基因目前有22个亚型，其中包括我们指定的6R-6v亚型。我们已经对16个亚型和2个等价物的第一批完整基因组进行了测序。我们还发现了16个变异，可能代表14个新的亚型和36个变异，显示出巨大的遗传差异。这项应用的总体目标是通过对将从这些变异中确定的一组完整的基因6型完整序列进行详细分析，来确定丙型肝炎病毒的遗传变异和病毒学特性。基于这些序列，我们将组装全长的丙型肝炎病毒基因组，并用于开发新的丙型肝炎病毒细胞培养模型。其基本原理是，澄清这些丙型肝炎病毒序列将使许多生物信息学分析成为可能，这些分析将基础研究与患者护理联系起来。其具体目标是：(1)确定71个基因6变种的完整基因组，显示出实质性的遗传变异；(2)确定由最古老的6个变种所代表的丙型肝炎病毒的流行动态；以及(3)利用(1)中定义的独特的丙型肝炎病毒基因组，建立丙型肝炎病毒6型细胞培养模型。假设丙型肝炎病毒基因6代表“活的病毒化石”，将最好地反映病毒过去的历史，并且作为所有丙型肝炎病毒基因中最复杂的，基因6变种可能具有更高的差异性，这可能使它们更适合细胞培养。为了实现这些目标，我们将采用改进的DNA步行策略，仅从100份L血清中就可以对完整的丙型肝炎病毒基因组进行测序。我们将使用Beast软件，它结合了各种合并方法。新的策略将被用来从最具传染性的病毒颗粒中获得全长的丙型肝炎病毒基因组。在野生型或与JFH1分离株的嵌合体中，这些基因组将被用于开发新的丙型肝炎病毒细胞培养模型，将有许多应用。这些研究的成功完成将有助于对丙型肝炎病毒流行动力学、其基因变异的生物信息学定义以及发展新的丙型肝炎病毒细胞培养模型的新方法的缺失和基础知识的了解。这些带有序列的模型将对基础的丙型肝炎研究、患者护理和公共卫生至关重要。公共卫生相关性：这些研究的成功完成将有助于建立新的病毒序列和细胞培养模型，以了解丙型肝炎病毒的遗传变异、流行病学行为和病毒学机制。这些信息和衍生的模型对于更好的丙型肝炎诊断和治疗策略以及抗病毒药物和疫苗的改进设计至关重要。

DESCRIPTION (provided by applicant): Among six genotypes, the genotype 6 represents the oldest, most diverse and genetically most complex viruses. This genotype currently has 22 subtypes, including 6r-6v we designated. We have sequenced the first complete genomes of 16 subtypes and 2 equivalents. We have also discovered 16 variants that may represent 14 new subtypes and 36 variants that show great genetic differences. The overall goal of this application is to define the genetic variation and virology properties of HCV by conducting detailed analyses on a full panel of genotype 6 complete sequences that will be determined from these variants. Based on the sequences, we will assemble full length HCV genomes and use for developing new HCV cell culture models. The rationale is that clarifying these HCV sequences will enable many bioinformatics analyses that have applications linking to basic research to patient care. The Specific Aims are to: (1) determine the complete genomes of 71 genotype 6 variants showing substantial genetic variations; (2) determine the epidemic dynamics of HCV that is represented by the oldest genotype 6 variants; and (3) develop HCV genotype 6 cell culture models using unique HCV genomes that are defined in (1). It is hypothesized that HCV genotype 6 represents a "living viral fossil" that will best reflect the past history of the virus and that, being the most complex among all HCV genotypes, the genotype 6 variants may have higher divergent properties that may make them more suitable for cell cultures. To accomplish these aims, we will employ an improved DNA walking strategy that can sequence a complete HCV genome from only 100 ¿l of serum. We will use the BEAST software that combines various coalescent methods. Novel strategies will be recruited to obtain the full length HCV genomes from the most infectious virus particles. In wild type or in chimeras with the JFH1 isolate, these genomes will be used to develop new HCV cell culture models that will have many applications. The successful completion of these studies will contribute the missing and fundamental knowledge to the epidemic dynamics of HCV, bioinformatic definition of its genetic variations, and the novel approaches in developing new HCV cell culture models. These models with the sequence will be vital for basic HCV research and for patient care and public health. PUBLIC HEALTH RELEVANCE: successful completion of these studies will contribute new viral sequences and cell culture models to understand the HCV genetic variation, epidemiology behavior, and virology mechanisms. This information and the derived models are vital for better strategies of HCV diagnosis and treatment, and the improved designs of antivirals and vaccines.