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

7.项目总结/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聚糖， 进入并引发感染在这类病原体中是甲型流感病毒（IAV），它继续在 每年都有大流行的风险，并给美国经济带来沉重的财政负担。快速检测 IAV对不同宿主聚糖类特异性的表型变化可以提供早期指示 病毒传播性的评估和在爆发初期对感染潜力的评估。目前，技术- 将表型适应与直接基因组变化联系起来的方法是时间、成本和劳动密集型的， 导致对意外的新病毒株的鉴定的显著延迟。这种拖延可能会妨碍及时 由卫生官员和机构生产疫苗和制定有效的应对措施，往往与疾病有关， 裤子效果在这里，我们提出了一种新的聚糖阵列平台，以快速识别潜在的遗传改变。 原代分离株中IAV的聚糖特异性发生明显变化。我们计划实现所需的高灵敏度， 通过在合成多价配体中呈递聚糖， 增强了对IAV的亲和力，并通过开发新的检测试剂，利用病毒性神经炎的活性， 用于信号放大的数据酶。最后，我们将开发微阵列基底， 以及基于其独特的聚糖结合表型对捕获的IAV进行测序。我们预计查拉克- 通过其聚糖结合表型驱动的IAV群体的化，避免了病毒扩增的需要， 在分析前使用阳离子将提供更有效和准确的病毒株测定， 流行病风险。所提出的聚糖阵列和检测方法将提供一种通用工具， 迅速扩展到利用聚糖相互作用进入其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.