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

0905713 Wiggington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。国际研究奖学金计划使美国科学家和工程师能够在国外进行9到24个月的研究。 该计划的奖项提供了联合研究的机会，以及使用独特或互补的设施，专业知识和国外的实验条件。Wigginton与瑞士洛桑联邦理工学院的Tamar Kohn博士合作。太阳能消毒是一种有效、低成本和可持续的处理方法，可用于去除废水沃茨和饮用沃茨中的病原体。 对于阳光照射的沃茨，微生物污染物的失活可以通过直接和间接机制发生。在清水中，对生物体的直接伤害？的核酸发生的UVB光。在高度着色的沃茨中，如典型的废物稳定池（WSP），UVB光仅穿过透光层的顶层。因此，直接灭活的微生物污染物在水溶性悬浮物是唯一显着的薄层附近的表面的池塘。另一方面，UVA和可见光可以进一步渗透到透光区并引发间接光灭活。当存在氧气时，存在于微生物内的敏化剂的光激发（例如，卟啉、某些氨基酸）或水柱中的活性氧物质（ROS）可导致形成活性氧物质（ROS），例如单线态氧（1 O2）、羟基自由基、过氧化氢等。这些物质随后可与病原体的细胞成分反应并导致其失活。尽管太阳能消毒被广泛使用，但关于表面沃茨中ROS和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预测和优化太阳能病毒消毒效率的能力有限。 Kohn等人先前的工作检查了在天然存在的敏化材料存在下用太阳能处理的噬菌体MS 2（一种常用的人类病毒替代物）的灭活。 结果表明，单线态氧（1 O2）是在这些条件下MS 2灭活的最重要的过程。科恩博士最近的工作？研究小组的研究表明，在1 O2灭活MS 2几个对数后，MS 2衣壳内的病毒RNA大部分被保留下来。这表明，通过1 O2的MS 2的光灭活发生在病毒衣壳蛋白的修饰，而不是由于基因组材料的氧化。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灭活。 具体而言，用1 O2处理MS 2噬菌体和脊髓灰质炎病毒3型，并用标准平板法监测灭活。 使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改变。MALDI-MS和ESI-MS/MS已成为病毒学研究中的关键工具，并且先前已用于研究病毒蛋白质以及整个病毒的结构。对私家侦探？这是第一次用质谱技术研究病毒蛋白的氧化。 与质谱分析同时，用表面增强拉曼光谱（Sers）检查蛋白质衣壳光氧化。 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试图使用这些技术来开发一种预测工具，用于测量病毒的viability.To确认所观察到的结果发生在真实的沃茨中，将在废物稳定池样品中进行平行实验。 将真实的沃茨的结果与实验室水实验结果进行比较。 此外，两种研究的病毒类型的灭活途径将进行比较，然后用于预测1 O2灭活途径和动力学的一些水性人类病毒。

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.