A Luciferase Reporter Minigenome System for Quantifying Respiratory Syncytial Virus Replication

1. Transient Transfection Results 3000 2500 2000 RLU 1500 1000 500 0 0 0.2 0.4 0.6 0.8 1 DNA Transfected (μg) Hep-2M5.4 800 RSV 600 Reovirus RLU 400 200 0 Mock 1E-4 1E-3 0.001 0.01 0.1 1 Log RSV MOI A Luciferase Reporter Minigenome System for QuantifyingRespiratory Syncytial Virus Replication Melanie J. Aston1, Michael H.Chi1, Monica K. Deterding1, Matthew M. Huckabee1,Martin L. Moore2, and R. Stokes Peebles, Jr.2 1Department of Biomedical Engineering, Vanderbilt University and 2Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN Solution Abstract Methods Novel Plasmid Based Reporter System We propose a plasmid system that expresses an RSV minigenome under the control of the CMV promoter. This minigenome contains a luciferase reporter gene under the control of RSV transcriptional elements. • Generation and Confirmation of Stably Transfected HEp-2 Cell Line • Perform transfection of pRSVlucM5 into HEp-2 cells • Apply Neomycin to select for only HEp-2 cells stably transfected • Test stably transfected cell line candidates • Infect with known concentrations of RSV • Perform luciferase assay • Choose cell line candidates that perform as expected Respiratory Syncytial Virus is the leading cause of respiratory tract infection in infants in the United States and worldwide. There is currently no vaccine available to treat RSV. The current method to determine RSV titer in the laboratory is the viral plaque assay, a labor, materials, and time intensive procedure. There is a need for a high throughput, inexpensive, and highly sensitive method to quantify infectious RSV. We engineered a cell line that emits bioluminescnce in response to RSV infection. An RSV minigenome containing a firefly luciferase gene was cloned into a DNA plasmid. The plasmid was introduced into an epithelial cell line (HEp‑2) via transfection, and stably-transfected cell lines were selected. Luciferase bioluminescence was measured 48 hours after RSV infection in a 96-well plate. Luciferase activity was RSV-specific and dose-dependent. Methods Creation and Function of Plasmid Minigenome (pRSVlucM5) Results/Discussion Fabrication Isolation Fabrication Transient Transfection We conducted three replicate experiments to determine the effectiveness of our plasmid construct. Relative light units increased from approximately 240 with no DNA transfected to 2100 with 0.8µg of DNA transfected (Figure 3). This is significant because it demonstrates that a transfection of our engineered plasmid is possible in HEp-2 cells and that the cells express luciferase in response to RSV infection. Stable Transfection Stably transfected cell lines were generated and luciferase assays were conducted at 24, 48, and 72 hours post infection with a serial dilution of RSV. We detected a significant bioluminescence at an RSV MOI of 0.1 48 hours post infection that was specific to RSV (Figure 4). Conclusion We successfully designed and created a luciferase reporter minigenome system that responds to RSV infection. The specificity of the system could be further improved in order for this system to replace the plaque assay. Leader Luciferase Trailer Background • RSV • The most common cause of bronchiolitis and pneumonia in children under one year of age • Currently there are two methods to confirm RSV infection: • Viral isolation from culture • Direct antigen test • There are currently no vaccines or drugs available to prevent or treat RSV. • Viral Plaque Assay • Current method for obtaining viral titer in the laboratory • Involves: • Culturing, infecting, and staining cells • Can take anywhere from 7-8 days • Counting the plaques by eye and manually calculating viral titer Leader Luciferase Trailer Luciferase Trailer Leader Drug Resistance pRSVlucM5 Figure 3. The results from three replicate transient transfection experiments with infection at an RSV MOI of 1. An increase in luminescence is shown. The Problem A high throughput, inexpensive system to quantify infectious RSV is needed to rectify the major shortcomings of the viral plaque assay. AAAAA 3’ 5’ RSV 5’ 3’ AAAAA Culture Cells Wait For Cells to Grow Inoculate Cells with Virus Wait for Cells to become Infected Figure 4. The results of stably transfected cell line #4. A significant bioluminescence was detected at an RSV MOI of 0.1 Overlay Cells with Methyl- Cellulose Luciferase • Generation and Confirmation of pRSVlucM5 • Determine sequences necessary to create RSV minigenome (Leader: leader region, NS1 gene start signal, NS1 nontranslated region; Trailer: L nontranslated region, L gene end signal, trailer region) • Design ends of Leader and Trailer regions to enable future ligation • Acquire DNA leader and trailer regions using minigene and oligonucleotides • Remove luciferase gene from pGem-luc; prepare pcDNA3.1 • Ligate all pieces together • Cut final plasmid candidates with SphI • Choose plasmid candidates that match predicted cut patterns • Generation and Confirmation of Transiently Transfected HEp-2 Cells • Perform transfection of pRSVlucM5 into HEp-2 cells • Test transiently transfected cells • Infect with known concentration of RSV • Perform luciferase assay 3 days 1 hour Allow Plaques To Form Stain Cells with Hematoxylin and Eosin Count Plaques Calculate Viral Titer Figure 1. The flow diagram shows the major delays for the plaque assay (highlighted in red). Figure 2. Our engineered luciferase system and the plaque assay were compared. 5 days Acknowledgements Vanderbilt Pulmonary Medicine: Kasia Goleniewska, Kirk Lane Vanderbilt Pediatrics: Jim Crowe Others: Peter Collins (NIAID) References 1. Grosfeld H, Hill M, Collins P. RNA Replication by Respiratory Syncytial Virus (RSV) Is Directed by the N, P, and L Proteins; Transcription Also Occurs under These Conditions but Requires RSV Superinfection for Efficient Synthesis of Full-Length mRNA. Virology: 69. Sept. 1995, page 5677-5686