Determination of Markers of High Pathogenicity in Influenza

描述（由申请人提供）：甲型流感中特定的突变蛋白可能是与大流行毒株毒力相关的重要诊断标记。对PB1-F2序列的分析发现了一个单一突变（N66S），该突变在香港1997年H5N1高致病性流感和1918年大流行流感毒株中都是保守的。其他研究表明，N1甲型流感病毒的NA柄区也影响毒力。快速筛选这些和其他尚未发现的毒力因素的能力，将大大提高我们识别具有高死亡率风险的新菌株的能力，并使公共卫生专业人员能够有效应对，防止广泛感染和/或死亡。不幸的是，目前还没有一种快速、多路的方法来筛选毒力因子。我们实验室最近的研究表明，一种基于气相沉积的纳米制造技术可以产生银纳米棒阵列，当它们用作表面增强拉曼光谱（SERS）衬底时，它们表现出极高的电磁场增强。我们的初步结果表明，这些新的SERS底物可以作为诊断工具，快速、灵敏地检测RNA和DNA病毒的拉曼光谱。SERS光谱有效地充当分子指纹，提供有关目标的特定和定量信息。利用这种方法，我们已经证明，病毒株、血清型、基因型和寡核苷酸之间的区分是可能的，仅基于它们的内在SERS谱。本研究项目的长期目标是开发一种快速、灵敏、特异的多路复用方法来检测和量化流感毒力因子。当前提案的目标是开发一种基于寡核苷酸的捕获阵列来检测流感RNA，从而允许在没有扩增或标记的情况下进行毒性评估。这项研究将为开发人类流感感染风险评估所急需的新筛查方法提供科学基础。该项目将：1)利用合成寡核苷酸开发毒力因子杂交策略；2)鉴定实验室病毒中毒力相关的PB1-F2突变；3)鉴定实验室病毒中毒力相关的NA柄基序；4)在临床样本中验证基于sers的流感毒力因子检测。该提案的创新之处在于开发了一种新的纳米制造方法，以生产一种高灵敏度、快速和可重复的平台技术，用于检测流感毒力因子，这是目前尚不存在的能力。该提案将利用明确定义的SERS底物平台、成熟的SERS传感技术以及物理和生物研究者的综合专业知识。在这个项目结束时，我们期望我们将开发并验证一种纳米光学方法，作为检测流感毒力因子的有力工具。该项目最终将证明生物纳米技术与振动光谱学相结合能够增强对新发病毒感染的检测和诊断，并具有临床应用价值。

DESCRIPTION (provided by applicant): Specific mutated proteins in influenza A may be important diagnostic markers related to the virulence of pandemic strains. Analysis of the PB1-F2 sequence identified a single mutation (N66S) that was conserved for both the Hong Kong 1997 H5N1 highly pathogenic influenza as well as the 1918 pandemic strain. Additional studies have demonstrated that the NA stalk region of N1 influenza A viruses also affects virulence. The ability to rapidly screen for these, and other yet-to-be- discovered virulence factors, would significantly improve our ability to identify emerging strains with risk of high mortality, and allw public health professionals to effectively respond to prevent widespread infection and/or death. Unfortunately, a rapid, multiplexed method to screen for virulence factors does not exist. Recent research in our laboratories has demonstrated that a nanofabrication technique based on vapor deposition produces Ag nanorod arrays that exhibit extremely high electromagnetic field enhancements when they are used as surface-enhanced Raman spectroscopy (SERS) substrates. Our initial results suggest that these novel SERS substrates can be used as diagnostic tools to rapidly and sensitively detect the Raman spectra of RNA and DNA viruses. SERS spectra effectively act as molecular fingerprints that provide specific and quantitative information about the target. Using this method, we have shown that differentiation is possible between virus strains, serotypes, genotypes and oligonucleotides based solely on their intrinsic SERS spectra. The long-term goal of this research project is to develop a rapid, sensitive, and specific multiplexed method to detect and quantify influenza virulence factors. The objective of the current proposal is to develop an oligonucleotide-based capture array to detect influenza RNA, thus allowing for virulence assessment, without amplification or labeling. This research will provide the scientific foundation for development of new screening methods that are critically needed for risk assessment in human influenza infections. The project will: 1)develop a hybridization strategy for virulence factors using synthetic oligonucleotides, 2) identify the virulent-associated PB1-F2 mutation in laboratory viruses, 3) identify the virulent- associated NA stalk motif in laboratory viruses, and 4) validate the SERS-based assay for influenza virulence factors in clinical samples. The innovative aspect of this proposal lies in the development of a novel nanofabrication method to produce a high sensitivity, rapid and reproducible platform technology for detection of influenza virulence factors, a capability that does not currently exist The proposal will take advantage of a well-defined SERS substrate platform, proven SERS sensing technology, and the integrated expertise of physical and biological investigators. At the conclusion of this project, it is our expectation that we will have developed and validated a nano-optical method for use as a powerful tool to detect influenza virulence factors. This project will ultimately demonstrate that bio-nanotechnology combined with vibrational spectroscopy has the ability to enhance detection and diagnosis of emerging viral infections with clinical applications. PUBLIC HEALTH RELEVANCE: There are several highly pathogenic influenza strains that are able to infect humans and are considered as potential precursors for future influenza pandemics. Unfortunately, to date there is no rapid and multiplexed method to screen for the biochemical markers of influenza virulence. The successful development of a rapid and sensitive method for screening of highly pathogenic influenza virulence factors would significantly improve our ability of identify emerging strains with a risk of high mortality, and allow public health professional to effectively respond to prevent widespread infection and/or death.