Glycan array for phenotype-driven capture and genotyping of viruses in primary isolates

7.项目摘要/摘要 许多病原体不断进化以利用大量分布在靶组织上的宿主多糖来 进入并引发感染。在这类病原体中有甲型流感病毒(IAV)，它继续 构成年度大流行风险，并给美国经济带来沉重的财政负担。快速检测 不同宿主多糖类别的IAV特异性的表型变化可以提供早期迹象 病毒可传播性和暴发时感染潜力的评估。目前，科技- 将表型适应与直接基因组变化联系起来的难题是时间、成本和劳动力密集型， 导致对意想不到的新病毒株鉴定的显著延误。这样的延误可能会妨碍及时 由卫生官员和机构生产疫苗和制定有效的应对措施，通常包括-- Trous效果。在这里，我们提出了一种新的糖链阵列平台来快速识别潜在的基因改变 在初级分离株中IAV的糖链特异性发生了明显的变化。我们计划达到所需的高度敏感度 检测这些阵列中的低丰度病毒，方法是将合成的多价配体中的葡聚糖与EN- 提高了对IAV的亲和力，并开发了利用病毒神经细胞活性的新检测试剂- 用于信号放大的数据库酶。最后，我们将开发使收集成为可能的微阵列底物 以及基于其独特的糖结合表型对捕获的IAV进行测序。我们期待着这个角色- IAV种群由其多糖结合表型驱动的端化，消除了病毒扩增的需要。 阳离子分析前将提供更有效和准确的病毒株检测增加 大流行风险。建议的多糖阵列和检测方法将提供一种通用工具，可以 迅速扩展到利用多糖相互作用进入宿主生物体的其他类别的病原体。

7. Project Summary/Abstract Many pathogens continuously evolve to exploit host glycans that are abundantly distributed on target tissues to gain entry and initiate infection. In this class of pathogens is the Influenza A virus (IAV), which continues to pose annual pandemic risks and exerts a heavy financial burden on the US economy. Rapid detection of emerging phenotypic changes in IAV specificity for distinct host glycan classes can provide an early indication of virus transmissibility and an assessment of infection potential at the onset of an outbreak. Currently, tech- niques that connect a phenotypic adaptation to a direct genomic change are time-, cost- and labor-intensive, causing significant delays in identification of unexpected new viral strains. Such delays may preclude timely production of vaccines and formulation of effective responses by health officials and agencies, often with disas- trous effects. Here we propose a new glycan array platform to rapidly identify genetic alterations underlying distinct shifts in glycan specificity in IAVs in primary isolates. We plan to achieve the high sensitivity required to detect low abundance viruses in these arrays by presenting glycans in synthetic polyvalent ligands with en- hanced avidity toward IAVs and by developing new detection reagents that utilize the activity of viral neurami- dase enzymes for signal amplification. Finally, we will develop microarray substrates that enable the collection and sequencing of captured IAVs based on their unique glycan-binding phenotype. We anticipate that charac- terization of IAV populations driven by their glycan binding phenotype that obviates the need for virus amplifi- cation prior to analysis will provide more efficient and accurate determination of viral strains with increased pandemic risks. The proposed glycan array and detection methods will provide a general tool that could be rapidly extended to other classes of pathogens that utilize glycan interactions to enter their hosts organism.