Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Biological Science

Committee Chair

Jason K. Lanman

Committee Member 1

Richard J. Kuhn

Committee Member 2

Cynthia V. Stauffacher

Committee Member 3

R. Claudio Aguilar


All of the known positive-sense RNA viruses cause alterations to the membranes of their host cells. These modifications are thought to be involved in replication and assembly of the viruses. Alphaviruses are a genus of viruses that can elicit the formation of two distinct membrane structures, which are derived from the host cell membranes. These structures are referred to as cytopathic vacuole 1s (CPV- 1s) and 2s (CPV-2s). CPV-1 generally form early during infection and are typified as vacuoles that contain small (approx. 50 nm in diameter) invaginations that are contiguous with the cytoplasm termed spherules and are the sites of viral replication. CPV-2s occur later during infection and are associated with assembly and egress of the nucleocapsids (NCs) out of the cell. Morphologically they appear as vacuoles with NCs around the cytoplasmic face of their membranes and are associated with glycoprotein transport. A large portion of this work is understanding the variability of these structures with various alphaviruses and with different cell types, as well as, examining them at different levels of resolution. In this work large areas of an alphavirus-infected, specifically vaccine-strain Chikungunya virus (CHIKV), mammalian cells with high levels of induced CPV-2 structures were imaged in three-dimensions (3D) through large-area electron tomography (ET). From the 3D volume, qualitative and quantitative data was derived from the CPV-2 membrane structures. Two different types of CPV-2s were identified: singlemembraned (SM-CPV2s) and double-membraned (DM-CPV2s). The DM-CPV2s also had NCs inside which were bound to the inner lea et of the inner membrane. Measurements such as surface area and volume indicated that a portion of the SMCPV2s may be able to transition into DM-CPV2s. Additionally, these structures appeared to be derived from highly modified Golgi that had altered morphology from what is seen in uninfected cells. All of this suggested a model where the Golgi stacks start breaking down their native structure, NCs interact with the resulting remnants of Golgi and depending on the size and shape of the starting piece form into SMCPV2s. After forming the SM-CPV2s a subset are have enough NC interactions to cause membrane curvature and also enough membrane to form into a DM-CPV2s. The DM-CPV2s are then trafficked to the plasma membrane where they expel their contents and deposit glycoproteins and NCs onto the plasma membrane. Since alphaviruses are arboviruses the membranous alphavirus formations were examined in the context of a mosquito (Aedes albopictus) cell infection in three different cell subclones (C6/36, C7-10, and U4.4). This examination was done through the use of the correlative light and electron microscopy (CLEM) technique to target only those cells that were infected with a uorophore bearing Sindbis virus in nsP3 (SINV-mCherry), saving time during EM screening. Spherules were observed dispersed among internal vesicles for every cell line and in some cases looked morphologically similar to CPV-1s observed in mammalian cells. Additionally, a CPV-1 structure was observed in U4.4 cells for the first time indicating that these structures form in the insect as well since this cells have been characterized as the most similar to the actual mosquito. Internal vacuoles were also observed that were filled with viral particles that were assumed to have budded into the vacuole. Vacuoles were also observed that contained both spherules and budded viral particles, suggesting that in mosquito cells replication and assembly may be more linked than in mammalian cells. The last chapter involves the collection of an entire whole BHK cell infected with Sindbis virus (SINV). This procedure was undertaken to establish how a cell of this size could be imaged through serial-section ET and to hopefully establish globally spatial relationships among organelles (ER, mitochondria, etc.) during an alphavirus infection. Steps and pitfalls in the process are addressed. However, the produced volume proved too noisy for a timely analysis. With the aim of producing a dataset with better contrast and resolution, an experiment with scanning transmission electron microscopy (STEM) was conducted. With this alternative imaging not only were vacuoles with spherules able to be observed, but the NCs as well while still having the entire cell in the field of view.