Browsing by Subject "Bunyavirales"

Rift Valley fever virus (RVFV) is a mosquito-borne virus of the order Bunyavirales with a tripartite (-)ssRNA genome. It infects humans and cattle, causing a febrile disease with symptoms ranging from mild to severe. Safe and efficient human vaccines have not yet been developed, which underlines the importance of gaining a clear understanding of the viral antigenic surface. One significant challenge for RVFV research is posed by the costly and time-consuming biosafety precautions warranted by the pathogenicity of the virus. The surface of RVFV, formed by the two viral envelope glycoproteins Gn and Gc, could also be studied with a non-pathogenic model, such as a virus-like particle (VLP). A VLP is a macromolecular complex that resembles the virus, especially with respect to its outer structure, but lacks the viral genome. In this work, RVFV VLPs were produced by transient transfection of mammalian cells with genes encoding RVFV glycoproteins Gn and Gc. The objective was to design an optimized production and purification pipeline for RVFV VLPs to elucidate their structure by cryo-electron microscopy. To optimize the VLP production and purification, the effect of sample harvest times and DNA-to-cell ratios of transfection on RVFV glycoprotein expression was examined. Several methods were tested for VLP sample concentration and purification. VLPs were successfully detected in the purified samples by immuno-electron microscopy. Despite some challenges related to sample purity and scarcity of VLPs in samples, which prevented analyses by cryo-electron microscopy, the expression system described in this thesis has great potential to streamline RVFV VLP sample preparation for electron microscopy and to accelerate vital research into the structural properties of this emerging pathogen.