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Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Group 14: Oral Report 4, 3/13/08 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee Background on RSV in the Clinic Human Respiratory Syncytial Virus is the most common cause of bronchiolitis and pneumonia in children under 1 year of age (CDC) About 800000 children die per year worldwide due to RSV infection (~91 per hour) There are currently two methods for the clinical confirmation of RSV infection Viral isolation from culture (gold standard, requires several days) Direct antigen test (tests range from 20-75 mins) There are currently no vaccines or drugs available to prevent or treat RSV VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Background on RSV in the Lab Ongoing RSV research: Understand mechanisms of RSV pathogenesis in order to develop drugs Test vaccine candidates Mouse models are commonly used The current method to quantify RSV titer in mice is the plaque assay VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Current Method: Viral Plaque Assay Culture Cells Wait For Cells to Grow Inoculate Cells with Virus Wait for Cells to become Infected 3 days Overlay Cells with MethylCellulose 1 hour Allow Plaques To Form Stain Cells with Hematoxylin and Eosin 5 days Count Plaques VUSE Senior Design Calculate Viral Titer Oral Report 4 Thursday March 13th, 2008 The Problem Viral plaque assay is Labor intensive Costly Time consuming Partially subjective Need high throughput, inexpensive system to quantify infectious RSV VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Our Solution Novel plasmid based reporter system Luciferase plasmid Cell line Luminesce upon infection with RSV VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Comparison Plaque Assay Luciferase System Detection Method Staining/Counting Luminescence Objectivity Partial Yes Time (work/total) 10 hours/7 days 2.5hrs/2 days Materials Cost $8 $1 Throughput 30 samples/experiment 240 samples/experiment VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Comparison: Evaluation Chart Plaque Assay Criteria Weight (1-5) Value Luciferase System Product Value Product Quick 5 2 10 4 20 Low Cost 3 2 6 4 12 Objective 3 4 12 5 15 Efficient 4 3 12 5 20 Total VUSE Senior Design 40 Oral Report 4 67 Thursday March 13th, 2008 Methods RSV Genome NS1NS2 N 3’ SH P M M2 G F L 5’ RSV Genome (truncated) NS1 3’ L NS1 Start 5’ L Stop pcDNA (Synthesized) Methods Luciferase Gene (luc) L Stop NS1 Start luc pRSVlucM5 selection pRSVlucM5 VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Development Costs Item Cost pcDNA3.1 vector $361 pGEM-luc $83 Trailer minigenome plasmid $274 Leader oligonucleotides 2x at $78 and 2x at $98 Cloning discs 2x at $29 Misc. chemicals and disposable lab equip. $750* TOTAL $1878* * Approximate value VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Alternate Solutions PCR - polymerase chain reaction Proven to work for the detection and quantification of viruses Limitations: Measures amount of nucleic acid (cannot differentiate between live virus and dead virus) Low throughput Costly VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Project Status Completed: Design of all plasmid constituents in silco Ligation of the plasmid constituents Screening selection Demonstration that the plasmid works as designed Stable transfection of cells with plasmid Submitted Information Disclosure Form to Office of Tech Transfer VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Luminescence Data RLU vs. Amount of DNA Transfected 3000 2500 RLU 2000 (-) control 2,1 1500 2,2 3,1 1000 3,2 500 0 0 0.2 0.4 0.6 0.8 1 Amt of DNA Transfected (ug) VUSE Senior Design Oral Report 4 Thursday March 13th, 2008 Project Status In Progress: Test stable transfection Future Work: Optimize the system Final write-up VUSE Senior Design Oral Report 4 Thursday March 13th, 2008