Michael H. D'Souza, University of Lethbridge

Michael H. D'Souza,a Ash Mosabeb-Omran,a Sophie Metheral,a Higor S. Pereira,a Jason Luddu,a Temi Agunlejika,a Meshva Patel,a Gunjan Vasudeva,a Carmen Agenbacht,a M. Quadir Siddiqui,a Scott Tersteeg,a and Trushar R. Patel.a,b,c a Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada b Department of Microbiology, Immunology and Infectious Disease, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada c Li Ka Shing Institute of Virology and Discovery Lab, University of Alberta, Edmonton, AB T6G 2E1, Canada

Fighting & Responding

Venezuelan Equine Encephalitis Virus (VEEV) is a re-emerging enveloped, mosquito-borne, single-stranded, positive-sense RNA virus. Within the Togaviridae Family genus of Alphaviruses, VEEV is highly pathogenic, inducing 90% mortality rates in equine animals, including horses, donkeys, zebras, and mules, while engendering 1% mortality in humans with 14% of infected persons suffering severe neurological diseases. Historically, VEEV was engineered as a biological agent during the Cold War, intended to target developing, agrarian-oriented countries that primarily relied on equines for industrial and agricultural production. VEEV is considered as a CDC Biosafety Level 3 pathogen and a NIAID Category B weapon of biowarfare and bioterrorism due to its ability to target the central nervous system and be aerosolised, and its high pathogenicity. We intend to design a VEEV antiviral using Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Systems (CRISPR-Cas), particularly RfxCas13d which is a bacterial antiviral endonuclease. Cas13d is a Type VI-D CRISPR, single effector protein unique for its RNA-guided, RNA-targeting knockdown effects. We employ Cas13design to computationally produce guide-RNAs that will complementarily bind to VEEV's genomic RNA-dependent RNA-polymerase (RdRp). Mammalian expression vectors are employed to express eGFP-bound RdRp in cell culture, with fluorescence intensities reported to measure RNA degradation. Future validation of ideal Anti-VEEV-RdRp RfxCas13d-gRNA candidates will be performed on live VEEV TC-83 strain with efficacy measured in reduced viral titres. A novel but effective CRISPR-based strategy has serious implications for biological agent and bioterrorism threat mitigation, as few USA FDA-approved medical countermeasures exist for VEEV and other deadly Alphaviruses.