1. Luciferase Based Plasmid Reporter System for the Detection and Quantification of Human Respiratory Syncytial Virus Group 14: Oral Report 2, 1/24/2008 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)  ~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 Background Thursday, January 24, 2008VUSE Senior DesignOral 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 Thursday, January 24, 2008VUSE Senior DesignOral Report 2

5. Comparison: Evaluation Chart Plaque AssayLuciferase System CriteriaWeight (1-5)ValueProductValueProduct Quick5210420 Low Cost3210420 Objective3420525 Efficient4315525 Total5590 Thursday, January 24, 2008VUSE Senior DesignOral Report 2

6. Comparison Plaque AssayLuciferase System Detection MethodStaining/CountingLuminescence ObjectivityPartialYes Time (work/total)10 hours/7 days2.5hrs/2 days Materials Cost$8$1 Efficiency30 samples/experiment240 samples/experiment Thursday, January 24, 2008Oral Report 2VUSE Senior Design

7. Methods  Remove luciferase gene from pGEM-luc Thursday, January 24, 2008VUSE Senior DesignOral 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 region s Thursday, January 24, 2008VUSE Senior DesignOral Report 2

9.  Cut pcDNA3.1. Ligate luciferase, additional sequences, and pcDNA3.1 together Methods Thursday, January 24, 2008VUSE Senior DesignOral Report 2

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

11.  Transfect cells with plasmid Methods Plasmid Thursday, January 24, 2008VUSE Senior DesignOral Report 2

12.  Infect cells with various RSV concentrations Methods mRNA Luciferase mRNA Luciferin Thursday, January 24, 2008VUSE Senior DesignOral Report 2

13.  Measure luminescence Methods Plate Reader Thursday, January 24, 2008VUSE Senior DesignOral Report 2

14. Development Costs ItemCost pcDNA3.1 vector$361.00 pGEM-luc$83.00 Trailer minigenome plasmid$274 Leader oligonucleotides2x at $78.00 and 2x at $97.50 Cloning discs2x at $29.30 Misc. chemicals and disposable lab equip. $750* TOTAL$1877.60* Thursday, January 24, 2008Oral Report 2VUSE Senior Design * Indicates an approximate value, many supplies are for general lab use

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 Thursday, January 24, 2008Oral Report 2VUSE Senior Design

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 Thursday, January 24, 2008VUSE Senior DesignOral 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 Thursday, January 24, 2008VUSE Senior DesignOral 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 Thursday, January 24, 2008VUSE Senior DesignOral Report 2

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 Thursday, January 24, 2008VUSE Senior DesignOral Report 2