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Automated Methods for the Quantification and Infectivity of Human Noroviruses in WaterEPA Grant Number: R833831
Title: Automated Methods for the Quantification and Infectivity of Human Noroviruses in Water
Investigators: Straub, Timothy M. , Bartholomew, Rachel , Bruckner-Lea, Cindy , Ozanich, Richard
Institution: Battelle Memorial Institute, Pacific Northwest Division
EPA Project Officer: Klieforth, Barbara I
Project Period: July 1, 2008 through June 30, 2010 (Extended to June 30, 2011)
Project Amount: $592,140
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens and Cyanobacteria and Their Toxins in Drinking Water (2007) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
This project will develop and demonstrate an automated system approach for large volume concentration, purification, and quantitative detection of CCL viruses in drinking water. For systems testing we will use bacteriophages MS2 and PRD1 as surrogates, but final testing and analysis of drinking water samples shall be conducted with human noroviruses (hNoV). Whole virus capture within this system will also allow us to employ our recently published 3-D cell culture method to assess hNoV infectivity when these viruses are found in drinking water.
Optimization of water sample collection, sample processing, quantitative detection, and assessment of infectivity of hNoV in drinking water supplies will provide valuable occurrence information for both public health officials and the water industry.
Our research approach will comprise 5 Specific Aims. Specific aim 1: Evaluate the automated concentration of viruses from large volumes of drinking water using hollow fiber ultrafiltration (HFF) in the context of a) engineering into an automated sampling system, and b) providing efficient and reproducible concentration of viruses within an automated system. Specific aim 2: Develop and validate fluidic secondary capture and purification of intact viruses from the retained volumes in HFF to allow analysis by PCR and cell culture. Specific aim 3: Optimize fast, quantitative reverse transcription real-time PCR (qRT real-time PCR) for the detection of the purified hNoV in drinking water. Specific aim 4: Optimize our 3-D cell culture infectivity assay for hNoV, and test this assay for identification of infectious hNoV in spiked and unspiked drinking water samples. Specific aim 5: Design and test process integration of HFF and secondary capture and purification, and evaluate the system performance. For the purposes of this project, the concentrated samples will be split such that half of the purified sample will be evaluated offline by qRT real-time PCR and the other half evaluated for infectivity in the 3-D cell culture.
Development of an approach for automated waterborne viral monitoring is expected to significantly improve reproducibility compared to manual methods. Secondary concentration using novel carbohydrate and lectin affinity reagents may additionally provide broad agent secondary capture and purification (simultaneous capture of viral, bacteria, and protozoan pathogens) such that the volumes delivered and evaluated in PCR represent 100 – 1,000 L of water. The qRT real-time PCR detection component will provide a rapid (hours) and quantitative assessment of hNoV in drinking water; and novel 3-D cell culture techniques will allow, for the first time, evaluation of human NoV infectivity in environmental samples. Overall, the products of this research will provide rapid, sensitive, and specific detection of potential contamination of drinking water by pathogenic microorganisms. This will allow development of more accurate microbial risk assessment models to better protect public health.