Detection technologies and physical methods used for separation of complex molecules can be effective tools in research when applied to bioparticles including, but not limited to, bacteria, viruses, and proteins. Dielectrophoresis (DEP) is a technique that has been used in microfluidics for separation and concentration of bioparticles, with the benefits of not requiring custom primers, utilizing small sample sizes, and relatively quick separation times for rapid identification of pathogens such as viruses. As demonstrated in this study, a DEP device using polydimethylsiloxane (PDMS) as an insulator was used for the identification and separation of a mouse hepatitis coronavirus (MHV), a model coronavirus that only infects mice. Results indicate that, using 10 microliters of MHV test sample diluted in buffer, the virus can be identified and separated within 30 seconds using DC voltage of 800 V.

The need of organs for transplantation has become an increasing medical need due to a limited donor organ supply. Many organs fail within 10 years due to acute and chronic rejection. Acute or antibody mediated rejection leads to decreased long term graft survival and increases the need for a repeat transplant. In prior work, reducing endothelial heparan sulfation and blockade of chemokine-glycosaminoglycan (GAG) interaction with Myxomavirus-derived protein, M-T7, reduced aortic and renal graft vascular inflammation and rejection. Conditional endothelial Ndst1 deficiency and inhibition of chemokine-GAG interaction reduces early allograft damage and suggest new therapeutic options for graft rejection. Here acute renal rejection was examined in grafts with conditional endothelial N-deacetylase-N-sulfotransferase-1 knockout (Ndst1-/-) and in wildtype (WT) C57Bl6/J grafts treated with saline, M-T7, antisense oligonucleotides (ASO) for Ndst1 or a scrambled ASO control. Viruses have a highly adaptive ability to evade hosts defense and immune response. The immunomodulatory proteins derived from viruses provide potential therapeutic uses to alleviate this need for organs. The Myxoma virus derived protein M-T7 is a promising therapeutic for reducing kidney transplant rejection. Orthotopic transplantations in mice are extremely difficult and costly because they require a highly trained microsurgeon. This kidney to kidney subcapsular and subcutaneous transplant model is a practical and simpler method that requires fewer mice, one kidney can be used for transplants in 6 or more mice and there is much lower morbidity, pain and mortality. Heterotopic transplantation of allografts is a simple model for preliminary testing of treatments for early inflammation, ischemia, and graft rejection. Subcapsular kidney transplantation provides a first step approach to test virus-derived proteins as potential treatments to reduce transplant rejection and inflammation. This project reports on a broadly applicable platform on which to rapidly and conveniently test new treatments for transplant rejection. This finding will significantly lower the barrier to entry for labs which are interested in translating their laboratory findings to animal models of organ transplantation which is a complex surgical procedure, and thus accelerate the bench-to-bedside translation of novel, putative treatments for transplant rejection as an initial screening tool.

Structural details about viruses and their components are important for understanding the many steps in a virus life-cycle, including entry into host cells, replication, assembly, and release of progeny virions. X-ray crystallography and electron microscopy, including cryo-EM, have been used extensively for virus structural studies. Recent advances with cryo-EM have significantly advanced the field with near-atomic resolution structures of viruses being achievable. X-ray free-electron lasers (XFELs) are a novel, developing method to solve structures for non-crystalline single particle targets like viruses. Diffraction patterns can be collected directly from particles at room temperature. High quality, homogeneous virus preparations are critical for both cryo-EM and XFEL studies. Thus, optimization of virus growth and sample preparation are important steps in virus structural studies. The work described in this thesis focused on optimization of protocols for growth and purification of bacteriophage PR772 for XFEL and complementary cryo-EM studies. PR772 is one of several model viruses used in the single particle initiative (SPI) experiments at the SLAC National Laboratory Accelerator Laboratory Linac Coherent Light Source (LCLS). SPI is a collaborative international effort that works towards identifying and solving challenges of high-resolution single particle imaging using XFELs. Single particle diffraction snapshots were collected from PR772 particles prepared with optimized protocols. PR772 preparations were also used for cryo-EM imaging, with the goal to obtain a high-resolution structure of the virus. The optimization and characterization employed to assure samples suitable for XFEL and cryo-EM are detailed, along with data collected with both approaches.

Recent studies have shown that human papillomavirus (HPV) plays a role in development of cancers, one of which is head and neck cancer. There is strong and consistent molecular evidence demonstrating that human papillomavirus (HPV) is an etiological cause of these oropharyngeal cancers. Despite the introduction of HPV vaccines, there is still an increase in human papillomavirus associated OPC (HPVOPC) and it is expected that the incidence of head and neck cancer, specifically oropharyngeal cancer (OPC) will increase. The aim of this study is to utilize human papillomavirus (HPV) seropositivity for rapid detection of HPV early specific antigen-antibodies using a lateral flow assay.

Human papillomavirus (HPV) 16 proteins of interest, E7, E6 and CE2 were expressed and purified in E. coli for detection of specific antibodies using lateral flow assay because viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer. 17 samples and 5 controls with already known antibody reactivity from ELISA analysis were selected for HPV serologic responses. The lateral flow strip was evaluated for its color band intensity using Image J software. Peak area was used to quantify the color intensity of the lateral flow strip. Out of the 17 samples, 11 (64.7%) showed high antibody levels to E7, 12 (70.6%) showed high Ab levels to E6 and 6 (35.3%) showed high Ab levels to CE2. Correlation coefficient between antibody detection by sight and ELISA for E7, CE2 and E6 were 0.6614, 0.4845 and 0.2372 respectively and correlation coefficient between lateral flow assay and ELISA for E7, CE2 and E6 were 0.3480, 0.1716 and 0.1644 respectively. This further proves patients or samples with HPV 16 oropharyngeal cancer have detectable antibodies to early E7, E6 and E2 proteins, which are potential biomarkers for HPV-associated oropharyngeal cancer.

Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV.

Coronaviruses are a medically significant group of viruses that cause respiratory and enteric infections in humans and a broad range of animals. Coronaviruses assemble at the internal membranes of the endoplasmic reticulum- Golgi intermediate compartment (ERGIC). While there is a basic understanding of how viruses assemble at these membranes, the full mechanistic details are not understood. The coronavirus envelope (E) protein is a small multifunctional viroporin protein that plays a role in virus assembly but its function is unknown. The two goals of this study were : 1. To identify and analyze the localization of MHV E and 2. To identify the functions of conserved residues in the tail of the E protein. This study closely examined the localization, dynamics and mobility of the mouse hepatitis virus (MHV) E protein to gain insight into its functions. The results from the first aim of this study showed that the MHV E protein localizes at the site of assembly in the ERGIC-Golgi region based on analysis by immunofluorescence and correlative electron microscopy. A novel tetra-cysteine tagged MHV E protein was used to study the dynamics of the protein in cells. A recombinant MHV E Lumio virus was used to study the trafficking and mobility of the E protein. Live cell imaging and surface biotinylation confirmed that the E protein does not traffic to the cell surface. Fluorescence recovery after photo-bleaching (FRAP) analyses revealed that the E protein is mobile at the site of localization. As a part of the second aim, conserved prolines and tyrosine in the tail of the protein were targeted by site directed mutagenesis and analyzed for functionality. While none of the residues were absolutely essential for localization or virus production, the mutations had varying degrees of effect on envelope formation, protein stability and virus release. Differential scanning calorimetry data suggests that the proline and tyrosine residues enhance interaction with lipids. A wild type (WT) peptide contained the conserved residues was also able to significantly reduce the hexagonal phase transition temperature of lipids, whereas a mutant peptide with alanine substitutions for the residues did not cause a temperature shift. This suggests that the peptide can induce a negative curvature in lipids. The E protein may be playing a role as a scaffold to allow membrane bending to initiate budding or possibly scission. This data, along with the localization data, suggests that the E protein plays a mechanistic role at the site of virus assembly possibly by remodeling the membrane thereby allowing virus budding and/or scission.

Coronaviruses are medically important viruses that cause respiratory and enteric infections in humans and animals. The recent emergence through interspecies transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) strongly supports the need for development of vaccines and antiviral reagents. Understanding the molecular details of virus assembly is an attractive target for development of such therapeutics. Coronavirus membrane (M) proteins constitute the bulk of the viral envelope and play key roles in assembly, through M-M, M-spike (S) and M-nucleocapsid (N) interactions. M proteins have three transmembrane domains, flanked by a short amino-terminal domain and a long carboxy-terminal tail located outside and inside the virions, respectively. Two domains are apparent in the long tail - a conserved region (CD) at the amino end and a hydrophilic, charged carboxy-terminus (HD). We hypothesized that both domains play functionally important roles during assembly. A series of changes were introduced in the domains and the functional impacts were studied in the context of the virus and during virus-like particle (VLP) assembly. Positive charges in the CD gave rise to viruses with neutral residue replacements that exhibited a wild-type phenotype. Expression of the mutant proteins showed that neutral, but not positive, charges formed VLPs and coexpression with N increased output. Alanine substitutions resulted in viruses with crippled phenotypes and proteins that failed to assemble VLPs or to be rescued into the envelope. These viruses had partially compensating changes in M. Changes in the HD identified a cluster of three key positive charges. Viruses could not be recovered with negatively charged amino acid substitutions at two of the positions. While viruses were recovered with a negative charge substitution at one of the positions, these exhibited a severely crippled phenotype. Crippled mutants displayed a reduction in infectivity. Results overall provide new insight into the importance of the M tail in virus assembly. The CD is involved in fundamental M-M interactions required for envelope formation. These interactions appear to be stabilized through interactions with the N protein. Positive charges in the HD also play an important role in assembly of infectious particles.