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Abstract

Inhibition of Equine Infectious Anemia Virus using RNA Interference

Devony Wingo*, Mike Peoples, Sarah Canterberry, Ph.D., Kim Tessanne, Charles Long, Ph.D.

Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University.

ABSTRACT

Objective: To use RNA interference to significantly decrease prevalence of Equine Infectious Anemia Virus in horses.

Animals: This research involves the use of fetal equine kidney cells to develop a disease resistant cell line through integration of a short-hairpin RNA delivered by a lentiviral vector.

Procedure: We will develop four shRNAs, two targeting each of two different genes of EIAV. The shRNAs targeting EIAV genes and a non-targeting shRNA control will be cloned into a lentiviral vector carrying a red fluorescent protein marker. Each of the four EIAV shRNAs and a control non-targeting shRNA will be delivered into fetal equine kidney (FEK) cells using a lentiviral vector. Initial integration rates will be determined by dsRed fluorescence, but stable populations of FEK cells expressing the shRNAs will be produced by drug selection (neomycin resistance carried in with the shRNA). Transgenic cell lines will be tested by challenging with a laboratory strain of EIAV, (EAIV19) (Payne, Lim et al. 2005). Cells in which EAIV19 replicates will shed viral particles into the media and can be detected using a reverse transcriptase (RT) assay.  Cell lines that inhibit viral replication transiently will be tested for mutation of the virus at the shRNA-targeting site by clonal propagation of virus, PCR amplification and sequencing of the viral target site. 

Results: Three of the four designed shRNA segments were successfully inserted into carrier dsRed plasmid.  Large plasmid volumes were generated and transfected to construct a lentiviral vector.  Infection into fetal equine kidney cells is pending.

Conclusions: The results of this work are expected to further understanding of EIAV in terms of its mutation rates and invariant regions, while aiding owners and veterinarians in its prevention.  The results of this research will also contribute to the further understanding of HIV-1 and development of human treatments.