Group 14: Oral Report 2, 1/24/2008 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 2, 1/24/2008 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 due to RSV infection, which is about 91 per hour • There is no current vaccine available for RSV • Current method for quantification of infectious RSV: Plaque Assay Oral Report 2

3. The Problem Viral plaque assay is Labor intensive Costly Time consuming Partially subjective Need high throughput, inexpensive system to quantify infectious RSV

4. 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 Oral Report 2

5. Comparison: Evaluation Chart VUSE Senior Design Oral Report 2 Thursday, January 24, 2008

6. Comparison VUSE Senior Design Oral Report 2 Thursday, January 24, 2008

7. Methods • Remove luciferase gene from pGEM-luc Oral Report 2

8. Methods • Ligate luciferase and additional sequence together • Blue: leader, NS1 gene start, and non-coding regions • Red: non-coding, L gene end, and trailer regions Oral Report 2

9. Methods • Cut pcDNA3.1. Ligate luciferase, additional sequences, and pcDNA3.1 together Oral Report 2

10. pRSVlucM5 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008

11. Methods • Transfect cells with plasmid Plasmid Oral Report 2

12. Methods • Infect cells with various RSV concentrations mRNA mRNA Luciferase Luciferin Oral Report 2

13. Methods • Measure luminescence Plate Reader Oral Report 2

14. Development Costs * Indicates an approximate value, many supplies are for general lab use Oral Report 2

15. 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 • Possible E. coli rejection of RSV sequences • Sensitivity relative to plaque assay Oral Report 2

16. Alternate Solutions • Try other E. coli strains • 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 Oral Report 2

17. Current Progress • Completed: • Design of leader and trailer sequences • Design of final plasmid construct in silco • Purified pcDNA3.1 vector and luciferase insert • In Progress: • Preparation of leader and trailer inserts • Gel purification of leader and trailer inserts Oral Report 2

18. Setbacks 1/18/08 Failure of oligonucleotide ligation due to unknown factors Failure of trailer double digest due to unknown factors 1/21/08 Confirmation of ligation failure due to lack of 5’ phosphorylation Success of trailer double digest 1/18 1/21 VUSE Senior Design Oral Report 2 Thursday, January 24, 2008

19. Future Work • Phosphorylate and ligate leader insert parts • Cut out trailer insert from minigene plasmid • Quantify all four sequences • Ligate three sequences into pcDNA3.1vector • Transform e. coli with plasmids • Screen colonies with minipreps • Maxiprep correct colony to obtain high yield of final plasmid • Stably transfect cells with final plasmid • Test luminescence of cells using varying amounts of RSV • Optimize the system Oral Report 2