Group 14: Oral Report 3, 2/12/2008 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

1. Luciferase Based Plasmid Reporter System for the Detection and Quantification ofHuman Respiratory Syncytial Virus Group 14: Oral Report 3, 2/12/2008 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

2. Background • Human Respiratory Syncytial Virus is the most common cause of bronchiolitis and pneumonia in children under 1 year of age (CDC) • ~800000 children die per year (~91 per hour) due to RSV infection • There is no current vaccine available for RSV • Current method for quantification of infectious RSV: Plaque Assay VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

3. 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 3 Tuesday, February 12, 2008

4. Oral Report 3 Our Solution • Novel plasmid based reporter system • A luciferase plasmid and cell line that will luminesce when infected with RSV • Stable transfection of plasmid into cell • Optimization of system protocol

5. Comparison VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

6. Comparison: Evaluation Chart VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

7. Methods NS1 NS2 SH M2 P N M G F L 3’ 5’ NS1 L 3’ 5’ NS1 Start L Stop pcDNA RSV Genome RSV Genome (truncated) (Synthesized)

8. Methods Luciferase Gene (luc) L Stop NS1 Start luc pRSVlucM5 selection

9. pRSVlucM5 VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

10. Development Costs * Indicates an approximate value, many supplies are for general lab use VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

11. Factors Affecting Success • There are 5 possible plasmids resulting from the combination of our four DNA molecules; we must screen for the correct one: pRSVlucM5 • Unforeseen problems with designed sequences • Sensitivity relative to plaque assay VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

12. 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 3 Tuesday, February 12, 2008

13. Current Progress • Completed: • Design of all plasmid constituents in silco • Purified all plasmid constituents by gel electrophoresis • Quantify all four sequences • Ligate three sequences into pcDNA3.1vector • Transform e. coli with plasmids • Screen colonies with minipreps • In Progress: • Maxiprep correct colony to obtain high yield of final plasmid • Submit Information Disclosure forms to Office of Tech Transfer VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

14. Cut with SphI Screening 1146bp 795bp 574bp 4908bp 72bp VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008

15. Future Work • Stably transfect cells with final plasmid • Test luminescence of cells using varying amounts of RSV • Optimize the system VUSE Senior Design Oral Report 3 Tuesday, February 12, 2008