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

[0905713] wigginton该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。国际研究奖学金项目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。该计划的奖励为联合研究提供了机会，并利用独特或互补的设施、专业知识和国外的实验条件。该奖项将支持Krista R. Wigginton博士与瑞士洛桑联邦理工学院的Tamar Kohn博士进行为期24个月的研究。太阳能消毒是从废水和饮用水中去除病原体的一种有效、低成本和可持续的处理选择。对于阳光照射的水体，微生物污染物的失活可以通过直接和间接的机制发生。在清澈的水中，会对生物体造成直接伤害吗？s的核酸是由UVB光产生的。在高度着色的水域中，如典型的废物稳定池（WSPs）， UVB光仅穿过光区的顶层。因此，wsp中微生物污染物的直接失活仅在池塘表面附近的薄层中显着。另一方面，UVA和可见光可以进一步渗透到光区并引起间接的光失活。当氧气存在时，存在于微生物内部（如卟啉、某些氨基酸）或水柱中的敏化剂的光激发可导致活性氧（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发生反应并导致其失活。尽管太阳能消毒已被广泛使用，但对于地表水中活性氧与微生物污染物之间发生的反应仍然知之甚少。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等人之前的研究研究了噬菌体MS2的失活，MS2是一种常用的人类病毒替代品，在天然致敏材料存在的情况下，采用太阳能处理。结果表明，在这些条件下，单线态氧（1O2）是MS2失活的最重要过程。Kohn博士最近的研究？s的研究小组已经证明，在被1O2灭活几次后，MS2衣壳内的病毒RNA大部分被保存了下来。这表明MS2通过1O2的光失活发生在病毒衣壳蛋白的修饰上，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位点，表征与ROS的反应机制，并确定哪些氧化位点负责病毒失活。具体而言，用1O2处理MS2噬菌体和3型脊髓灰质炎病毒，并用标准电镀方法监测灭活情况。利用基质辅助激光解吸/电离质谱法（MALDI-MS）和电喷雾电离串联质谱法（ESI-MS/MS）鉴定和监测衣壳蛋白的变化。MALDI-MS和ESI-MS/MS已成为病毒学研究的重要工具，并已被用于研究病毒蛋白结构和整个病毒。到PI？这是MS技术第一次被用于研究病毒蛋白的氧化。在质谱分析的同时，用表面增强拉曼光谱（SERS）检查了蛋白质衣壳的光氧化。活病毒和灭活病毒的SERS光谱与多元统计工具相结合，最终，PI寻求使用这些技术开发一种测量病毒活力的预测工具。为了证实观察结果在真实水域的正确性，将在废物稳定池样品中进行平行实验。实际水的结果将与实验室水实验结果进行比较。此外，两种病毒类型的失活途径将被比较，然后用于预测一些水传播的人类病毒的1O2失活途径和动力学。

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.