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

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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.