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About Dengue
Dengue is one of the most important mosquito-born viral diseases affecting humans. Viral life cycle involves humans and the mosquito vector Aedes aegypti.
In the U.S. it has been found that the mosquito Aedes albopictus also transmits the DEN virus.
The disease is caused by 4 serotypes of the Dengue virus, a member of the genus Flavivirus: DEN-1, DEN-2, DEN-3, DEN-4. Infection with the DEN virus can result in Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS).
DEN-2 Serotype
Strain 16681 from Thailand. DEN virus is an enveloped, 10.75 kb, positive, single-stranded RNA virus. 1 ORF, 380 kDa. Structure contains a 5 cap and a 3 stem-loop structure (no 3 -poly(A) tail). Has the ability to replicate in mosquitoes and primate cells.
Risks for this include the possibility of attenuation reversal of a vaccine strain resulting in mutations that might increase gene expression.
Identify features in the 5 and 3 regions of DEN-2 RNA genome that control translation. This will be done using a sensitive luciferase reporter mRNA.
Determine whether the translation of DEN RNA is altered in the presence of viral proteins. Understand the regulation of replication.
Identify features in the 5 and 3 regions of DEN-2 RNA genome that control translation. This will be done using a sensitive luciferase reporter mRNA.
Determine whether the translation of DEN RNA is altered in the presence of viral proteins. Understand the regulation of replication.
Experimentation:
Series
Experimentation:
Series
3 UTR Series
Experimentation:
Series
3 UTR Series:
Luciferase Constructs
Controls:
Constructs:
Experimental:
General Design
LUC
2. In Vitro run-off Transcription by T7 RNA Polymerase (with cap analog) 1. Linearize Plasmid
WWC
3. RNA Electroporation
WWC
WWC & AC
4. Cell Lysis
WWC & AC
Lysate 5. Luciferase/Protein Assays
AC
Luciferase Assay
When in the presence of the substrate LAR (Luciferase Assay Reagent), luciferase will undergo an enzymatic reaction that emits light.
Problem: This assay does not take into account the total amount of cells that were lysed.
Protein Assay
The protein present in the lysates cause the Protein Assay Reagent to turn blue. Light absorbance at 595 nm is measured and used as a reflection on the total amount of protein present in the lysates.
Results from the protein assay are measured in mg protein/L of lysate. These values are then used to normalize the results from the Luciferase Assay (RLU/mg protein).
Analysis:
Luciferase Expression
1.0E+10
10 8
Capped GCLGpolyA
8.69
RLU/mg protein
8.0E+09
6.0E+09
4.0E+09
4 2
2.0E+09
Maximum Accumulation illustrates the RNAs ability to be expressed inside the cell.
0
(hr)
2.17
Analysis:
Functional Life
1.0E+10
10 8
Capped GCLGpolyA
RLU/mg protein
8.0E+09
6.0E+09
4.0E+09
4 2
2.0E+09
4
3.57 hr
(hr)
2.17
T1/2=1.40 hr
Functional Life shows the change over time of the RNAs relative efficiency to be used as a template for translation.
Analysis:
Accumulative Life
(x109) 1.0E+10
10 8
Capped GCLGpolyA
8.69
RLU/mg protein
8.0E+09
6.0E+09
4.0E+09
4.35
4 2
2.0E+09
1
0.83
(hr)
T1/2 = 1.46 hr
Accumulative Life shows the amount of time it takes for the mRNA to reach of the maximum LUC expression.
Results:
3UVR
3 UTR Series
DB2 DB1
3CS SLB SLA
Results:
Life Analysis
DCL DCLG /NcoI UVR DB1+2 DB2 DB1 SLB
SLA
GCLGpA DCLD
Time (hrs)
Results:
CS1 Mutation Series
Results:
Life Analysis
DCmLDm
DCLDm
DCmLD
DCLD
Time (hrs)
A Look Ahead
Cap/no cap 5 UTR series.
Examining cap dependent/independent translation. Possible interactions between viral/cellular proteins and how they affect translation of DEN-2 genome.
Acknowledgements
Dr. Dreher Wei-Wei Chiu Kevin Ahern
HHMI
NSF