Location: Foreign Arthropod Borne Animal Disease Research
Project Number: 3022-32000-024-023-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2026
End Date: Jul 31, 2028
Objective:
Development of translational approaches to combat vector-borne disease is hampered by host- and virus-specific differences in responses that condition vector competence. Culex and Aedes species transmit important exotic zoonotic arboviruses, e.g. Rift Valley fever virus (RVFV), of potential concern to U.S. animal and public health. The objective of this work is to identify genomic regulatory regions in mosquitoes that contribute to vector competence. Commercial antibodies specific for histone modifications and others targeting to transcription factors, e.g., sterol regulatory element binding protein (SREBP), will be used to capture DNA fragments of interest, which will then be sequenced and then validated using transcriptomic data from matched samples.
Our previous work showed evidence for coordinated changes to gene expression and H3K27ac (histone 3 acetylated lysine 27) and H3K9me3 (H3 trimethylated lysine 9) marks at 3 days post-RVFV MP12-exposure in Aedes aegypti. However, we were not able to build support for a direct association between these changes and the effects of virus, due to the low competency of Aedes aegypti for RVFV. Therefore, we will also study the more competent vector Cx tarsalis.
To address mechanisms underlying transcriptional regulatory changes that occur in mosquitoes during arbovirus infection, we will compare genomic regulatory sequences identified using CUT&RUN chromatin-immunoprecipitation-sequencing for mosquito midguts after exposure to virulent (ZH501) and non-virulent strains of RVFV (MP12 and DDVax, an NSs/NSm double deletion mutant). When analyzed alongside coordinated gene expression changes, this approach is powerful, because it will lend support to our hypothesis that transcriptional regulatory changes occur in RVFV-exposed mosquitoes. It will also provide hints about the role of RVFV NSs in genomic regulatory changes during midgut infection. Cx tarsalis will be a challenge due to the limited annotation of the genome. Therefore, our progress will also require improvement of genome annotation resources for this organ.
Approach:
Culex tarsalis KNWR will be maintained under standard insectary conditions. Adult female mosquitoes 3-7 days of age will be maintained on 10% sucrose and fasted 12h before exposure to an artificial bloodmeal RVFV MP-12, ZH501 or DDVax. Artificial blood meals will contain about 6.0 log PFU/ml freshly grown RVFV or 1:1 dilution of media in blood for mock-infected controls. Engorged females with visible abdominal blood will be collected and provided with 10% sucrose solution. Chromatin marks of interest include H3K27ac, H3K9me3 and the transcription factor SREBP, which regulates lipid metabolism. Midguts will be collected at 1 or 3 days post feeding for chromatin-immunoprecipitation-sequencing (n=3 pools of 20 midguts per replicate for 3 marks at 2 timepoints for a total of 96 libraries). Subsequently, NGS library preparation, DNA NGS sequencing and RNA-Seq will be used. Annotation and differential gene expression data will be collated with DNA peak patterns to identify genomic regions of interest that are modulated during virus infection.