Using Semliki Forest virus as a vaccine platform against Bovine Respiratory Syncytial Virus / Attenuated replication of Semliki Forest virus in potassium channel inhibitor treated cells

Abstract

This study includes two subjects linked by Semliki Forest virus (SFV). The first is to examine the potential impairment of the replication of SFV through the use of ion channel inhibitor drugs. The second is to use SFV to make a potential Bovine Respiratory syncytial virus (BRSV) vaccine. The Alphavirus genus contains dangerous viruses for human health such as Chikungunya virus (CHIKV). SFV is a safe model and the results can potentially be applied to other alphaviruses. Viruses have a strong ability to adapt to or develop resistance to antiviral drugs in order to maintain replication and spread. This is due to the high rate of genetic mutation normally associated with RNA viruses in particular. So to potentially overcome this there may be opportunities to target the host rather than the virus with drugs, that will ultimately impact on the virus. In the first results chapter of this study, potassium channels were inhibited using a broad-spectrum inhibitor, potassium chloride and cells were also treated with another, more specific inhibitor, Spermine, which targets 2pK potassium channels. Viability assays and immunostaining were preformed to demonstrate the attenuated replication of SFV. In an attempt to identify the specific channel involved, siRNA knockdowns of the individual 2pK ion channels, were performed but results were inconclusive. Secondly, there is a noticeable economic impact in the agricultural sector as a result of livestock infection with Bovine Respiratory Syncytial Virus (BRSV). There are vaccines available for this disease, for example, a prophylactic live-attenuated vaccine provided by Pfizer, but BRSV continues to be a major problem in the industry thus raising questions around the effectiveness of these current vaccines. So, the idea of producing a more-effective vaccine is appealing. To take advantage of the genetic expression potential of SFV, a gene that translated to a protein that causes an immune response was inserted into SFV using a plasmid- based genome system. As part of this project, tests were conducted to verify the genetic expression efficacy of the plasmid and the ability to generate virus replicon particles (VRPs). Western blotting was performed to detect proteins that were produced after transfection confirming successful cloning of the BRSV gene and infection studies confirmed generation of VRPs that could express the BRSV protein successfully.