Department of Veterinary Medicine Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/2762

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End date: 2019Subject: search.filters.subject.Veterinary medicine

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    Ecology and Molecular Epidemiology of Avian and Swine Influenza A viruses in Guatemala
    (2015) Gonzalez Reiche, Ana Silvia; Perez, Daniel R.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The greatest diversity of Influenza A viruses (IAV) is found in waterfowl species from numerous geographic locations. In addition, multiple IAV are, and continue to be, perpetuated in swine populations around the globe. Due to the zoonotic potential of IAV and to respond more effectively to potential agricultural and public health threats, there is a need to increase surveillance in avian and swine hosts in understudied geographical regions. In Latin America, avian influenza surveillance has been scarce, localized only to places where outbreaks in poultry have occurred. Similarly, active swine influenza surveillance was implemented only after the emergence of the 2009 pandemic strain (pH1N1). The project presented here was aimed at investigating the circulation of IAV in wild birds and pigs in Guatemala. Over 2200 birds were sampled during six consecutive migration seasons from 2007 to 2013 in different locations. Virus prevalence detected by rRT-PCR in positive species ranged from 5.2% to 38%. Preliminary data indicates temporal variation of IAV prevalence in migratory waterfowl. Eighty-three viruses were recovered with 22 different subtype combinations. Through phylogenetic inferences and the analysis of virus genotypes and gene constellations of 60 fully sequenced genomes, we provide a detailed description of the genetic structure of avian IAV circulating in Guatemala. Our results suggest that the virus diversity in this location is sourced from multiple migration flyways from North America. Overlap of these flyways, in a natural geographical bottleneck such as the Neotropics, may contribute to the patterns of extensive genetic reassortment observed at a continental scale. In addition, the results from two nationwide multistage random surveys in pigs demonstrated circulation of swine influenza in commercial and peridomestic herds in Guatemala. Herd prevalence of IAV was 36.3% in 2010 and 34.6% in 2011. Viruses of the H1N1 and H3N2 subtypes and antibodies against viruses of distinct genetic lineages of these subtypes were detected. Our results indicate that human-animal contact likely plays a role in the IAV epidemiology in local swine populations. The findings from this research constitute the most abundant data on the ecology and epidemiology of animal influenza currently available for Central America.
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    METABOLIC CHANGES ASSOCIATED WITH ANDROGEN INDEPENDENT GROWTH IN A MOUSE MODEL OF PROSTATE CANCER
    (2014) Martin, Philip Lloyd; Samal, Siba; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    PTEN and TP53 loss are common molecular alterations in aggressive prostate cancer that progresses to castrate resistant prostate cancer (CRPC). PTEN/TP53 loss contributes to regulation of self-renewal and differentiation in prostate progenitor cells, the presumptive tumor and metastasis initiating cells for prostate cancer. TP53 plays an important role in regulating normal cellular metabolism, and loss of function is responsible for metabolic alterations in tumor cells, including increased aerobic glycolysis. We use a novel model of Pten/Tp53 deleted prostate cancer to investigate properties of tumor and metastasis initiating cells, and metabolic alterations that contribute to the evolution of CRPC. We employed a genetically engineered mouse model of Pten-/-Tp53-/- prostate cancer to develop an orthotopic model derived from a clonal cell line from the parental heterogeneous prostate carcinoma. We used histopathology and immunohistochemistry to characterize the orthotopic primary tumors and metastases. We performed metabolomic screening followed by focused analysis of HK II enzyme levels, activity, and cellular distribution in androgen replete and androgen deprived tumors. We also compared HK II levels in primary and metastatic human prostate cancer. Tumor heterogeneity was due to transformation of tumor and metastasis initiating cells with biphenotypic potential capable of basal and luminal differentiation. There was epithelial-to-mesenchymal transition (EMT) in cells of the luminal lineage. The model was capable of androgen independent growth, which influenced the differentiation of metastasis initiating cells. CRPC had increased reliance on glycolysis with increased cytoplasmic and mitochondrial-associated HK II. These metabolic adaptations afforded CRPC increased ability to withstand metabolic stress. HK II levels in human metastases were markedly increased compared to primary tumors. Pten/Tp53 loss in prostate cancer contributes to lineage plasticity in both tumor and metastasis initiating cells, contributing to heterogeneity observed in primary tumors and metastases. Increased glycolysis due to increased total and mitochondrial HK II is a metabolic adaptation that contributes to the evolution of aggressive disease, with progression to androgen independence, providing increased energy and carbon precursors for anabolic processes. Mitochondrial bound HK II blocks apoptosis and contributes to survival in the androgen deprived environment. Targeting this metabolic adaptation may provide improved treatment for this deadly disease.
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    REGULATION OF MACROAUTOPHAGY BY VITAMIN A/ RETINOIDS
    (2013) Rajawat, Yogendra Singh; Bossis, Ioannis; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Retinoic acids (RAs) have diverse biologic effects and regulate several cellular functions. Here, we investigated the role of RA on autophagy by studying its effects on autophagosome (AUT) maturation, as well as on upstream regulators of autophagosome biogenesis. Our studies, based on the use of pH-sensitive fluorescent reporter markers, suggest that RA promotes AUT acidification and maturation. By using competitive inhibitors and specific agonists, we demonstrated that this effect is not mediated by the classic Retinoic Acid Receptor (RAR) and Retinoid X Receptors (RXR). RA did not affect the protein expression levels of upstream regulators of autophagy, such as Beclin-1, phospho-mTOR, and phospho-Akt1, but induced redistribution of both endogenous cation-independent mannose-6-phosphate receptor CI-MPR and transiently transfected GFP and RFP full-length CI-MPR fusion proteins from the trans-Golgi region to acidified AUT structures. Those structures were found to be amphisomes (acidified AUTs) and not autophagolysosomes. The critical role of CI-MPR in AUT maturation was further demonstrated by siRNA-mediated silencing of endogenous CI-MPR. Transient CI-MPR knockdown resulted in remarkable accumulation of nonacidified AUTs, a process that could not be reversed with RA.These results suggest that RA induces AUT acidification and maturation by regulating CI-MPR subcellular location, a process critical in the cellular autophagic mechanism.
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    IMPROVED INFECTIOUS LARYNGOTRACHEITIS VIRUS VACCINES USING NEWCASTLE DISEASE VIRUS VECTOR
    (2013) Kanabagatte Basavarajappa, Mallikarjuna; Samal, Siba K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Infectious laryngotracheitis (ILT) is a highly contagious acute respiratory disease of chickens for which safe and efficacious vaccines are not available currently. In the present study, we have generated three recombinant Newcastle disease viruses (rNDV's) expressing three major envelope glycoproteins gB, gC and gD of ILTV individually. A single oculonasal inoculation of chickens with rNDV's elicited detectable level of systemic antibodies specific to ILTV. Following challenge with virulent strain of ILTV, chickens immunized with the rNDV's displayed partial protection with reduced clinical signs and shorter duration of disease compared to the control group. Our data suggested that NDV vectored ILTV vaccines are useful against ILTV infection, but might require augmentation by a second dose or require modification of ILTV glycoproteins which allow them to incorporate into the mature rNDV virions for better induction of humoral and cell mediated immune responses.
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    Review of the Molecular Biology and Epidemiology of Infectious Laryngotracheitis (Gallid Herpesvirus-1)
    (2012) Menendez, Kimberly Rae; Tablante, Nathaniel L; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    A review of the molecular biology and epidemiology of avian infectious laryngotracheitis (ILT) is conducted due to the outdated state of current ILT review material. The objective of this review is to include updated information on the molecular biology of Gallid herpesvirus 1 (GaHV-1), the causative agent of ILT, and to present the latest information on the molecular epidemiology of ILT. Recent developments in molecular biology specific to GaHV-1 have been made and are highlighted in this review, and the role of current and historical use of live-attenuated vaccines is associated with the global and molecular epidemiology of ILT. Also, target genes for detection and strain differentiation are compiled by region of the world, and the global distribution of ILT is illustrated. Additionally, the field of epigenetics related to virus-host interactions is reviewed, and the molecular, epidemiologic, and epigenetic factors investigated are related to prospects for future eradication of ILT.
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    DETERMINATION OF GENETIC FACTORS INVOLVED IN THE VIRULENCE OF NEWCASTLE DISEASE VIRUS
    (2012) Paldurai, Anandan; SAMAL, SIBA K; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Newcastle disease is economically the most important disease of poultry. The causative agent Newcastle disease virus (NDV) is a large, enveloped virus containing single stranded non-segmented negative-sense RNA genome. The genome of NDV contains six genes in the order of 3'Leader-N-P-M-F-HN-L-5'Trailer. NDV has at least three different genome size categories: 15,186, 15,192 and 15,198 nucleotides (nt) in length. The virulence of NDV is considered to be contributed by multiple genes. The importance of genome lengths and the roles of individual genes in virulence of NDV in its natural host, chickens, have not been determined. In this study, the effects of naturally occurring nucleotide insertions in NDV genome and roles of individual genes in the virulence of NDV in chickens were determined. To achieve this goal, reverse genetic systems for two strains of NDV were established for a highly virulent strain Texas GB (GBT) and a moderately virulent strain Beaudette C (BC). Both GBT and BC are isolated from chickens and belong to genotype II of class II NDV strains and have the genome length of 15,186 nt. The 6- and 12-nt insertions in the backbones of rBC and rGBT showed little attenuation in virus replication and in pathogenicity of the parental recombinant viruses. The reciprocal swap between NDV strains BC and GBT for the genes, nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN) and large polymerase (L) protein genes, showed that F protein gene is most important for NDV virulence, followed by the L protein gene. M, HN, N and P genes appeared not to affect the pathotypes of their parental recombinant viruses in chickens. The observations of the present study paves the way for future directions: to use the naturally occurring insertion site in the coding region of the phosphoprotein gene for insertion of potential marker sequences; to determine the amino acid residues important in fusion protein and polymerase protein for replication and pathogenesis of NDV.
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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.