Development of High-Throughput and Real-Time Methods for the Detection of Infective Enteric VirusesEPA Grant Number: R833008
Title: Development of High-Throughput and Real-Time Methods for the Detection of Infective Enteric Viruses
Investigators: Chen, Wilfred , Mulchandani, Ashok , Myung, Nosang V.
Current Investigators: Chen, Wilfred , Mulchandani, Ashok , Myung, Nosang V. , Yates, Marylynn V.
Institution: University of California - Riverside
EPA Project Officer: Page, Angela
Project Period: August 31, 2006 through August 30, 2009 (Extended to August 26, 2011)
Project Amount: $600,000
RFA: Development and Evaluation of Innovative Approaches for the Quantitative Assessment of Pathogens in Drinking Water (2005) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
The main goal of this research is to improve on the current analytical methods for quantitative detection of infective enteric viruses, specifically the non-polio enteroviruses (NPEV), in drinking water. The overall objective of the research is to develop methods to provide high-throughput real-time detection and quantitation of infective enteric viruses from contaminated water. The specific objectives of this research are to: (1) Develop a new generation of molecular beacons (MBs) based on quantum dots as the fluorophore and gold nanoparticles as the quencher for improved sensitivity and multiplexing capability; (2) Develop a real-time method to probe and quantify infective enteric viruses using TAT- or transferrin-modified nuclease-resistant MBs in infected cell lines without permeabilization; (3) Develop a genetically engineered cell line to probe and quantify infective enteric viruses by generating a protease-sensitive FRET protein pair using an improved CFP-YFP pair; (4) Evaluate the use of flow cytometry for high-throughput sample processing; and (5) Evaluate the above methods to rapidly detect and quantify the presence of infective NPEV in environmental water samples.
The research approach will include in-vivo real-time monitoring of the virus replication in (1) infected BGMK cells using improved nuclease-resistant molecular beacons with quantum dots as the donor and gold nanoparticles as the quencher delivered uniformly to the entire cell population by either TAT or transferrin cell penetrating proteins that will eliminate the need for cell fixing and permeabilizing, (2) genetically engineered cell lines with protease-sensitive protein FRET pair to detect only infective/culturable viruses. We will also explore the use of fluorescence-activated cell sorter for high-throughput, sensitive, selective and rapid analysis, in less than 4 h. The developed protocols will be tested on seeded and unseeded drinking water samples to enable an assessment of the sensitivity of the methods to inhibitors present in environmental waters. The specificity and quantitative capability of the protocols will be assessed by comparing results to standard quantitative cell culture analysis.
The lack of standardized methods that can be routinely performed to detect and quantify infective enteric viruses has limited the amount of information available on the occurrence of these viruses in drinking water and other environmental samples. Thus, an assessment of the risk of enteric virus infection as a result of exposure to drinking water is not possible at this time. The methods developed in this study should improve our ability to provide rapid and efficient results for the detection and quantitation of infective viruses in samples from environmental waters. In addition, the use of flow cytometry will facilitate the analysis of larger numbers of samples in a shorter period of time than is possible using current methods. This will enable the collection of occurrence data in drinking water, which can then be used to assess the potential public health risks from these organisms. More importantly, the methods can be adapted to facilitate the detection of other microorganisms in water, improving our ability to calculate risks from those organisms as well. It may also be used to determine the efficacy of virus inactivation by disinfectants and to monitor virus survival in water.