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NCER Grantee Research Project Results

Development of a method for rapid virus detection using a viral bioreporter cell line

EPA Grant Number: F5D71330
Title: Development of a method for rapid virus detection using a viral bioreporter cell line
Investigators: Hwang, Yu-Chen
Institution: University of California - Riverside
EPA Project Officer: Zambrana, Jose
Project Period: September 1, 2005 through September 1, 2007
Project Amount: $105,119
RFA: GRO Fellowships for Graduate Environmental Study (2005)
Research Category: Academic Fellowships

Description:

Objective:

Enteric viruses can be easily transmitted through the fecal-oral route and cause a diverse array of clinical manifestations . Recent outbreaks associating with enteric viral contamination in aquatic environment have called for the development of a more efficient and accurate virus monitoring system. Conventional cell culture-based assays are too time-consuming and labor-intense for widespread monitoring. Studies have been directed toward applying biomolecular tests, such as RT-PCR, for the more sensitive and rapid detection of viral genome. However, PCR-based methods fail to identify the viability of the viruses. Improved methods that can be applied for rapid detection of infectious viruses are essential to provide quick and robust information for public health risk assessment. The proposed research is to investigate the possibility of using in vivo monitoring system to detect infectious viruses.

The objective of this research is to develop a rapid, in vivo detection system for infectious enteric viruses from environmental water samples, using poliovirus as a representative.

Approach:

Poliovirus, a member of enteric viruses, is a non-enveloped virus with a single-stranded RNA genome, which is translated into a single polypeptide upon infection; the polypeptide is subsequently cleaved by viral proteases 2A (2Apro) and 3C (3Cpro). 2Apro serves an excellent target for viral detection because it is highly expressed immediately after infection and the proteolysis of 2Apro is extremely efficient and selective. It is proposed to develop a fluorescent reporter system in which genetically engineered cells express a hybrid fluorescent indicator composed of a linker peptide, which is exclusively cleaved by the 2Apro, flanked with a cyan fluorescent protein and a yellow fluorescent protein undergoing fluorescence resonance energy transfer (FRET). The covalent linkage between two fluorophores is disrupted due to 2Apro activity upon viral infection, which leads to spatial separation of the fluorescent protein pair and results in a loss of FRET.

Expected Results:

The method developed in this study should improve our ability to provide efficient and reliable results for the detection of infectious viruses in environmental samples. This will enable the collection of occurrence data in environmental water, which can then be used to assess public health risks of these organisms. The ultimate goal of this research will create an innovative approach for determining occurrence and concentration of infectious viruses in water and soil samples. This method should be widely applicable to all enteric viruses monitoring in source water, treatment and distribution systems, and will serve as a model for development of bioreporters for the other RNA viruses.

Supplemental Keywords:

Enteric viruses, FRET, viral protease, bioreporter cell line, detection method,, RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Monitoring/Modeling, Biochemistry, Environmental Monitoring, aquatic ecosystem, detection system, water quality, water monitoring, enteric pathogens, bioreporter cell lines

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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