Dengue (DEN; Flaviviridae) viruses continue to pose health problems for human populations in tropical regions of the world. DEN hemorrhagic fever and DEN shock syndrome are threatening large proportions of the urban and rural populations in Asia and the Americas. Concomitant with the spread of disease is the rapid proliferation of the DEN virus mosquito vector, Aedes aegypti. The lack of effective vaccines against DEN, as well as the emergence of pesticide resistant vectors, has had a major negative impact on developing conventional strategies for controlling the disease. The long term goal of this research is to develop molecular strategies that reduce or eliminate virus replication in the vector and prevent virus transmission. We have shown that expression of sense or antisense RNA of the DEN prM gene from a Sindbis (SIN; Togaviridae) virus vector in mosquito cells or adult Ae. Aegypti effectively interferes with replication of superinfecting DEN virus. In this proposal, we will use SIN transducing viruses to express second generation anti- DEN virus agents in Aedes aegypti. These agents will include: 1)sense and antisense RNAs of improved design that can be targeted to all 4 serotypes of DEN virus, 2) mutated DEN replicase gene NS5, and 3) anti-DEN ribozymes. Rationally designed, DEN sense and antisense RNAs should complement and potentially enhance RNA mediated resistance strategies that have already been shown to be effective. Initially, SIN transducing viruses will be used to express a model ribozyme targeted to trans-cleave a Beta- galactosidase gene expressed in transformed mosquito cells. The utilization of ribozyme technology to reduce or ablate the competency of mosquitoes for DEN viruses is a natural extension of the antisense strategies already in use. The expression in mosquitoes of mutated forms of the DEN polymerase (NS5) gene represents important strategies for ablating virus replication in the vector and its efficacy as an antiviral agent needs to be assessed. Additionally, promising second-generation anti virus agents will be analyzed in transformed mosquito cell culture to study the efficacy of these agents when delivered from a DNA expression system. Finally, promising interference strategies developed from SIN virus transduction and DNA expression of the anti-virus agents will be tested in vivo using Aedes densonucleosis (AeDNV; Parovoviridae) virus. In toto, this work should give us significant understanding of which anti-DEN virus strategies are important for ultimately generating transgenic mosquitoes that are resistant to DEN virus infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI034014-06
Application #
2886828
Study Section
Special Emphasis Panel (ZRG5-TMP (01))
Program Officer
Meegan, James M
Project Start
1993-04-01
Project End
2002-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
6
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Microbiology/Immun/Virology
Type
Schools of Veterinary Medicine
DUNS #
112617480
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Blair, Carol D; Olson, Ken E (2015) The role of RNA interference (RNAi) in arbovirus-vector interactions. Viruses 7:820-43
Olson, Ken E; Blair, Carol D (2015) Arbovirus-mosquito interactions: RNAi pathway. Curr Opin Virol 15:119-26
Blair, Carol D; Olson, Ken E (2014) Mosquito immune responses to arbovirus infections. Curr Opin Insect Sci 3:22-29
Bernhardt, Scott A; Simmons, Mark P; Olson, Ken E et al. (2012) Rapid intraspecific evolution of miRNA and siRNA genes in the mosquito Aedes aegypti. PLoS One 7:e44198
Blair, Carol D (2011) Mosquito RNAi is the major innate immune pathway controlling arbovirus infection and transmission. Future Microbiol 6:265-77
Brackney, Doug E; Scott, Jaclyn C; Sagawa, Fumihiko et al. (2010) C6/36 Aedes albopictus cells have a dysfunctional antiviral RNA interference response. PLoS Negl Trop Dis 4:e856
Scott, Jaclyn C; Brackney, Doug E; Campbell, Corey L et al. (2010) Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells. PLoS Negl Trop Dis 4:e848
Bernhardt, S A; Blair, C; Sylla, M et al. (2009) Evidence of multiple chromosomal inversions in Aedes aegypti formosus from Senegal. Insect Mol Biol 18:557-69
Black Iv, W C; Bernhardt, S A (2009) Abundant nuclear copies of mitochondrial origin (NUMTs) in the Aedes aegypti genome. Insect Mol Biol 18:705-13
Sanchez-Vargas, Irma; Scott, Jaclyn C; Poole-Smith, B Katherine et al. (2009) Dengue virus type 2 infections of Aedes aegypti are modulated by the mosquito's RNA interference pathway. PLoS Pathog 5:e1000299

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