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- Title
- Identifying the underlying mechanisms of Marek's disease vaccine synergy
- Creator
- Umthong, Supawadee
- Date
- 2019
- Collection
- Electronic Theses & Dissertations
- Description
-
Marek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more...
Show moreMarek’s disease virus (MDV; Gallid herpesvirus 2, aka, serotype 1) is a ubiquitous and highly oncogenic α-herpesvirus that causes Marek’s disease (MD), a lymphoproliferative disorder affecting chickens with estimated annual costs to the poultry industry of ~$2 billion worldwide. Since 1970, MD has been largely controlled through widespread vaccination. While MD vaccines are very successful in preventing tumors, they do not prevent viral replication and spread. As a consequence, new and more virulent MDV strains have repeatedly emerged in vaccinated flocks. Thus, there is a need to understand how MD vaccines work in order to design future vaccines that are more protective, especially against more virulent MDVs. One promising insight for vaccine development is based upon protective synergism, a phenomenon where two vaccines when combined provide greater protection compared to either original vaccine when administered alone as a monovalent vaccine. The mechanism that underlines the synergistic effect between SB-1 (a Gallid herpesvirus 3, aka, serotype 2 strain) and HVT (herpesvirus of turkey, aka, Meleagrid herpesvirus 1 or serotype 3), two of the most widely used MD vaccines, has never been investigated, and thus, provides a highly relevant and useful model to explore. To investigate the mechanisms of protective synergy of SB-1 and HVT, we used three approaches. First, we investigated how monovalent SB-1 or HVT replicates when they were alone in the host or together as a bivalent vaccine. We observed that the replication patterns of SB-1 and HVT were different with respect to time after administration into the bird and the organs that they were found to replicate in regardless if the other vaccine were present. Based on the observation that HVT replicated primarily early in the bursa, we found that this organ was necessary for protection using both HVT and bivalent HVT + SB-1 vaccines. Second, we measured the effects of CD8 T cells in monovalent SB-1, HVT, and bivalent SB-1+HVT vaccine treatment. Specifically, we reduced CD8 T cells to see their effect of CD8 T cells on MD incidence and vaccinal protection by injecting the chickens with a monoclonal antibody directed against chicken CD8 T cells. In this study, we found that CD8 T cells were necessary for protection induced by vaccines. Third, we identified the cytokine profiles induced by SB-1, HVT, and the bivalent vaccine to see if cytokine synergy could be one of the mechanisms to explain protective synergy. We found that SB-1 induced an innate anti-viral response typified by IFN-α, IFN-β, IL-1β, T-cell proliferation cytokine IL-21, and Th2 cytokine IL-5, while HVT suppressed TGF-β3 and TGF-β4. The early stimulation of IL-1β and IL-21 (IFN-γ-promoting cytokines) at 4 days post vaccination (DPV) by SB-1 combined with the suppression of TGF-β (IFN-γ- suppressing cytokine) at 1 day post challenge (DPC) by HVT could result in the strong induction of IFN-γ found in the bivalent vaccine at 10 DPC. The induction of IFN-γ supports the synergistic effect of cytokines by a cooperative action mechanism where multiple cytokines work together to enhance the signal. Based on these findings, we propose a model to explain bivalent SB-1 and HVT vaccine synergy, which combines the replication of vaccines, T cell response to vaccinations, and cytokine synergy between SB-1 and HVT vaccine. Our proposed mechanism provides insights on how to generate rationally designed MD vaccines.
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- Title
- Identifying the genetic basis of attenuation in Marek's disease virus via experimental evolution
- Creator
- Hildebrandt, Evin
- Date
- 2014
- Collection
- Electronic Theses & Dissertations
- Description
-
Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, causes up to $2 billion in loses a year due to Marek's disease (MD). Therefore control of this economically important disease is critical. The primary method to control MD is vaccination. Attenuated, or weakened, strains of MDV have been generated via repeated in vitro serial passage to generate avirulent MDV strains that have been used as successful MD vaccines. Despite introduction of several vaccines since the 1970's,...
Show moreMarek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, causes up to $2 billion in loses a year due to Marek's disease (MD). Therefore control of this economically important disease is critical. The primary method to control MD is vaccination. Attenuated, or weakened, strains of MDV have been generated via repeated in vitro serial passage to generate avirulent MDV strains that have been used as successful MD vaccines. Despite introduction of several vaccines since the 1970's, more virulent strains of MDV have evolved to break vaccinal protection. Therefore, development of new MD vaccines is necessary. To address this concern, we sought to better understand the molecular basis of attenuation in MDV to provide information that may assist in the rationale design of MD vaccines. Three attenuated replicates of a virulent MDV were serially passed in vitro for over 100 passages. DNA and RNA from attenuated viruses were deep sequenced using Illumina next-generation sequencers to identify changes in DNA sequence or expression following attenuation. Top candidate mutations identified via sequencing were used to generate seven recombinant viruses using red-mediated recombineering for mutations within UL42, UL46, UL5, two involving LORF2 and two mutations within ICP4. These recombinant viruses were tested in vivo to determine the impact of these mutations on MD incidence, in vivo replication and horizontal transmission. Point mutations within UL42, UL46, LORF2-Promoter and ICP4 did not cause observable phenotypic changes compared to the parental virus. A single point mutation within LORF2-Intron and a double mutant involving ICP4 both resulting in 100% MD in challenged birds but failed to transmit horizontally to uninfected contact birds. Finally, a point mutation within UL5 reduced MD incidence by over 90%, significantly reduced in vivo replication, and eliminated horizontal transmission. Further characterization of this UL5 point mutation determined that it increased in vitro replication in growth curves, yet head-to-head competition of the Mut UL5 virus versus parental virus showed the parental virus outcompeted the mutant virus. Furthermore, serial passage of Mut UL5 in vivo did not result in increased in MD incidence, in vivo replication or result in reversion or compensatory mutations to UL5 after passage through birds. Trials testing vaccinal protection of the Mut UL5 virus showed the virus provided partial protection against challenge with virulent MDV, yet did not exceed protection achieved through use of traditional vaccines. Therefore, use of this point mutation in combination with other candidate mutations was tested. Addition of the UL5 mutation with Delta Meq, a candidate vaccine with high protection and replication but also induces bursal-thymic atrophy (BTA), resulted in a recombinant virus that replicated at low levels and did not cause BTA, yet reduced levels of vaccinal protection, indicating an intricate relationship between replication levels, BTA and vaccinal protection. This study shows that a variety of genes are mutated during attenuation, and particularly mutations within DNA replication genes, such as UL5, appear to play an important role in attenuation. We also determined that experimental evolution is a process that not only can identify mutations involved in attenuation, but also offer protection as a vaccine to provide information for further development of MD vaccines.
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- Title
- Understanding the mechanisms of oncogenicity by Marek's disease virus : role of Meq oncoprotein
- Creator
- Subramaniam, Sugalesini
- Date
- 2013
- Collection
- Electronic Theses & Dissertations
- Description
-
Marek's disease (MD) is one of the most economically significant diseases in chickens. It is caused by a highly oncogenic, alpha-herpesvirus named Marek's disease virus (MDV). Currently, the main strategy to control MD is vaccination. However, accumulating evidence points to increase in virulence among MDV field isolates over time, which implicates that new strains of the virus are evolving and could break vaccine protection. This necessitates better understanding of MDV-host interactions,...
Show moreMarek's disease (MD) is one of the most economically significant diseases in chickens. It is caused by a highly oncogenic, alpha-herpesvirus named Marek's disease virus (MDV). Currently, the main strategy to control MD is vaccination. However, accumulating evidence points to increase in virulence among MDV field isolates over time, which implicates that new strains of the virus are evolving and could break vaccine protection. This necessitates better understanding of MDV-host interactions, not only to elucidate the events in pathogenesis but also develop strategies for newer and more effective vaccines. One of the major unanswered questions in this area is the mechanism of tumor formation by MDV. The main objective of this project is to gain a comprehensive understanding of host genes that are transcriptionally regulated by Meq, the major oncoprotein of MDV and their relevance in genetic resistance to MD. MDV oncogenicity is largely attributed to the bZIP transcription factor Meq. Although it was discovered in the 1990s, only a few of host target genes have been described. This knowledge gap has impeded our understanding of Meq-induced tumorigenesis. Using a combination of state-of-the-art genomic techniques including ChIP-Seq and microarray analysis, a high confidence list of Meq binding sites and a global transcriptome of genes regulated by Meq was generated. Given the importance of Meq in MDV pathogenesis, we next explored the role of Meq in genetic resistance to MD. Two highly inbred chicken lines, varying in MD resistance, were infected with a virulent strain of MDV, Md5 or a mutant virus lacking Meq, Md-deltaMeq. Analysis of differentially expressed genes provided a list of Meq-dependent genes that are involved in MD resistance and susceptibility. Pathway analysis indicated that MD resistant lines were enriched for positive regulation of cell death whereas the susceptible cell lines were enriched for regulation of cell proliferation. In addition, some of the Meq-regulated pathways like ERK/MAPK signaling and Jak-STAT pathways were also involved in differential MD susceptibility. Taken together, our study provides a comprehensive analysis of how Meq interacts with cellular pathways involved in oncogenesis. In addition, this study forms the basis for selection of candidate genes that might be involved in genetic resistance to Marek's disease.
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- Title
- A genomic investigation of Marek's disease lymphomas
- Creator
- Steep, Alexander Cordiner
- Date
- 2019
- Collection
- Electronic Theses & Dissertations
- Description
-
"Meq, a bZIP transcription factor and the viral oncogene for pathogenic strains of Marek's disease virus (MDV), is required to induce CD4 T cell lymphomas that characterize Marek's disease (MD) in chickens. However, Meq is not sufficient for neoplastic transformation as not all birds infected with pathogenic strains of MDV developed Marek's disease. We hypothesize that additional drivers or somatic mutations in the chicken genome are required for MDV-induced transformation. Using and...
Show more"Meq, a bZIP transcription factor and the viral oncogene for pathogenic strains of Marek's disease virus (MDV), is required to induce CD4 T cell lymphomas that characterize Marek's disease (MD) in chickens. However, Meq is not sufficient for neoplastic transformation as not all birds infected with pathogenic strains of MDV developed Marek's disease. We hypothesize that additional drivers or somatic mutations in the chicken genome are required for MDV-induced transformation. Using and integrating DNA and RNA genomic screens of Marek's disease tumors from genetically-defined experimental layers, our analyses reveal 0.3 somatic mutations per megabase consisting primarily of somatic single nucleotide variants (SNVs) and small insertions and deletions (Indels). Somatic deletions, insertions, and point mutations were enriched in IKZF1 (Ikaros), the first driver gene of Marek's disease lymphomas. Ikaros, a Zn-finger transcription factor and the master regulator of lymphocyte development, is a known tumor suppressor in human and murine acute leukemias and lymphomas. In our surveyed Marek's disease tumors, 41% of the samples had somatic mutations in key N-terminal Zn-finger binding domains, strongly suggesting perturbed Ikaros function in its ability to bind DNA and regulate transcription. Somatic mutations in IKZF1 were preferentially found in tumors of gonadal tissues as well as their metastatic clones. IKZF1 mutant Marek's disease tumors revealed gene expression profiles indicative of Ikaros perturbation. In addition to IKZF1, other putative somatic mutations reside in ZNF384, EFNA5, CLDND1, FOXD1, ROBO1, and ROBO2 and warrant evaluation. Our results suggest MDV-induced tumors are driven by both Meq expression and IKZF1 somatic mutations that in combination lead to unregulated proliferation, increased cell adhesion, increased migration, and dedifferentiation."--Pages ii-iii.
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- Title
- Contribution of the T cell repertoire to resistance in Marek's disease
- Creator
- Hearn, Cari Jacqueline
- Date
- 2019
- Collection
- Electronic Theses & Dissertations
- Description
-
Marek's disease (MD) is an alpha-herpesvirus-induced lymphoproliferative disease of chickens which results in CD4+ T cell lymphomas in multiple organ systems, as well as peripheral and central nervous system disorders. Genetic studies of MD-resistant and susceptible chicken lines have identified the major histocompatibility complex (MHC) locus as the most important disease resistance locus in chickens; however, the contribution of the T cell receptor (TCR) repertoire to MD resistance mediated...
Show moreMarek's disease (MD) is an alpha-herpesvirus-induced lymphoproliferative disease of chickens which results in CD4+ T cell lymphomas in multiple organ systems, as well as peripheral and central nervous system disorders. Genetic studies of MD-resistant and susceptible chicken lines have identified the major histocompatibility complex (MHC) locus as the most important disease resistance locus in chickens; however, the contribution of the T cell receptor (TCR) repertoire to MD resistance mediated by the peptide-MHC-TCR synapse has not yet been characterized, in contrast to the extensive TCR repertoire studies that have been performed in human herpesviral infections. In this study, we identified differences in the TCR Vbeta repertoire of MD-resistant and susceptible chicken lines, and sought to determine the genetic basis of these differences. Additionally, we studied the contribution of thymic tolerance to MD neuropathogenicity in a non-oncogenic MD model, identifying a potential role of adaptive immune dysregulation in the acute disease. Model pairs of genetically MD-resistant and susceptible chickens that are either MHC-matched or MHC congenic were studied in order to characterize the T cell response, particularly the TCR Vbeta repertoire, during Marek's disease virus (MDV) infection. In contrast to previous models of T cell-mediated genetic resistance which suggested that resistant birds might have less-easily activated CD4+ T cells and thus be resistant to transformation, we were unable to find differences in in vitro mitogen response within the CD4+ T cell populations of MHC-matched MD-resistant and susceptible chickens. However, TCR Vbeta repertoire in vivo differed between MD-resistant and susceptible birds, and shifts towards higher TCR Vbeta-1 usage in response to MDV-infection could be identified within the CD8+ T cell populations, most notably within MD-resistant birds, consistent with CTL-mediated resistance. Chickens resistant to MD showed higher usage of Vbeta-1 TCRs, in both the CD8 and CD4 subsets in the MHC-matched model, and in the CD8 subset only in the MHC-congenic model. Using Illumina sequencing and PacBio long-read sequencing, we characterized the TCR beta locus of the MHC-matched lines, and found that the MD-resistant line expressed a greater number of Vbeta-1 TCRs and an increased number of Vbeta-1 CDR1 loops with a Trp-45 residue. TCR Vbeta-1 CDR1 usage in MHC-matched F1 birds was studied with Illumina RNA-seq, and usage of a susceptible line CDR1 variant was disproportionately high, suggesting that selection for resistance in the MHC-matched model has optimized the TCR repertoire away from dominant recognition of one of the MHC molecules. We also studied TCR down-regulation by MDV infection, and found that in vitro down-regulation could be mediated by viral reactivation independently of TCR activation or apoptosis, suggesting a TCR-targeting immune evasion strategy by MDV. Lastly, we studied a potentially immune-mediated phenotype associated with MD, acute transient paralysis, using an MDV virus which lacks the Meq oncogene. We describe a fatal neuropathy of chicks induced by injection of Meq-deleted MDV in ovo during the thymic tolerizing window (prior to 15 days of embryogenesis), which induces severe bursa and thymic atrophy as well as mild lymphocytic peripheral nerve lesions. This establishes that oncogenicity is not absolutely required for the acute neuropathic syndrome, and suggests that vaccine strains may be capable of inducing neuropathic disease in T-cell-immunity-disregulated birds.
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- Title
- 2,3,7,8-tetrachlorodibenzo--dioxin (TCDD)-elicited steatosis : the role of aryl hydrocarbon receptor (AhR) in lipid uptake, metabolism, and transport in Scd1+/+, Scd1-/-, and C57BL/6 mice
- Creator
- Angrish, Michelle Manente
- Date
- 2012
- Collection
- Electronic Theses & Dissertations
- Description
-
Metabolic syndrome (MetS) and its associated disorders such as obesity, type II diabetes, non-alcoholic fatty liver disease (NAFLD), and hypertension are epidemic in Western countries including the United States. Conventional thoughts hold excess energy consumption accountable for MetS phenotypes, and although Western diet and culture is characterized by too many calories consumed and too little calories burned, environmental endocrine disrupting chemicals (EDCs) have emerged into the...
Show moreMetabolic syndrome (MetS) and its associated disorders such as obesity, type II diabetes, non-alcoholic fatty liver disease (NAFLD), and hypertension are epidemic in Western countries including the United States. Conventional thoughts hold excess energy consumption accountable for MetS phenotypes, and although Western diet and culture is characterized by too many calories consumed and too little calories burned, environmental endocrine disrupting chemicals (EDCs) have emerged into the spotlight for their role in positive energy balance. Dioxin-like compounds (DLCs) including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are environmentally ubiquitous and persistent EDCs that alter energy balance and lipid metabolism in animals and humans. TCDD elicited hepatic steatosis involves aryl hydrocarbon receptor (AhR) activation and is marked by increased triglycerides, free fatty acids, inflammatory cell infiltration, and increased serum alanine aminotransferase levels. Hepatic steatosis in the absence of alcohol consumption is a cryptic, yet significant manifestation of MetS and its associated diseases and may precede cirrhosis as well as other extrahepatic effects. Stearoyl-CoA desaturase 1 (Scd1) catalyzes the rate-limiting step in monounsaturated fatty acid (MUFA) biosynthesis, its deficiency protects mice form diet-induced steatosis, and it is a target for the treatment of metabolic related disorders. In this report the role of AhR regulation of lipid uptake, metabolism, and transport in TCDD-elicited steatosis was characterized using Scd1 null mice, diet, and 14C-lipid uptake studies. Collectively, these studies showed that 1) AhR regulation of Scd1 contributed to the hepatotoxicity of TCDD, 2) dietary fat is the primary source of lipid in TCDD-elicited steatosis, 3) TCDD increases the uptake of dietary lipids, and 4) AhR mediates not only altered hepatic lipid composition, but also systemic lipid composition. This work indicates that AhR activation results in a systemic response that involves coordinated interactions between the digestive tract, circulatory system, and liver with important health implications for individuals at risk for metabolic disease.
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