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Translational efficiency of Dengue Virus(DEN) and DEN variants in cells. . Andrew Regan - Microbiology. DREHER LAB - ALS1065. Dengue Virus. Vectored by mosquito. Dengue Virus. DEN infection causes: Dengue Fever (DF) fever, headache, rash, muscle pain .
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Translational efficiency of Dengue Virus(DEN) and DEN variants in cells.
Andrew Regan - Microbiology
DREHER LAB - ALS1065
Vectored by mosquito
DEN infection causes:
Dengue Fever (DF) fever, headache, rash, muscle pain
Four serotypes (DEN1,DEN2, DEN3, DEN4)
Co-infection by two serotypes can cause:
Dengue Hemorrhagic Fever (DHF) and/or Dengue Shock Syndrome (DSS)
can be fatal, especially in children
Positive-strand RNA virus
Viral genome is ssRNA around 11kb contains the codons for synthesis of the viral polyprotein
The 5’Untranslated Region (5’UTR) and 3’UTR are expected to control translational efficiency as they do with typical cellular mRNA
To assay changes in translational efficiency between DEN2 and DEN2 variants in cells
STRATEGY: DEN2-luciferase(luc) assay system
Assay was established by doctoral student Wei-Wei Chiu
Clone DEN2-luc (wt) and DEN2-luc variants into dsDNA plasmids
Linearize plasmids, cutting at the 3’-terminal cleavage site
Transcribe constructs with T7 RNApol
Transfect RNA into Vero cells (monkey kidney)
Measure Luciferase activity in cell lysates using a luminometer
Luciferin + Luciferase + ATP + O2
Oxyluciferin + Luciferase + AMP + Light
Live virus vaccines utilize attenuated strains
Examples: Polio, Measles, Smallpox, etc…
DEN(S1) is an attenuated strain of DEN2
An attenuated virus has accumulated mutations that disable its ability to cause severe disease
Two 11-nt sequences are conserved among all DEN serotypes, as well as all close relatives
(e.g. West Nile Virus, Yellow Fever Virus, etc…)
Shiyun, You & R. Padmanabhan. A Novel in vitro Replication System for Dengue Virus. J Biological Chem. 1999. 274:45 33714-33722
Khromykh AA, Meka H, Guyatt KJ & EG Westaway. Essential role of cyclization sequences in flavivirus RNA replication. J Virology. 2001. 75:6719-28.
CS1 and cCS1 allow intramolecular circularization of the viral genome
The model has similarities to the protein-facilitated circularization of highly expressed cellular mRNA
40S subunit scans 5’ 3’ for the initiation codon
Nucleotides +6 through -4 are known to be important in halting ribosomal migration
Wei-Wei Chiu - OSU Microbiology
Dr. Theo W. Dreher - OSU Microbiology - OSU Center for Gene Research
Dr. Kevin G. Ahern - OSU Biochemistry & Biophysics
Howard Hughes Medical Institute