Location: Zoonotic and Emerging Disease Research
Project Number: 3022-32000-021-021-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 15, 2025
End Date: Sep 14, 2026
Objective:
Orthonairoviruses such as Crimean Congo Hemorrhagic Fever Virus (CCHFV) and Nairobi Sheep Disease virus (NSDV or Ganjam virus) pose a threat to agriculture. CCHFV infection can result in severe, lethal disease in humans but remains largely asymptomatic when infecting cattle. Work with NSDV results in a mild to moderate flu-like disease in humans but results in an 80-90% lethal hemorrhagic viral diarrhea when infecting sheep. Both viruses are vectored by multiple species of ticks. As has occurred with the Asian Longhorned tick, Orthonairovirus-transmitting ticks could be accidently imported into the United States which raises concerns about these foreign vectors becoming endemic here, and their ability to spread viral diseases like CCHF or NSDV in the U.S.. The objective of this work is to develop minigenome and plasmid launch virus rescue systems to advance countermeasures, understand pathogenesis, and model consequences of accidental or purposeful introduction of these dangerous pathogens into the United States. Minigenome systems are useful for small molecule inhibitor studies, determining host-pathogen interactions, and gain insight into viral lifecycle mechanisms. Plasmid launch virus rescue systems are necessary to avoid tissue culture adaptations that may result in poor data quality and inaccurate modeling of pathogenesis. The virus rescue systems allow clonal expansion of a virus based on the consensus sequence rather than selection of a subpopulation in the viral population swarm that selects for fitness in cell culture rather than fitness and virulence in vivo.
Approach:
Orthonairoviruses are trisegmented negative strand viruses meaning that introduction of the genetic material alone is insufficient for virus growth, other viral proteins must be expressed to drive the infectious cycle. The lab will generate reverse genetics systems for CCHFV and NSDV. Minigenome systems consist of the viral proteins and genomes necessary for transcription and replication in complementary DNA (cDNAs) copies and a reporter such as green-fluorescent protein or luciferase, these systems are non-infectious and safe for use at biosafety level-2 because they lack the structural proteins needed to generate an infectious viral particle. Each viral protein will be in its own expression vector and the viral minigenome will have an insertion of a reporter protein that replaces a viral protein, which will ensure that only viral proteins are provided in-trans and the product is non-infectious. Expression of the viral proteins is driven by a host promoter and they will interact with each other and the viral genome to drive expression of the reporter protein in the minigenome viral RNA. Minigenome RNA will be expressed under the control of a T7 or Sp6 DNA dependent RNA polymerase. The polymerase will be supplied as an expression plasmid. The plasmid launch viral rescue system will consist of the same expression plasmids and each segment of the viral genome cloned under the control of the T7 or Sp6 DNA dependent RNA polymerase. NSDV rescue will be performed at BSL-3 in permissive cells and CCHFV will be rescued in BSL-4 through existing agreements with UTMB. NSDV will also be rescued in vivo in sheep by NBAF personnel through existing agreements with Kansas State University. Source material for the cDNAs will be obtained through collaborators from non-infectious cell extracts derived from methods shown to be effective and render the pathogens noninfectious or from vendors that make custom expression vectors. All materials generated will be in compliance with Institutional Biosafety Committee approved protocols, FSAP guidelines, and NIH Guidelines for Research Involving Recombinant DNA molecules.
NSDV requires biosafety level (BSL) 3 while CCHF requires BSL4 facilities. Inclusion of both a BSL3 and BSL4 orthnairoviruses will development of expertise and protocols for eventual work at BSL4 at NBAF.