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Research Project: Identifying Flavivirus Determinants of Entry and Replication and Design of Potential Vaccine Candidates

Location: Foreign Arthropod Borne Animal Disease Research

Project Number: 3022-32000-025-014-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 15, 2022
End Date: Sep 14, 2027

Flaviviruses like Dengue (DENV), Zika (ZIKV), Yellow Fever (YFV), and Japanese Encephalitis (JEV) viruses are transmitted by mosquitos causing debilitating diseases in vertebrate hosts, and substantial loss of live-stock. Although similar in genomic and structural properties, flaviviruses display specificity towards susceptible hosts and transmission dynamics. While Dengue, Zika viruses spread by Aedes mosquitos to human hosts, and YFV by Aedes and Haemogogus mosquitos, JEV is spread from birds and pigs to humans by Culex mosquitos. Another group of flaviviruses are spread by ticks, which include Powassan and Tick-borne encephalitis viruses. Very little is known about the virus determinants of virus entry and replication that defines host and vector specificity. Understanding these molecular determinants could lead to more effective antivirals and vaccine strategies. The objectives of this study are: 1. Follow virus life cycle in different cell-lines in real time to inform on molecular aspects of virus and host specificity. 2. Determining virus and host factors involved in virus entry and replication. 3. Generation of attenuated or chimeric vaccine candidates of flaviviruses.

To achieve these objectives, cDNA clones will be generated from genetic material of both tick-borne and mosquito-borne flaviviruses. The clones will be created with fluorescent tags for live-cell imaging of virus infection in various different cell lines. To identify host factors important for differences in virus replication and specificity, viral proteins will be tagged by site-directed mutagenesis. These tags will be used to pull the viral protein along with the host proteins. The interacting proteins will be analyzed and identified using affinity purification-mass spectrometry. The identified candidates will be confirmed by knock-down studies using gene-editing technology such as siRNA and CRISPR/CAS9. Attenuated viruses will be created using site-directed mutagenesis on the cDNA plasmids. In vitro experiments will characterize the entry and replication of the generated chimeric virus.