Role of nonstructural protein in limited genetic diversity of yellow fever 17D vaccine virus

摘要 许多成功的减毒活疫苗(Lav)都是根据经验得出的，对它们的情况知之甚少。 衰减的机制。这些LAV病毒的最大特点是脊髓灰质炎病毒已经减弱 定位于5‘NCR和衣壳基因。加深对许可证衰减机制的理解 LAV将有助于未来LAV的合理发展。黄热病是通过使用一种 减毒活疫苗，17D株，来自野生型(WT)株Asibi，相差20个氨基酸； 结构基因和非结构基因(NS)中的11个。我们的总体目标是了解 17D疫苗的减毒作用，人们对此知之甚少。下一代测序(NGS)技术已经 在疫苗开发和LAV的质量控制和安全性方面有很好的应用。我们已经比较了WT Asibi 和17D疫苗，发现Asibi是一种典型的RNA病毒，具有准种种群， 令人惊讶的是，17D疫苗几乎没有准种的证据，我们认为这可能有助于 疫苗病毒的减毒表型。我们已经证明了17D疫苗病毒中的限制性准种 是由于突变(S)在NS蛋白的复制复合体中作为一个整体，而不是依赖于核糖核酸 RNA聚合酶(RdRp)单独。此外，利巴韦林是一种将突变引入rna的抗病毒药物。 由于病毒RdRp的不保真度，在复制过程中的基因组。我们已经证明了WT Asibi病毒是 对利巴韦林敏感，而17D疫苗病毒相对耐药，提示高保真复制 复合体可能与17D疫苗的减毒表型有关。我们认为，对这一事件的调查 17D疫苗病毒中有限准种的机制可能对理解 17D疫苗和其他LAV减毒的分子基础以及未来黄病毒LAV的发展。在这 应用：我们将鉴定对17D疫苗有限准种有贡献的病毒基因。我们假设 17D疫苗病毒的限制性准种是由于病毒NS蛋白的多个突变所致 复制复合体作为一个整体，而不是单独的RdRp，编码一种减弱的表型，并且 突变导致了极低的毒力复原率。这项提议的目标是履行 有针对性的研究，以确定哪些YFV NS基因对17D疫苗的限制性准种有贡献 提出一个假说，研究NS基因在17D疫苗减毒机制中的作用。这 将通过三个具体目标实现：目标1将调查准种种群和利巴韦林敏感性 ASIBI/17D突变体以确定NS基因中有助于限制准种种群的残基和 17D疫苗对利巴韦林的抗药性；AIM 2将产生对利巴韦林抗药性的Asibi突变体，鉴定 负责抗药性的核苷酸变化，并表征突变体的准种种群；以及 目的3将研究AG129小鼠中ASIBI/17D突变的表型，以确定利巴韦林耐药性 和/或缺乏准种与减弱的表型相关。

ABSTRACT Many successful live attenuated vaccines (LAVs) were derived empirically and little is known about their mechanisms of attenuation. The best characterized of these LAVs is poliovirus where attenuation has been mapped to the 5’NCR and capsid genes. Increased understanding the mechanism of attenuation of licensed LAVs will help in the rational development of future LAVs. The disease yellow fever is controlled by the use of a live attenuated vaccine, strain 17D, derived from wild-type (WT) strain Asibi, and differ by 20 amino acids; 9 in the structural genes and 11 in the nonstructural (NS) genes. Our overall goal is to understand the mechanism of attenuation of 17D vaccine, which is poorly understood. Next Generation Sequencing (NGS) technology has great applications to vaccine development and quality control and safety of LAVs. We have compared WT Asibi and 17D vaccine by NGS and found that Asibi is a typical RNA virus with a quasispecies population while, surprisingly, 17D vaccine has very little evidence of quasispecies, and we believe that this may contribute to the attenuated phenotype of the vaccine virus. We have shown that the restricted quasispecies in 17D vaccine virus is due to mutation(s) in the NS proteins of the replication complex as a whole, rather than the RNA dependent RNA polymerase (RdRp) alone. In addition, ribavirin is an antiviral drug that introduces mutations into RNA genomes during replication due to the lack of fidelity of the viral RdRp. We have shown that WT Asibi virus is sensitive to ribavirin while 17D vaccine virus is relatively resistant suggesting that a high fidelity replication complex potentially contributes to the attenuated phenotype of 17D vaccine. We believe that investigation of the mechanism of limited quasispecies in 17D vaccine virus may have important applications to understanding the molecular basis of attenuation of 17D vaccine, other LAVs, and development of future flavivirus LAVs. In this application we will identify the viral genes that contribute to limited quasispecies of 17D vaccine. We hypothesize that the restricted quasispecies in 17D vaccine virus is due to multiple mutations in the NS proteins of the replication complex as a whole, rather than the RdRp alone, encodes an attenuated phenotype, and multiple mutations contribute to the very low rate of reversion to virulence. The objective of this proposal is to perform targeted studies to identify which YFV NS genes contribute to the restricted quasispecies of 17D vaccine to propose a hypothesis to investigate the role of NS genes in the mechanism of attenuation of 17D vaccine. This will be achieved via three specific aims: Aim 1 will investigate the quasispecies population and ribavirin sensitivity of Asibi/17D mutants to identify residues in NS genes that contribute to restricted quasispecies population and ribavirin resistance of 17D vaccine; Aim 2 will generate Asibi mutants that are resistant to ribavirin, identify the nucleotide changes responsible for resistance, and characterize the quasispecies population of the mutants; and Aim 3 will investigate the phenotype of Asibi/17D mutants in AG129 mice to determine if ribavirin resistance and/or lack of quasispecies correlates with an attenuated phenotype.