Novel aptamer-based diagnostics and antiviral therapeutics for animal influenza viruses

禽流感感染对动物健康构成重大威胁，是人类流感大流行的潜在起源。最近的一个例子是猪中出现了一种新的大流行性流感病毒（h1n1 - pdm），目前正在造成广泛感染和一些人死亡。同样，H9N2亚型禽流感病毒以及高致病性H5N1和H7 （H7N1、H7N3和H7N7）亚型禽流感病毒在许多国家的家禽中持续流行，这突出表明需要改进监测和控制措施。这将需要快速、易于使用的人工智能诊断，能够在不需要大量实验室资源的情况下区分这些高度流行的流感病毒的亚型。此外，改进用于治疗和预防的抗病毒药物也将有助于尽量减少这些病毒对人类和动物的影响。考虑到这些当前和未来的需求，该项目将利用被称为“适体”的合成单链核酸分子来开发下一代流感病毒的诊断和可能的治疗分子。由于甲型流感病毒存在许多亚型，这种通用技术可作为敏感和特异性诊断分析的基础，可同时检测不同亚型的流感病毒（鉴别诊断分析）。此外，还可以检查对流感病毒显示出有希望结合亲和力的适体作为抗病毒治疗药物的潜力，例如针对H1N1（猪流感）和H5N1（禽流感）流感病毒。将生产适配体，特异性和选择性地结合流感病毒表面糖蛋白血凝素（HA）和神经氨酸酶（NA）（赋予亚型特异性）以及核蛋白（NP）和基质（M1）蛋白（在所有AI病毒中都是保守的）的选定亚型，然后与基于金纳米颗粒的高灵敏度检测技术相结合。这些适体偶联物有望在临床样品中与病毒结合形成核壳纳米结构。适体病毒纳米结构可以通过光学或电学方法进行量化。这些aptananosensor装置的检测灵敏度可以在临床样品中检测到100到1000个病毒颗粒，并且可以在30分钟或更短的时间内检测到。aptananosensor应该是经济的，便携的和简单的使用高效和快速的笔侧诊断。最重要的是，它将为鉴别诊断感染人类和动物的流感病毒提供必要的高灵敏度和特异性。由于所选择的适体对病毒包膜蛋白具有高亲和力，因此它们也可以阻止病毒复制。在这个项目中，我们还将在体外试验和动物模型中探索选定的适体抗流感病毒感染的治疗特性。这可以为开发新一代治疗人类或动物流感病毒感染的疗法提供一个平台。第一步将是评估组织培养中选定的高亲和力适配体对选定的H1N1和H5N1病毒的抗病毒特性。在体外显示出有希望的结果的适体体将在体内进行测试。选定的哺乳动物和禽类动物模型将被选定的H1N1和H5N1病毒株感染，并将评估适体的保护作用，以评估其预防感染和控制感染的能力。与未治疗的感染动物相比，将通过将疾病症状的发作和病毒滴度的降低相关联来评估动物的保护功效。人工智能适配体将为诊断和治疗平台提供共同的分子实体，以对抗这种始终存在但不断变化的威胁。

Avian influenza (AI) infections pose a major threat to animal health and are potential progenitors for human influenza pandemics. A recent example is the emergence of a new pandemic influenza virus (H1N1pdm) from pigs which is currently causing widespread infection and some mortality in humans. Similarly, the continued prevalence of avian influenza viruses of the H9N2 sub-type, and the highly pathogenic H5N1 and H7 (H7N1, H7N3 and H7N7) subtypes in poultry in many countries highlights the need for improved surveillance and control measures. These will require rapid, easy to use AI diagnostics that can differentiate subtypes of these highly prevalent influenza viruses without the need for extensive laboratory resources. Additionally, improved antiviral drugs for treatment and prophylaxis would also help to minimise the impact of these viruses in both humans and animals. Considering these immediate and future requirements, this project will exploit synthetic single stranded nucleic acid molecules known as 'aptamers' to develop the next generation of diagnostic and possibly therapeutic molecules for influenza viruses. Since Influenza A viruses exist in many subtypes this versatile technology can be used as the basis of sensitive and specific diagnostic assays that can detect different subtypes of influenza viruses simultaneously (differential diagnostic assays). In addition the aptamers that show promising binding affinity for influenza viruses can also be examined for their potential as antiviral therapeutics, for example against H1N1 (swine flu) and H5N1(bird flu) influenza viruses. Aptamers will be produced that specifically and selectively bind to selected subtypes of influenza virus surface glycoproteins heamagglutinin (HA) and neuraminidase (NA) (which confer subtype specificity) and the nucleoprotein (NP) and matrix (M1) protein (which is conserved in all AI viruses) and then combined with highly sensitive detection technologies based upon gold nanoparticles. These aptamer conjugates are anticipated to bind to viruses in clinical samples to form core-shell nanostructures. The aptamer-virus nanostructures than can be quantified by either optical or electrical methods. The detection sensitivity of these aptananosensor devices would be such that 100 to 1000 virus particles in a clinical sample and could be detected within 30 minute or less. An aptananosensor should be economical, portable and simple to use for efficient and rapid pen-side diagnosis. Above all it will provide the high sensitivity and specificity necessary for differential diagnosis of influenza viruses infecting both humans and animals. Since the selected aptamers will have a high affinity for viral envelope proteins they could also block virus replication. In this project, we will also explore the therapeutic properties of selected aptamers against influenza virus infection in both in vitro assays and in animal models. This could provide a platform to develop new generation of therapeutics against influenza virus infections in humans or animals. The first step will be to assess the antiviral properties of selected high affinity aptamers in tissue culture against selected H1N1 and H5N1 viruses. The aptamers that show promising results in vitro will then be tested in vivo. The selected mammalian and avian animal models will be infected with selected H1N1 and H5N1 virus strains and the protective effects of the aptamers will be assessed in their ability to prevent infection and also to control infection. The protective efficacy in animals will be assessed by correlating the onset of disease signs and reduction in viral titres compared with infected untreated animals. AI aptamers will provide common molecular entities for both diagnostic and therapeutic platforms in the fight against this ever present, yet ever changing threat.