Grantee Research Project Results
2007 Progress Report: Development of High-Throughput and Real-Time Methods for the Detection of Infective Enteric Viruses
EPA Grant Number: R833008Title: Development of High-Throughput and Real-Time Methods for the Detection of Infective Enteric Viruses
Investigators: Chen, Wilfred , Myung, Nosang V. , Mulchandani, Ashok
Current Investigators: Chen, Wilfred , Mulchandani, Ashok , Yates, Marylynn V. , Myung, Nosang 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 Period Covered by this Report: August 31, 2006 through August 30, 2007
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
Objective:
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 fluorescence resonance energy transfer (FRET) protein pair using an improved cyan fluorescent protein-yellow fluorescent protein (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.
Progress Summary:
Research Accomplishments in Year 1
Detection of Hepatitis A Virus (HAV) Using a Combined Cell Culture–MB Assay. Although we have already demonstrated the use of MB for the detection of infective coxsackievirues, we were interested in whether the same approach could result in similarly rapid detection of HAV, which do not usually produce plaque in less than 1 week. An MB, HAV1, specifically targeting a 20-base pair (bp) 5’ non-coding region of HAV, was designed and synthesized. These MBs were introduced into fixed and permeabilized fetal rhesus monkey kidney (FrhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were easily visualized using a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB HAV1. A non-specific MB, which is not complementary to the viral RNA sequence, produced no visible fluorescence signal (Figure 1a). To investigate whether this approach can be used to rapidly detect low doses of infectious HAV, cultures infected with 1 plaque-forming unit (PFU) of HAV were analyzed from 6 to 48 hours post-infection (P.I.) in order to investigate the minimum time required to consistently detect a positive fluorescent signal. As shown in Figure 2, fluorescent cells can be visualized even within 6 h P.I., and the number of fluorescent cells increased with increasing infection time (Figure 1b). To investigate whether this 6-hour infection window could be used to quantify the number of infectious HAV, cells were infected with 1 to 1000 PFU HAV, and the average number of fluorescent cells was determined. A linear correlation was obtained by plotting the number of fluorescent cells versus log PFU (Figure 2), indicating that this MB-based assay can be used as a quantification tool for detecting infectious HAV. A detection limit of 1 PFU was obtained at 6 hours P.I., a 32-fold reduction in detection time compared with the 8-day conventional plaque assay.
Figure 1. (a) Visualization of Uninfected or Infected FrhK-4 Cells by Introducing MBs. I. Uninfected cells with 5 μM MB HAV1. II. Highly infected (107 PFU) cells at 72 hours P.I. with 5 μM nonspecific MB. III. Uninfected cells with 5 μM MB HAV1/oligonucleotide hybrids. IV. Highly infected (107 PFU) cells at 72 hours P.I. with 5 μM MB HAV1. (b) Visualization of FrhK-4 cells infected with 1 PFU at various P.I. time points. Scale bar = 20 μm.
Figure 2. (a) A Correlation Between the Fluorescent Cells and the Corresponding PFU at 6 hours P.I. (b) Comparison of the conventional 8-day plaque assay and the fluorescence assay. FrhK-4 cells infected with unknown viral dosages for 6 hours were fixed and permeabilized before 1 hour incubation with 5 μM MB HAV1. The number of fluorescent cells of 27 fields within the chosen chamber well (y) was recorded, and the log-PFU values (x) were calculated by the calibration equation obtained above. In parallel, the viral dosages were independently determined using the 8-day plaque assay.
Evaluate the Use of Flow Cytometry for High-Throughput Detection. A real-time detection method, employing mammalian cells expressing fluorescent proteins exhibiting FRET, has recently been developed for the detection of enteroviral infection (Hwang, et al., 2006). Even though fluorescent microscopy can be used to detect infected cells, this procedure is tedious and is not amenable to automation. We therefore used fluorescent-activated cell sorter (FACS) to detect infected cells, with the hope of achieving an automated high-throughput sample processing. In this study, we used Poliovirus 1 (PV1) as the model system for FACS analysis.
Experiments were conducted to evaluate if FACS was a sensitive method for detecting PV1 infection. Cells were infected with PV1 at a multiplicity of infection (MOI) of 0.5. Cells were harvested at a certain time point and analyzed by FACS. Representative FACS scatter plots from PV1 infected cells are shown in Figure 3. Increasing numbers of infected cells were detected as the infection progressed, indicating that the FACS assay was suitable for following the kinetics of infection.
Figure 3. Time Course of PV1 Infection of the BGM-PV Cells. BGM-PV cells grown in 6-well plates were infected with 10-1 dilution of PV1 (1.6 X 106 PFU/ml; MOI = 0.5; 20 minutes absorption). Cells were trypsinized, washed with 1X TBSS + 5 mM EDTA, pH 8.0, resuspended in the same buffer, and then subjected to FACS. Cells were gated according to their size and granularity (x axis, forward-scattered cells; y axis, side-scattered cells) to identify intact, single cells. The gated cells were displayed on dot plots in which the x axis is the CFP intensity, and y axis is CFP/YFP intensity. The percentage of infected cells was determined by counting the number of cells without FRET signal divided by the total number of cells.
Future Activities:
- In the second year, we will extend our approach by modifying the backbone of MBs to make them more nuclease resistant. In addition, a cell-penetrating TAT peptide will be added to enable intracellular delivery without permeabilization. The efficiency of the approach for real-time detection will be demonstrated using coxsackievirus B6.
- We will construct genetically engineered BGMK cell lines expressing the FRET substrates specific for HAV and investigate their utility for HAV detection.
- Continue the flow cytometry investigation to evaluate the lowest detection limit.
Journal Articles:
No journal articles submitted with this report: View all 7 publications for this projectSupplemental Keywords:
RFA, PHYSICAL ASPECTS, Scientific Discipline, INTERNATIONAL COOPERATION, Water, POLLUTANTS/TOXICS, Health Risk Assessment, Microbiology, Physical Processes, Drinking Water, Microorganisms, enteric viruses, health effects, measurement method, monitoring, pathogens, human health effects, microbiological organisms, exposure, viruses, waterborne pathogens, drinking water monitoring, drinking water contaminantsProgress and Final Reports:
Original AbstractThe 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.