1. 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

2.  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 Background on RSV in the Clinic Thursday March 13 th, 2008Oral Report 4VUSE Senior Design

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

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

5. The Problem  Viral plaque assay is  Labor intensive  Costly  Time consuming  Partially subjective  Need high throughput, inexpensive system to quantify infectious RSV Thursday March 13 th, 2008Oral Report 4VUSE Senior Design

6. Our Solution  Novel plasmid based reporter system  Luciferase plasmid  Cell line  Luminesce upon infection with RSV Thursday March 13 th, 2008Oral Report 4VUSE Senior Design

7. Comparison Plaque AssayLuciferase System Detection MethodStaining/CountingLuminescence ObjectivityPartialYes Time (work/total)10 hours/7 days2.5hrs/2 days Materials Cost$8$1 Throughput30 samples/experiment240 samples/experiment Thursday March 13 th, 2008Oral Report 4VUSE Senior Design

8. Comparison: Evaluation Chart Plaque AssayLuciferase System CriteriaWeight (1-5)ValueProductValueProduct Quick5210420 Low Cost326412 Objective3412515 Efficient4312520 Total4067 Thursday March 13 th, 2008Oral Report 4VUSE Senior Design

9. Methods RSV Genome RSV Genome (truncated) NS1 L 3’5’ NS1 Start L Stop pcDNA (Synthesized) NS1NS2SHM2 3’5’ NMGLFP

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

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

12. Development Costs ItemCost pcDNA3.1 vector$361 pGEM-luc$83 Trailer minigenome plasmid$274 Leader oligonucleotides2x at $78 and 2x at $98 Cloning discs2x at $29 Misc. chemicals and disposable lab equip. $750* TOTAL$1878* * Approximate value Thursday March 13 th, 2008Oral Report 4VUSE Senior Design

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

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

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

16. Project Status  In Progress:  Test stable transfection  Future Work:  Optimize the system  Final write-up Thursday March 13 th, 2008Oral Report 4VUSE Senior Design