Transcript Viral Plaque Assay - Research

A Luciferase Reporter Minigenome System for Quantifying
Respiratory Syncytial Virus Replication
Melanie J.
1Department
1
Aston ,
1
H.Chi ,
1
Deterding ,
Michael
Monica K.
Matthew M.
2
2
Martin L. Moore , and R. Stokes Peebles, Jr.
1
Huckabee ,
of Biomedical Engineering, Vanderbilt University and 2Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN
Solution
Abstract
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
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
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
Creation and Function of Plasmid Minigenome (pRSVlucM5)
Fabrication
Isolation
Fabrication
Leader
Luciferase
Trailer
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.
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
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.
Transient Transfection Results
3000
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’
Wait for Cells
to become
Infected
3 days
Overlay Cells
with MethylCellulose
5’
0
3’ AAAAA
0.2
0.4
0.6
0.8
1
DNA Transfected (μg)
1 hour
Stain Cells with
Hematoxylin
and Eosin
Count
Plaques
Calculate
Viral
Titer
5 days
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.
Detection Method
Objectivity
Time (work/total)
Materials Cost
Throughput
500
0
Luciferase
Allow
Plaques
To Form
1500
Plaque Assay
Staining/Counting
Partial
10 hours/7 days
$8
30 samples/experiment
Luciferase System
Luminescence
Yes
2.5hrs/2 days
$1
240 samples/experiment
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
Figure 4. The
results of stably
transfected cell
line #4. A
significant
bioluminescence
was detected at
an RSV MOI of
0.1
Hep-2M5.4
800
RSV
600
RLU
Culture
Cells
Inoculate
Cells with
Virus
2000
1000
RSV
Wait
For Cells to
Grow
RLU
2500
Reovirus
400
200
0
Mock
1E-4
1E-3
0.001
0.01
0.1
1
Log RSV MOI
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