Presentation on theme: "Respiratory Syncytial Virus is the leading cause of respiratory tract infection in infants in the United States and worldwide. There is currently no vaccine."— Presentation transcript:
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. A Luciferase Reporter Minigenome System for Quantifying Respiratory Syncytial Virus Replication 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 Generation and Confirmation of Stably Transfected HEp-2 Cell Line 1.Perform transfection of pRSVlucM5 into HEp-2 cells 2.Apply Neomycin to select for only HEp-2 cells stably transfected 3.Test stably transfected cell line candidates 1.Infect with known concentrations of RSV 2.Perform luciferase assay 3.Choose cell line candidates that perform as expected Results/Discussion 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. Acknowledgements Vanderbilt Pulmonary Medicine: Kasia Goleniewska, Kirk Lane Vanderbilt Pediatrics: Jim Crowe Others: Peter Collins (NIAID) Solution 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. Methods Abstract The Problem A high throughput, inexpensive system to quantify infectious RSV is needed to rectify the major shortcomings of the viral plaque assay. 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. 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 Figure 4. The results of stably transfected cell line #4. A significant bioluminescence was detected at an RSV MOI of 0.1 Generation and Confirmation of pRSVlucM5 1.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) 2.Design ends of Leader and Trailer regions to enable future ligation 3.Acquire DNA leader and trailer regions using minigene and oligonucleotides 4.Remove luciferase gene from pGem-luc; prepare pcDNA3.1 5.Ligate all pieces together 6.Cut final plasmid candidates with SphI 7.Choose plasmid candidates that match predicted cut patterns Generation and Confirmation of Transiently Transfected HEp-2 Cells 1.Perform transfection of pRSVlucM5 into HEp-2 cells 2.Test transiently transfected cells 3.Infect with known concentration of RSV 4.Perform luciferase assay Creation and Function of Plasmid Minigenome (pRSVlucM5) Leader Luciferase Trailer Leader Luciferase Trailer Fabrication Isolation pRSVlucM5 AAAAA 3’ 5’ 3’ AAAAA 5’ RSV Luciferase Melanie J. Aston 1, Michael H.Chi 1, Monica K. Deterding 1, Matthew M. Huckabee 1, Martin L. Moore 2, and R. Stokes Peebles, Jr. 2 1 Department of Biomedical Engineering, Vanderbilt University and 2 Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN Hep-2M5.4 0 200 400 600 800 Mock1E-41E-30.0010.010.11 Log RSV MOI RLU RSV Reovirus Figure 3. The results from three replicate transient transfection experiments with infection at an RSV MOI of 1. An increase in luminescence is shown. Transient Transfection Results 0 500 1000 1500 2000 2500 3000 00.20.40.60.81 DNA Transfected (μg) RLU 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 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
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