Location: Foreign Arthropod Borne Animal Disease Research
Project Number: 3022-32000-062-010-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2023
End Date: Aug 30, 2025
Objective:
The USDA and the Cooperator are interested in collaborating on understanding the status of arborviruses circulating in Southwestern USA. The objective of this agreement is to develop and/or apply new ARS technology to detect and characterize arboviruses present in the Southwestern USA that could pose emerging animal disease threats and develop a baseline of virus population variability. The area to be investigated will include boundary areas between agricultural areas and regional ports.
Approach:
At least three targeted mosquito collection sites will be selected to represent different geographical areas. The areas where mosquitoes will be sampled will be on the boundary between urban and rural or sylvatic habitats. Mosquitoes will be sorted by species and deployable point-of-need tools will be developed to quickly identify and characterize viruses. The cooperative team will develop a panel of family specific RT-qPCR assays for alphaviruses, bunyaviruses, flaviviruses, orbiviruses and rhabdoviruses. Where scientifically reasonable and technically possible multiplex assays will be developed. For example: a mosquito-borne assay might include only the first three virus families. The initial development and validation will be done using standard laboratory real-time thermocyclers. Once developed the assay will be adapted by ARS and equivalency validated on the field-based system (eg. BioMeme). Positive samples will be subjected to further characterization by next generation sequencing technology. Briefly, at ARS, a virus capture isolation procedure will be developed to enhance the viral target RNA in the sample. Unbiased metagenomic sequencing will be used to generated sequence data using the Minion, Illumina Miseq and NextSeq platforms. The Cooperators, semi-agnostic pan-viral amplicon and tiled amplicon sequencing will be used to genomically characterize the detected target. The pan-amplicon data will be analyzed with an alpha version of a QIIME-based pan-viral bioinformatics plug-in that the Cooperator has recently developed, along with other standard workflows (e.g., reference-based alignment and processing with Bowtie2 or BWA and Samtools). The initial study was conducted with commercial software (such as CLC Genomics Workbench, Genetic). The data will also be subjected to more sophisticated computational virus population genetic analysis that is available through another ARS cooperative project and the NBAF high performance computer cluster. The data generated in this project will be used to provide baseline arbovirus prevalence that could emerge and threat animal and public health. In addition, the virus population variability will be used to inform risk models in future studies.
