Theses and Dissertations from UMD

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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM

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    HEPATITIS E VIRUS MODULATES HOST FACTORS TO GENERATE A CONDUCIVE ENVIRONMENT FOR REPLICATION
    (2020) lin, shaoli; Zhang, Yanjin; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV infection mainly presents as acute and self-limiting hepatitis in young adults. However, it can be exacerbated to fulminant hepatitis in HEV-infected pregnant women, resulting in up to 30% case fatality. Besides, chronic HEV infection with rapid progression in immunocompromised patients has been a challenge in many countries since it was reported years ago. HEV infection is zoonotic, and human HEV strains are grouped into four major genotypes in the genus Orthohepevirus A, the family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. Due to the lack of an effective cell culture system and a proper animal model, HEV biology, virus-cell interactions, and pathogenesis are understudied. HEV is known to inhibit the innate immune response by targeting type I interferon (IFN) signaling pathway via its ORF1 products. Nevertheless, it remains largely unknown how the virus manipulates host factors to facilitate its replication. The objective of these studies was to elucidate the mechanism of HEV manipulation of host factors to generate a conducive environment for replication. Our results show that the capsid protein of HEV inhibits the IFN production to dampen the antiviral response through its N-terminal arginine-rich motif. In addition to the impairment of innate immunity, HEV proliferation requires the presence of other host factors: DDX3, an RNA helicase, and oxysterol-binding protein (OSBP), a lipid transporter. The knockdown of these two factors led to a significant reduction of HEV replication, whereas the reconstitution of these two genes restores the HEV proliferation level. The capsid protein was found to interact with the C-terminal domain of DDX3. The HEV helicase was shown to interact with OSBP and block its translocation to the Golgi apparatus. These results indicate that HEV employs multiple strategies including blocking antiviral response and recruiting host factors for its invasion and proliferation. Our data provide insights into the HEV-cell interactions and may facilitate the development of novel antiviral strategies.
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    MOLECULAR PATHOGENESIS OF INFLUENZA IN SWINE AND ENGINEERING OF NOVEL RECOMBINANT INFLUENZA VIRUSES
    (2011) Pena, Lindomar Jose; Perez, Daniel R; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Influenza A viruses (IAVs) belong to the family Orthomyxoviridae and represent major pathogens of both humans and animals. Swine influenza virus is an important pathogen that affects not only the swine industry, but also represents a constant threat to the turkey industry and is of particular concern to public health. In North America, H3N2 triple reassortant (TR) IAVs first emerged in 1998 and have since become endemic in swine populations. In the first part of this dissertation, we focused on the role of surface glycoproteins and PB1-F2 to unravel their roles in the virulence of TR IAVs in this important natural host. We found that surface glycoproteins are necessary and sufficient for the lung pathology, whereas the internal genes play a major role in the febrile response induced by TR H3N2 IAVs in swine. With respect to PB1-F2, we found that PB1-F2 exerts pleiotropic effects in the swine host, which are expressed in a strain-dependent manner. Pathogenicity studies in swine revealed that the presence of PB1-F2 leads the following effects in context of three TR strains tested: no effect in the context of sw/99 strain; increases the virulence of pH1N1; and decreases the virulence of ty/04. Next, we developed temperature-sensitive live attenuated influenza vaccines for use in swine and shown that these vaccines are safe and efficacious against aggressive intratracheal challenge with pH1N1. Lastly, we rearranged the genome of an avian H9N2 influenza virus to generate replication competent influenza virus vectors that provide a robust system for expression and delivery of foreign genes. As a proof-of-principle, we expressed the hemagglutinin from a prototypical highly pathogenic avian influenza virus (HPAIV) H5N1 and shown that this vectored H5 vaccine retained its safety properties in avian and mammalian species, and induced excellent protection against aggressive HPAIV H5N1 challenges in both mice and ferrets. Taken together, these studies have advanced our understanding of molecular basis of pathogenesis of influenza in the swine host and have contributed to the development of improved vaccines and influenza-based vectors with potential applications in both human and veterinary medicine.
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    CHARACTERIZATION OF TWO HIGHLY CONSERVED POXVIRUS TRANSMEMBRANE PROTEINS OF UNKNOWN FUNCTION
    (2009) Sood, Cindy Leigh; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. I5 synthesis was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered. The vaccinia virus A43R open reading frame encodes a 168-amino acid protein with a predicted N-terminal signal sequence and a C-terminal transmembrane domain. Although A43R is conserved in all sequenced members of the orthopoxvirus genus, no non-orthopoxvirus homolog or functional motif was recognized. Biochemical and confocal microscopic studies indicated that A43 is expressed at late times following viral DNA synthesis and is a type-1 membrane protein with two N-linked oligosaccharide chains. Neither mature nor enveloped virions contained appreciable amounts of A43, which was detected in Golgi and plasma membranes. Loss of A43R expression had no discernible effect on plaque size or virus replication in cell culture and little effect on virulence in a mouse intranasal infection model. Although the A43 mutant produced significantly smaller lesions in the skin of mice than the control, the amounts of virus recovered from the lesions were similar.
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    Host Molecular Responses in Chickens Infected with an Avian Influenza Virus
    (2008-11-20) Ramirez-Nieto, Gloria Consuelo; Perez, Daniel R.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Avian influenza virus has a segmented RNA genome that allows the virus to evolve continuously and generate new strains. Wild birds serve as natural reservoirs of avian influenza virus and provide a potential source for emergence of new viruses, which traverse host barriers and infect new avian or mammalian species. The mechanisms involved in this process are not completely understood. Our main goal is to understand host-pathogen interactions involved in avian influenza pathogenicity. As part of our approach we studied the effect of pre-exposure of chickens to IBDV (infectious bursal disease virus) on host susceptibility to infection, disease progression, and host molecular responses to infection with a mallard H5N2 low pathogenic avian influenza (LPAI) virus. We found that prior exposure of chickens to IBDV led to increased susceptibility to infection with the mallard H5N2 LPAI virus compared to normal chickens. This increased susceptibility allowed us to further adapt the virus to chickens. After 22 passages (P22) in IBDV-pre-exposed chickens, the LPAI virus replicated substantially better than the wild-type (WT) mallard virus in both IBDV-exposed and normal chickens. Interestingly, the P22 virus showed similar levels of replication in the respiratory and intestinal tracts of both groups, although it caused exacerbated signs of disease and severe lesions in the IBDV-pre-exposed group. We suggest that prior IBDV exposure provides a port of entry for avian influenza in an otherwise resistant chicken population. Furthermore, adaptation of avian influenza (AI) in IBDV-exposed chickens may allow for the selection of AI virus strains with expanded tissue tropism. We also studied the effects of host response to H5N2 AI in normal and IBDV-infected birds using high-throughput gene expression analysis. We demonstrated that IBDV-exposed chickens showed less than optimal humoral responses to LPAI infection as well as alterations in local molecular pathways that eventually led to exacerbated disease and death. At the molecular level we found amino acid substitutions in the surface glycoprotein hemagglutinin (HA). Those changes suggest selection for a virus that binds to and replicates more efficiently in chickens. Taken together our results suggest that IBDV-pre-exposure may play a role in exacerbating AI-induced pathogenicity.