Location: Research Programs
Project Number: 3022-32000-018-033-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2023
End Date: Jul 31, 2025
Objective:
Virtually nothing is known of Cis-regulatory elements (CREs) associated with arbovirus infection or whether chromatin modifications modulate mosquito host responses to infection. Culex and Aedes species transmit important exotic zoonotic arboviruses, e.g. Rift Valley fever virus, of potential concern to U.S. animal and public health. The objective of this work is to identify CREs in mosquito genomes that contribute to vector competence.
Approach:
Aedes aegypti or Culex tarsalis will be used for these studies and will be maintained under standard insectary conditions. Adult female mosquitoes 3-6 days of age will be maintained on 10% sucrose and fasted 24 hours before exposure to an artificial bloodmeal for each treatment group. Artificial blood meals will contain at least 6.0 log PFU/ml of Rift Valley fever virus or media (mock-infected control). Engorged females with visible abdominal blood will be collected and provided with 10% sucrose solution. A Pilot study will be done to develop experimental and data analysis workflows for subsequent studies and will compare CREs and DEGs in exposed and unexposed mosquitoes. Aedes aegypti will be infected with RVFV MP-12, then midguts or whole bodies from will be collected at 7 d post feeding for chromatin-immunoprecipitation-sequencing using anti- H3K27ac (histone 3 acetylated lysine 27) or anti- H3K9me (histone 3, methylated lysine 9) antibodies (n=3 pools of 100 mosquitoes or midguts per replicate for a total of 36 libraries). Subsequently, DNA library preparation, DNA NGS sequencing and transcriptomics (n=3 pools of 20 mosquitoes or midguts/timepoint). For transcriptomic analysis, mRNA libraries will be constructed, sequenced and analyzed. Annotation and differential gene expression (DGE) data will be aligned to CREs to identify genomic regions of interest.
Subsequent studies will utilize virulent RVFV strains and the optimized tissue type determined in the pilot study. Under this portion of the work, legs/wings will be removed from mosquito bodies for validation of dissemination status prior to sample pooling. Mosquito tissues will be pooled according to phenotype (undisseminated, disseminated, unexposed) for subsequent sample preparation and analyses to identify CREs of importance.
