Department of Veterinary Medicine Theses and Dissertations
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- ItemSTRUCTURE-FUNCTION ANALYSES OF AN ESSENTIAL VIRULENCE DETERMINANT OF THE LYME DISEASE PATHOGEN(2022) Foor, Shelby Dimity; Pal, Utpal; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lyme Disease (LD) is a tick-borne disease caused by a group of gram-negative-like spirochetal pathogens called Borrelia burgdorferi sensu lato. The number of cases reported in the United States have dramatically increased with CDC estimating 476,000 cases annually. This multifaceted infection can spread throughout the entire body, causing clinical complications of the central nervous system, joint and heart. Early antibiotic treatment is available and effective; however, untreated patients can develop chronic symptoms, and even after antibiotics, symptoms of unknown etiology and pathogenesis can develop into post-treatment Lyme disease syndrome (PTLDS). The enzootic life cycle of B. burgdorferi is maintained typically between a small rodent and the Ixodes tick vector, where transmission occurs during tick feeding on a host. Infection establishes after B. burgdorferi is deposited in the dermis and undergoes the required shift in its protein expression profile necessary to support spirochete persistence and pathology, often highlighting protein targets for development of diagnostic, therapeutic, and preventative measures. Two such proteins identified, BB0238 and BB0323, serve as novel virulence determinants and are essential for mammalian infection. These two proteins directly interact, mutually stabilize each other post-translationally, and form an essential complex required for infection; however, their precise functions remain undetermined. In collaborative efforts, we predicted a two-domain structure of BB0238. The N-terminal domain was predicted by AI methods to harbor an antiparallel helix-turn-helix motif (HTH) followed by a third helix and a low-confidence predicted meandering segment. The C-terminal domain structure was determined by X-ray crystallography as well as predicted with high confidence to adopt an α+β fold that resembles closely that of the nuclear transport factor 2 (NTF2) superfamily. While full-length BB0238 lacks homology to singular proteins of known functions, the individual N- and C-terminal regions display structural homology to non-bacterial proteins, particularly to eukaryotic sorting, or transport proteins, suggesting that BB0238 supports an unconventional function in spirochetes. We discovered that BB0238 binds another borrelial protein annotated as BB0108, orthologs of two bacterial chaperones and foldases, the extracellular membrane anchored PrsA, and the periplasmic SurA. This identified interaction requires further investigation, however, may be important for BB0238 protein stability or assist with the novel BB0238 function discovered herein, which regulates proteolytic processing of BB0323. Furthermore, We show that key amino acid residues within the HTH stabilize BB0238 in an environment-specific manner, influence its oligomerization properties, and facilitate tick-to-mouse transmission by aiding spirochete evasion of host cellular immunity, underscoring BB0238’s ability to support microbial establishment during early mammalian infection. Together, these studies highlight the divergent evolution of multidomain spirochete proteins involved in multiplex protein-protein interactions, possibly facilitating multiple functions, which support pathogen survival and thus, represent novel targets for vaccine and therapeutic development against Lyme disease.
- ItemDEVELOPMENT OF AVIAN PARAMYXOVIRUS VECTORED VACCINES(2022) Elbehairy, Mohamed Adel; Belov, George A; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Avian Avulaviruses (formerly Avian Paramyxoviruses, APMV) are important pathogens of avian species and have been used as viral vectors for more than two decades. Among all APMVs, Newcastle disease virus (NDV or APMV-1) has been most extensively used as a vaccine vector for protection against avian and animal diseases, and as an oncolytic agent. For poultry vaccination, the preexisting maternal antibodies against NDV can neutralize APMV-1 vectors resulting in vaccination failure. Hence, there is a need to develop new vaccine vectors that would escape neutralization by the maternal antibodies. In the first part of my study, I created a reverse genetics system for Avian paramyxovirus-3 strain Wisconsin (APMV-3 Wisc.) which was proven to be nonpathogenic for day-old chicks and embryonated chicken eggs. The virus was used as a vector to express the enhanced green fluorescent protein (GFP) as a heterologous antigen. The recombinant APMV-3 Wisc. expressing GFP was compared with similarly constructed APMV-1 strain LaSota and APMV-3 strain Netherlands-based vectors for GFP expression and growth kinetics in vitro, and for immunogenicity, safety, and tissue tropism in day-old specific pathogen-free (SPF) chicks. APMV-3 strain Netherlands (APMV-3 Neth.) showed the highest growth rate and GFP expression in chicken fibroblast DF-1 cells, followed by APMV-1 LaSota and APMV-3 Wisconsin. In day-old chicks, APMV-3 Neth. spread to different organs, decreased feed intake and caused stunted growth. APMV-3 Wisc. and APMV-1 LaSota were confined to the respiratory tract and did not induce any pathogenic effects. All three constructs induced seroconversion of the vaccinated chicks for the vector antigens. Thus, the reverse genetics system created in this study for APMV-3 Wisc. allows the development of safe APMV vector antigenically different from NDV that can be used for day-old chicks vaccination. In addition, it provides a tool to study the molecular basis of APMV3 pathogenesis. In the second part of my study, I explored a novel approach for the expression of a foreign gene as an uninterrupted open reading frame (ORF) with a cognate gene of NDV vector. This approach is expected to promote the foreign gene expression stability. Avian influenza virus (AIV) hemagglutinin (HA) protein-coding sequence was fused in-frame with various proteins of NDV vector, with a 2A self-cleaving peptide, a furin cleavage site, or both, placed between the AIV and NDV sequences for separation of the two proteins. Among different constructs tested, we only recovered viable viruses with AIV HA fused C-terminally to the NDV HN gene. These viruses demonstrated a higher expression level of AIV HA than the vector constructed according to a traditional scheme of expressing the transgene as a separate transcriptional unit. Also, they showed increased stability of the transgene expression over multiple passages in embryonated chicken eggs. Our results demonstrate the advantages and limitations of this novel method of foreign gene expression that need to be considered for the development of NDV-based vaccine or therapeutic vectors.
- ItemHost Immune Responses to Fungal Pathogens(2022) Strickland, Ashley Brenda; Shi, Meiqing; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Fungal infections are an increasing threat to global public health. Worldwide, more than one billion fungal infections are diagnosed each year and result in 1.5 million deaths annually. This number is expected to rise as advances in medical care for immunocompromised patients continue to be made. Furthermore, limited antifungal treatment options, coupled with the emergence of new and resistant fungal species, is anticipated to exacerbate this issue. For these reasons, it is essential to understand the dynamic interactions between host and fungus that contribute to fungal pathogenesis. The following work examined the role of IL-27 on T cell regulation in the lungs during infection with Aspergillus fumigatus, and in the brain during infection with Cryptococcus neoformans. In contrast to most infection settings demonstrating that IL-27 is anti-inflammatory, we report that this cytokine is proinflammatory in both the lung and the brain during infection with A. fumigatus and C. neoformans respectively. Genetic ablation of IL-27Rα in mice resulted in higher fungal burdens in the lung during Aspergillus infection and in the brain during Cryptococcus infection. These were associated with reduced IFN-γ production, as well as a fewer CD4+ T cells in target organs. In the case of C. neoformans infection, IL-27 signaling enhanced T cell accumulation by promoting T cell proliferation early during infection, and inhibiting T cell death at later time points. These suggest that IL-27 promotes T cell responses required for fungal clearance during infection with A. fumigatus or C. neoformans. In addition, the work presented here also characterized lung resident macrophage responses during infection with C. neoformans. Here, we report that cryptococcal infection induced the accumulation of CD68hi macrophages in the lungs of infected mice. Approximately 10% of these cells were alveolar macrophages, while nearly 90% were interstitial macrophages. Both of these populations were observed to interact with fungi and upregulated their expression of arginase 1. The absence of either macrophage in transgenic mice, or following pharmacological depletion, significantly reduced fungal burdens in the lungs of mice, indicating that these cells enhance fungal growth and may represent attractive targets aimed at limiting the pathogenesis of C. neoformans.
- ItemIdentification and functional characterization of the GBF1-controlled network of host proteins supporting enterovirus replication(2022) Moghimi, Seyedehmahsa; Belov, George; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The genus Enterovirus of the Picornaviridae family contains many established and emerging pathogens. However, licensed vaccines are currently available only against poliovirus and enterovirus A71. No therapeutics have been officially approved to treat any enterovirus infections, although some are being developed. To find suitable targets for antivirals and control the infections, we need to understand the virus's life cycle better and identify the cellular factors involved in virus infection. Enterovirus genome replication occurs on the unique membranes known as replication organelles (ROs). A Golgi resident protein, GBF1, is recruited to the ROs by a viral protein 3A. GBF1 activates small GTPases Arf, which are critical regulators of the cellular secretory pathway. Here, we investigated the mechanistic details of GBF1-dependent Arf activation during enterovirus replication and characterized the proteome of the ROs in the vicinity of GBF1. We showed that Arf1 appeared to be the first to associate with the ROs, followed by other Arfs. Once activated and recruited to the ROs, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that they do not actively cycle between GTP- and GDP-bound states in infected cells. siRNA depletion studies demonstrated an increased sensitivity of polio replication to inhibition of GBF1 in Arf1-, and to a lesser extent, Arf6-depleted cells, indicating the importance of GBF1-mediated activation of these Arfs for the viral replication. Taking advantage of the GBF1 recruitment to the ROs and GBF1’s essential role in enterovirus replication, we used a GBF1 construct fused to APEX2 peroxidase to explore the proteome of the ROs by proximity biotinylation. Among the proteins biotinylated in infected cells were the known cellular factors recruited to the ROs, including PI4KIII, OSBP, and ACBD3, indicating that these proteins are localized close to GBF1. Among the viral proteins, the intermediate products of the polyprotein processing were overrepresented, suggesting that GBF1 is localized close to the sites of active polyprotein processing. About 85% of the proteins identified by MS have not been previously associated with enterovirus infection. Gene ontology analysis revealed a significant enrichment of RNA binding and mRNA metabolic processes, suggesting a close localization of GBF1 to the RNA replication complexes. siRNA knockdown functional analysis of the selected proteins showed the recruitment of both proviral and antiviral factors to the ROs. Collectively, our work revealed important details about the involvement of Arfs in the replication process, introduced a highly efficient system to investigate the proteome of the enterovirus ROs, and provided novel data about the protein composition of the GBF1-enriched environment in the replication sites.
- ItemDevelopment of avian paramyxovirus 3 as a vaccine vector against infectious bursal disease in one-day-old specific pathogen free chickens(2021) Varghese, Berin Parambethu; Zhu, Xiaoping; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACTInfectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease affecting young chickens, resulting in substantial economic losses to the poultry industry worldwide. Although strict hygienic measures and various vaccination strategies have been adopted, IBD remains a major problem for the poultry industry. The economic significance of this disease is exhibited in two ways. First, the disease can cause high morbidity and mortality. Second, severe prolonged immunosuppression of chickens when infected at an early age leads to susceptibility to other diseases and vaccination failure. Therefore, chicks should be protected from early infection, usually achieved by vaccinating breeder flocks and active immunization of the newly hatched chicks. Vaccines include live-attenuated, viral vectored recombinant, subunit, and inactivated vaccines currently available to control infectious bursal disease (IBD). The major flaws of available vaccines are the reversion to virulence, the generation of new variant viruses, inability to replicate in the presence of maternal antibodies, lack of proper delivery systems, recombination and integration into the host genome, and inadequate systemic immune response. To overcome these obstacles, we have evaluated avian paramyxoviruses (APMVs) as vaccine vectors to generate vaccines against IBD. In the present study, we constructed recombinant Newcastle disease virus (NDV) strain LaSota (rLaSota/VP2) and recombinant avian paramyxovirus-3 (APMV-3) strain Netherlands expressing VP2 protein (rAPMV-3/VP2), the immunogenic protein of IBDV, and to evaluate their protective efficacies following immunization of one-day-old specific pathogen-free (SPF) chicks. Our results showed that both recombinant viruses stably express the VP2 protein, and their in vitro growth characteristics were almost similar to their respective parental viruses. Immunization of one-day-old SPF chicks demonstrated that rAPMV-3/VP2 protein elicited IBDV specific neutralizing antibodies and provided complete protection against the IBDV STC challenge. In addition, the rAPMV-3/VP2 protects chickens from clinical signs, gross lesions, and histopathology even at lower vaccine doses. Moreover, rAPMV-3/VP2 provides slightly better protection than the commercial vaccine from histopathology lesion against IBD at four weeks of age. This study suggests that recombinant APMV-3 expressing VP2 protein could be used as a potential vaccine against IBD in field conditions where maternal antibodies exist.