High-throughput single-molecule analysis of the influenza A genome structure and assembly

流感病毒是引起人类和动物流感的微小病原体。我们建议使用特殊的显微镜方法来了解流感病毒遗传物质的结构，以及遗传物质之间的相互作用如何在流感大流行中变得重要。流感病毒的遗传物质或基因组由RNA(编码遗传信息的长链分子)组成，分为八个单独的片段。流感病毒的自然宿主是野鸟；然而，在一个被称为“重组”的过程中，不同的病毒株可以交换RNA片段，从而使我们能够感染其他宿主生物，包括人类。重新分类事件导致产生全新的病毒株，人类以前从未接触过这种病毒株。当人类对一种新型流感病毒没有现有的免疫力时，重组可能会导致致命的全球大流行，如1918年的西班牙流感大流行，导致全球5000多万人死亡，以及2009年的猪流感大流行。虽然H5N1禽流感毒株还没有重新组合到可以可靠地感染人类并导致大流行的程度，但禽类疫情已经对家禽业造成了毁灭性的破坏，因为数以百万计的禽类被扑杀以减少病毒传播，导致巨大的经济损失。尽管流感是研究最深入的病毒之一，但我们仍然不确定病毒颗粒内基因组片段如何准确地相互联系。我们对这一过程的大部分知识都依赖于使用方法获得的数据，这些方法一次测量数百万个分子(或粒子)的性质，从而报告所有分子的平均性质。我们计划使用先进的单分子技术，允许我们一次研究一个分子(或粒子)；这使我们能够看到每个分子(或粒子)的独特细节，而这些细节可能是传统的分析或生物学方法无法看到的。我们计划通过使用许多小片段的荧光DNA来进行单分子分析，这些DNA将沿着病毒基因片段一直结合在一起，以非常高的分辨率检测基因片段的存在并绘制出结构图。此外，荧光DNA将无法结合到与其他基因组片段相互作用的基因组片段(由于片段之间的密切接触，这些区域将无法访问)，从而使我们能够建立起病毒颗粒内关键的RNA-RNA相互作用的图景。为了在小DNA片段检测到病毒颗粒时对它们进行成像，我们将使用一种专门的“单分子荧光”显微镜，这种显微镜经过精心设计，可以检测和监测存在于检测区的单个荧光分子(与要求检测区存在数千或数百万个分子的传统显微镜不同)。我们预计，我们的研究将有助于科学界更好地了解流感病毒基因组片段是如何相互作用并导致大流行的，并有助于控制和阻止其传播。我们的发现还应有助于我们了解和控制对人类和动物造成危险的其他病毒，并有助于开发新的诊断测试，以快速和灵敏地检测流感和其他致病病毒。

The influenza (flu) virus is the microscopic pathogen that causes flu in humans and animals. We propose to use special microscopy methods to understand the structure of the genetic material of the flu virus, and how interactions between the genetic material can become important in flu pandemics. The genetic material, or genome, of the flu virus is made of RNA (a long chain of molecules that encodes genetic information) and is divided into eight individual segments. The natural host of the flu virus is wild birds; however, in a process called 'reassortment', different strains of the virus can swap RNA segments in a way that allows us to infect other host organisms, including humans. Reassortment events result in the generation of entirely new virus strains, to which humans have never previously been exposed. When there is no existing immunity in the human population to a novel influenza virus, reassortment can cause deadly worldwide pandemics, such as the Spanish flu pandemic in 1918, which was responsible for over 50 million deaths worldwide, and the 'swine flu' pandemic in 2009. Whilst H5N1 'bird flu' strains haven't yet reassorted to the extent that they can reliably infect humans and cause a pandemic, outbreaks in birds have been devastating for the poultry industry since millions of birds have been culled to reduce virus spread, resulting in huge economic losses.Despite flu being one of the best-studied viruses, we are still unsure how exactly the genomic segments contact each other inside virus particles. Much of our knowledge of this process has relied on data obtained using methods that measure the properties of millions of molecules (or particles) in one go, and therefore report on the averaged properties of all of the molecules. We plan to use advanced 'single-molecule techniques', which allow us to study one molecule (or particle) at a time; this allows us to see details unique to each molecule (or particle) that may be impossible to see using traditional analytical or biological methods. We plan to perform our single-molecule analysis by using many small pieces of fluorescent DNA, that will bind all the way along the virus gene segments, to detect the presence and map the structure of the gene segments at very high resolution. Further, the fluorescent DNAs will not be able to bind to sections of the genome that interact with other genomic segments (as these areas will not be accessible due to the close contacts between segments), thereby allowing us to build up a picture of crucial RNA-RNA interactions within virus particles. To image the virus particles as they are being detected by the small DNA pieces, we will use a specialised "single-molecule fluorescence" microscope, which is carefully designed to allow the detection and monitoring of individual fluorescent molecules present in a detection zone (as opposed to conventional microscopes that require thousands or millions of molecules to be present in a detection zone). We anticipate that our research will help the scientific community to better understand how the genome segments of the flu virus interact and cause pandemics, and help in efforts to control and stop its spread. Our discoveries should also help us understand and control other viruses that cause danger to humans and animals, and contribute towards development of new diagnostic tests for rapid and sensitive detection of influenza and other pathogenic viruses.