International Research Fellowship Program: Virus Inactivation in Sunlight-treated Waters: An Investigation on the Reactions Between Singlet Oxygen and Capsid Proteins

国际研究奖学金计划：阳光处理水中的病毒灭活：单线态氧与衣壳蛋白之间反应的研究

• 批准号: 0905713
• 负责人: Krista Wigginton
• 金额: $ 14.31万
• 依托单位: Wigginton Krista R
• 依托单位国家: 美国
• 项目类别: Fellowship
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2012-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0905713&HistoricalAwards=false
• 关键词: International; Research; Fellowship; Program; Virus

0905713WiggintonThis award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).The International Research Fellowship Program enables U.S. scientists and engineers to conduct nine to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad.This award will support a twenty-four-month research fellowship by Dr. Krista R. Wigginton to work with Dr. Tamar Kohn at Ecole Polytechnique Federale de Lausanne in Switzerland.Solar disinfection is an effective, low-cost, and sustainable treatment option for the removal of pathogens from wastewaters and drinking waters. For sunlit waters, the inactivation of microbial contaminants can occur by a direct and an indirect mechanism. In clear water, direct damage of the organism?s nucleic acid occurs by UVB light. In highly colored waters, like those typical of waste stabilization ponds (WSPs), UVB light extends only through the top layer of the photic zone. As a result, direct inactivation of microbial contaminants in WSPs is only significant in a thin layer near the surface of the ponds. UVA and visible light, on the other hand, can penetrate further into the photic zone and initiate indirect photo-inactivation. When oxygen is present, the photoexcitation of sensitizers present either within the microbe (e.g., porphyrins, certain amino acids) or in the water column can lead to the formation of reactive oxygen species (ROS) such as singlet oxygen (1O2), hydroxyl radical, hydrogen peroxide, etc. These species can subsequently react with cell constituents of the pathogens and lead to their inactivation. Despite the widespread use of solar disinfection, there is still little known regarding the reactions that occur between ROS and microbial contaminants in surface waters. As a result, drinking water and wastewater engineers are limited in their ability to predict and optimize the efficiency of solar virus disinfection. Previous work by Kohn, et al. examined the inactivation of bacteriophage MS2, a commonly used surrogate for human viruses, with solar treatment in the presence of natural occurring sensitizing materials. The results demonstrated that singlet oxygen (1O2) was the most important process in MS2 inactivation under these conditions. More recent work by Dr. Kohn?s group has shown that after several logs of MS2 inactivation by 1O2, the viral RNA inside the MS2 capsid is mostly preserved. This suggests that photoinactivation of MS2 via 1O2 occurs from modifications on the viral capsid proteins and is not due to oxidation of the genomic material. The aim of this research is to identify sites of photooxidation on viral capsids, characterize the reaction mechanisms with ROS, and determine which oxidation sites are responsible for virus inactivation. Specifically, MS2 bacteriophage and poliovirus type 3 are treated with 1O2 and inactivation is monitored with standard plating methods. Alterations in capsid proteins are identified and monitored using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). MALDI-MS and ESI-MS/MS have become critical tools in virology studies and have been previously used to study the structure of virus proteins as well as entire viruses. To the PI?s knowledge, this is the first time MS techniques have been used to study the oxidation of virus proteins. Concurrent with the mass spectral analyses, protein capsid photooxidation is examined with surface enhanced Raman spectroscopy (SERS). SERS spectra of live and inactivated virus are combined with multivariate statistical tools and ultimately, the PI seeks to use these techniques to develop a predictive tool for measuring virus viability.To confirm that the observed results occur in real waters, parallel experiments in waste stabilization pond samples will be conducted. The results from real waters will be compared with the laboratory water experiment results. Additionally, inactivation pathways of the two studied virus types will be compared and then used to predict 1O2 inactivation pathways and kinetics for a number of waterborne human viruses.

该奖项由2009年美国复苏和再投资法案(公法111-5)资助。国际研究奖学金计划使美国科学家和工程师能够在国外进行9至24个月的研究。该项目的奖项提供了联合研究的机会，并利用国外独特或互补的设施、专业知识和实验条件。该奖项将支持Krista R.Wigginton博士与瑞士洛桑联邦理工学院Tamar Kohn博士为期24个月的研究奖学金。太阳能消毒是去除废水和饮用水中病原体的一种有效、低成本和可持续的治疗选择。对于阳光充足的水域，微生物污染物的灭活可以通过直接和间接机制发生。在清水中，有机体的直接损伤？S核酸是在紫外线照射下发生的。在色彩浓郁的水域中，如典型的废物稳定塘(WSP)，UVB光只穿过光学区的顶层。因此，直接灭活可再生水中的微生物污染物只在靠近池塘表面的一层薄层中显著。另一方面，UVA和可见光可以进一步穿透到光学区，并启动间接光灭活。当氧气存在时，存在于微生物(例如，卟啉、某些氨基酸)或水柱中的敏感剂的光激发可导致活性氧物种(ROS)的形成，如单线态氧(1O2)、羟基自由基、过氧化氢等。这些物种随后可与病原体的细胞成分反应，导致其失活。尽管太阳能消毒已被广泛使用，但关于ROS与地表水中微生物污染物之间发生的反应，人们仍然知之甚少。因此，饮用水和废水工程师在预测和优化太阳能病毒消毒效率方面的能力有限。Kohn等人之前的工作。研究了噬菌体MS2的灭活情况，MS2是一种常用的人类病毒替代品，在自然产生的致敏材料存在的情况下进行太阳能处理。结果表明，在此条件下，单线态氧(1O2)是MS2失活的最主要过程。科恩？S博士的研究小组最近的工作表明，在ms2灭活1o2的几个日志之后，ms2衣壳内的病毒rna大部分被保存下来。这表明，MS2通过1O2的光灭活是通过对病毒衣壳蛋白的修饰而发生的，而不是由于基因组物质的氧化。本研究的目的是确定病毒衣壳上的光氧化部位，表征与ROS的反应机制，并确定哪些氧化部位是病毒灭活的原因。具体来说，用1O2处理MS2噬菌体和3型脊髓灰质炎病毒，并用标准的平板法监测灭活情况。使用基质辅助激光解吸电离质谱仪(MALDI-MS)和电喷雾电离串联质谱仪(ESI-MS/MS)鉴定和监测衣壳蛋白的变化。MALDI-MS和ESI-MS/MS已成为病毒学研究中的重要工具，并已被用于研究病毒蛋白质和整个病毒的结构。据皮S所知，这是首次用MS技术研究病毒蛋白的氧化。在进行质谱分析的同时，还用表面增强拉曼光谱(SERS)检测了蛋白质衣壳的光氧化。将活的和灭活的病毒的SERS光谱与多元统计工具相结合，最终，PI试图利用这些技术来开发一种预测工具来测量病毒的生存能力。为了确认观察到的结果是在真实水域中发生的，将在废物稳定塘样本中进行平行实验。实际水域的结果将与实验室的水实验结果进行比较。此外，将比较两种所研究的病毒类型的灭活途径，然后用于预测一些水传播的人类病毒的1O2灭活途径和动力学。