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Jordan Kraft

Fellow – Jordan Kraft

Mentor – Charles Long

Project – Blocking Equine Infectious Anemia Virus replication using RNA interference

Objective – The aim of the project was to use RNA interference to block replication of Equine Infectious Anemia Virus (EIAV) in vitro.

Procedure – A Feline Adenocarcinoma cell line (FEAs) and a Fetal Equine Kidney cell line (FEKs) were genetically modified to express short hairpin RNAs (shRNAs) targeted at the EIAV genome. To modify the cells an engineered lentivirus was produced in human embryonic kidney cells using a Nef-Red plasmid, a packing plasmid, and a non-structural protein plasmid. The Nef-Red plasmid contains a shRNA, a fluorescent marker, and a neomycin resistance gene. Modified cells were visualized using fluorescence microscopy and were selected for using a neomycin analog. Viral replication was evaluated using a reverse transcriptase (RT) assay. Supernatant was collected from cell culture every 3 – 5 days and run through the RT assay. Finally, the RT data was normalized using a modified cell line lacking shRNA expression.

Results – RT levels revealed knockdown of EIAV replication at Day 8 post-transduction in all FEA treatment groups. Pol2 and Rev2 shRNAs showed knockdown through day 15. Gp2 and Ser2 shRNAs were contaminated mid-experiment invalidating later data collected from those lines. FEK cells were successfully modified using the engineered lentivirus; confirmation was realized using fluorescence.

Conclusions – Equine Infectious Anemia Virus replication can be blocked using RNA interference. The data shows an across the board decrease in EIAV replication in treatment cell lines. Contamination problems noted in the results prevented a complete data set; however, repeating the experiment may provide more evidence to support the conclusions of this experiment.