Location: Zoonotic and Emerging Disease Research
Project Number: 3022-32000-027-028-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: May 1, 2025
End Date: May 31, 2027
Objective:
America’s agriculture and public health is under constant threat from the importation of known pathogens as well as the emergence or reemergence of novel pathogens. Increasing food demands will drive expanded and higher density agriculture increasing the risk for the introduction or emergence of threats that compromise foods safety and the food supply. The ability to rapidly detect, characterize, and work with these pathogens is a core function of the NBAF mission. This collaborative project tackles four objectives: 1. Viral inclusive single-cell RNA sequencing (viscRNA-seq); 2. Genome-wide screenings for host factors in viral pathogenesis. 3. Functional analysis of uORFs identified from Massively Parallel Reporter Assay; 4. pen side detection tools and a data platform for pathogen surveillance.
Approach:
For Objective One Virus-inclusive scRNA-Seq (viscRNA-Seq) combining a CATCH-based primer pool for the capture and enrichment of non-poly(A) viral sequences in scRNAseq 10x Genomics with an ISO-MAS-seq (Multiplexed Arrays Sequencing) to generate long-read scRNAseq libraries will be optimized and utilized. Optimization will include the development of inactivation procedures for high containment and maximum containment pathogens. For Objective two genome-wide knockout and knockdown survival screens with targeted Perturb-seq to identify conserved mechanisms of viral susceptibility or resistance across a viral genus will be utilized. A crawl, walk, run approach will be utilized with work first being done with a BSL2 agent before progressing to agents requiring higher levels of containment. Results will be validated using a CRISPR interference screen using the dual-sgRNA library from, followed by Perturb-seq to assess how gene suppression affects infections. For Objective 3, A Massively Parallel Reporter Assay platform to measure viral translation via a synthetic library will be fused to a Red Fluorescent Protein (RFP) reporter and integrated into cells via transduction. The library will be sorted into expression bins and use deep sequencing to generate RFP expression scores. The role of uORFs in natural viral infections will be assessed for their impact on translation and fitness through ribosome profiling and growth assays. For Objective 4 Under Objective 4 a CRISPR-based Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic Acids (CARMEN) platform will be utilized as it enables rapid, high-throughput screening of multiple pathogens. The flexibility of the platform and ability to rapidly adapt assays to keep pace with viral changes makes it an ideal platform for Agriculture use. Syndromatic panels will be developed and evaluated to aid in the rapid discrimination of threats. Data visualization platforms will be improved to facilitate near real time detection of pathogens.
