Group 14: Oral Report 4, 3/13/08 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 4, 3/13/08 Melanie Aston, Michael Chi, Monica Deterding, Matt Huckabee

2. 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

3. 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

4. Current Method: Viral Plaque Assay Culture Cells Wait For Cells to Grow Inoculate Cells with Virus Wait for Cells to become Infected 3 days 1 hour Overlay Cells with Methyl- Cellulose Allow Plaques To Form Stain Cells with Hematoxylin and Eosin 5 days Count Plaques Calculate Viral Titer VUSE Senior Design Oral Report 4 Thursday March 13th, 2008

5. 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

6. 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

7. Comparison VUSE Senior Design Oral Report 4 Thursday March 13th, 2008

8. Comparison: Evaluation Chart VUSE Senior Design Oral Report 4 Thursday March 13th, 2008

9. 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)

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

11. pRSVlucM5 VUSE Senior Design Oral Report 4 Thursday March 13th, 2008

12. Development Costs * Approximate value VUSE Senior Design Oral Report 4 Thursday March 13th, 2008

13. 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

14. 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

15. Luminescence Data VUSE Senior Design Oral Report 4 Thursday March 13th, 2008

16. 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