Location: Foreign Animal Disease Research
Project Number: 8064-32000-059-07-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 15, 2020
End Date: Sep 14, 2023
The University of Texas Medical Branch (UTMB) will utilize the in vitro and in vivo models developed by UTMB to investigate the transmission of Crimean Congo Hemorrhagic Fever (CCHF) virus between ticks and animals. Also the research will support detection and identification of CCHF virus in ticks and animals by developing new assays. This project will leverage also the on-going collaborations between USDA and CDC as well as University of Califorina Davis. Specific objectives include: 1. The focus of the project is to improve the detection and understand the transmission of CCHF virus in ticks and livestock by utilizing experimental BSL4 models. 2. Train future National Bio and Agro- Defense Facility (NBAF) personnel on field studies for Bio Safety Level (BSL4) program.
Objective 1: Determining CCHFV vector competence of ticks in situ using an anti-strand PCR-It is hypothesized that by detecting actively replicating CCHFV in ticks will be able to make a preliminary statement on vector competence of that tick species. Develop a RT-PCR protocol that detected replication intermediates (anti-strand) using strand specific RT-PCRs (Sybr-Green assay). RNA standards have to be developed to quantify the genome equivalents. PCRs and standards for the S segments been tested at UTMB. The assay will be validated in field collected and experimentally infected ticks to determine CCHFV genome levels in competent tick species. Assays against the M and L segment will also be developed. Objective 2: Optimization of CCHFV detection and sequencing in ticks-It is hypothesized that by optimizing the tick homogenization, RNA extraction, and next-generation sequencing protocols will generate high quality CCHFV genome that can be used for full genome and viral population analysis. To improve RNA quality, evaluate the Cryoprep homogenizer, ribosomal tick RNA removal, ClickSeq technology and RACE PCR. The RNA quality after Cryoprep homogenization has been evaluated. Ribosomal RNA for different tick species has been sequenced, ribosomal removal kit needs to be designed and tested. ClickSeq technique will be tested as well. Use experimentally infected as well as field caught ticks to validate the protocols listed above. Objective 3. Characterize the transmission of CCHFV between ticks and domestic animals -It is hypothesized that goats and rabbits play a critical role in the transmission of CCHFV from infected to non-infected ticks. Tick colonies are already maintained at UTMB. Generate infected Hyalomma ticks by feeding them un rabbits that will be challenged with CCHFV. Infected ticks will then be fed on goats at BSL4 in close proximity to uninfected ticks. Transmission rates will be evaluated after ticks complete feeding. Animals will be investigated for virus in their blood stream. Co-feeding rates will be evaluated by virus isolation and by RT-qPCR.