Cryogenic Electron Microscopy of Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is an important livestock and human pathogen. It is also a potential bioweapon owing to its ability to spread by aerosols. It is an enveloped virus containing surface protrusions composed of two viral glycoproteins, Gc and Gn; the viral core contains ribonucleoprotein complexes. We describe the use of cryogenic electron microscopy (Cryo-EM) of single particles to visualize the three-dimensional (3D) organization of RVFV. The spherical RVFV virions have a diameter of 100 nm with icosahedral symmetry (T = 12) and 720 prominent protrusions on their surface.In this chapter we describe the general protocols to prepare virus suspensions for Cryo-EM, image acquisition of virus particles embedded in vitreous water, and their image processing. Because of RVFV being classified as a BSL3 and potentially as a select agent, we also describe the protocol of performing Cryo-EM grid preparation and imaging in our Cryo-EM BSL3 containment, along with the general etiquette of using BSL3 containment for research, including entry into containment, dressing up for work there, manipulation with the agent, grid preparation, transfer of the frozen grids into a microscope, etc., waste disposal, and finally exit from the containment. The microscope decontamination with ClO2 is briefly described as well because that technique is not widely used in the Cryo-EM field.