| Participant: | Grant, Ashley |
CRYO-ELECTRON MICROSCOPY OF JUNÍN CANDID#1
Ashley Grant1, M.Sherman, Ph.D.2, & CJ Peters, M.D.1,3
1Dept. of Pathology, 2Dept. of Biochemistry and Molecular Biology, 3Dept. of Microbiology and Immunology, UTMB
Background: Junín virus, the etiological agent of Argentine Hemorrhagic Fever (AHF), causes up to 30% mortality in humans and is a NIAID Category A Pathogen (Charrel et al, 2003). Arenaviruses are pleomorphic-enveloped viruses that contain a bisegmented ambisense single stranded RNA genome (Neuman et al, 2005). Arenaviruses code for a large (L) and a small (S) negative sense RNA segments. Candid#1 is the live attenuated vaccine strain that is currently licensed in Argentina. Methods: Candid#1 was grown at 35°C and 37°C over a ten-day period to determine when peak viremia was achieved. A 60°C kill curve was performed to show how long it takes to inactivate the virus. Candid#1 infection supernant was centrifuged to remove cell debris, polyethylene glycol was used to precipitate the virus and a potassium tartrate gradient was used to purify the virus. The virus was further concentrated on an Amicon ultrafiltration device. Particles were viewed by negative stain EM and cryo-electron microscopy. Results and Conclusions: We have revealed that Candid#1 is temperature sensitive growing better at 35°C than 37°C with a peak viremia at seven days post infection. The virus is completely inactivated at 60°C after 2 hours. Through electron microscopy we determined Junín is an enveloped virus approximately 200 nm in diameter with protruding glycoproteins. The virions are pleomorphic and therefore the reconstruction will be performed by cryo-electron tomography (cryo-ET). The viral glycoproteins are unambiguously organized on the surface of the Candid#1 particles. The negatively stained virions show variations in size, surface structure and surface units. The frozen hydrated Candid#1 particles viewed by cryo-EM show particles that are more spherical in shape but still pleomorphic. Supported by a training fellowship from the W.M. Keck Foundation to the Gulf Coast Consortia through the Keck Center for Virus Imaging.