Browsing by Author "Van Marle, Guido"

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    Activation of Toll-Like Receptor-Mediated Antiviral Response Against Infectious Laryngotracheitis Virus Infection
    (2023-09-21) Mohamed Abdul Cader, Mohamed Sarjoon; Abdul-Careem, Mohamed Faizal; van der Meer, Frank; Van Marle, Guido; Gomis, Susantha
    Infectious laryngotracheitis virus (ILTV) is a highly prevalent avian respiratory virus in Canada and globally, which can cause mild to severe respiratory illnesses. Although the live attenuated ILTV vaccines are commonly used for control, they pose challenges such as establishing lifelong latent infections, reactivating and shedding latent viruses, and regaining virulence in vaccine strains. Therefore, it is essential to develop novel control measures to address the limitations of current approaches. Inducing innate antiviral responses via the activation of toll-like receptors (TLRs) is a promising strategy for reducing ILTV replication. Endosomal TLRs in chickens, such as TLR7 and TLR21, recognize viral genetic materials, while surface TLRs (e.g., TLR4) primarily recognize bacterial molecules, but may also contribute to antiviral responses by recognizing viral proteins. Synthetic TLR ligands have been shown to induce antiviral responses against some avian viruses, such as avian influenza virus (AIV), infectious bursal disease virus (IBDV), ILTV, and infectious bronchitis virus (IBV). However, the impacts of in-ovo delivery of endosomal TLR7 and TLR21 ligands and surface TLR4 ligand (single-stranded (ss) RNA, cytosine-guanosine deoxynucleotides (CpG) DNA, and lipopolysaccharide (LPS), respectively) in reducing ILTV replication in chickens post-hatch through induction of antiviral responses is unknown. This thesis aimed to study the enhanced immune response following in-ovo treatment of these TLR ligands against ILTV infection in young chickens. Our hypothesis is that in-ovo delivery of these ligands will enhance antiviral immune responses and reduce ILTV replication in chickens post-hatch. Our results confirmed the following findings: Firstly, the in-ovo administration of synthetic TLR7 ligand, resiquimod, reduces ILTV shedding post-hatch, correlating with enhanced macrophage responses. Secondly, the in-ovo delivered CpG DNA stimulates cellular immune responses in multiple organs post-hatch, potentially reducing ILTV infection. Thirdly, the in-ovo LPS treatment stimulates protective antiviral responses against ILTV infection post-hatch, correlating with the expansion of macrophage population in the lungs. Overall, the studies provide insights into the mechanisms of host responses elicited following in-ovo delivery of these three TLR ligands against ILTV in chickens. The outcomes of the current studies can be helpful in fine-tuning the currently used vaccine strategies against ILTV in chicken to achieve maximum protection.
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    Comparative innate responses induced by Toll-like receptor (TLR)7 and 21 ligands against infectious bronchitis virus infection
    (2019-01-26) De Silva Senapathi, Yaseshwari Upasama; Abdul-Careem, Mohamed Faizal; Czub, Markus; Van Marle, Guido
    Toll-like receptor (TLR)7 and 21 ligands, resiquimod and cytosine-guanosine (CpG) oligonucleotides (ODNs) respectively have been used in ovo (pre-hatch) to enhance or prime an early immune response in chickens to provide protection against microbial pathogens. Yet, their protective efficacy against an infectious bronchitis virus (IBV) infection encountered post-hatch has not been evaluated. Thus, our objectives were to investigate the efficacy of resiquimod and CpG ODNs against a post-hatch IBV infection by delivering the ligands in ovo at embryo day (ED)18 and then, to determine possible mechanisms of protection. We found upregulation of interleukin (IL)-1β and interferon (IFN)-γ mRNA levels and considerable expansions of macrophage and cluster of differentiation (CD)8α+ T cell populations in lungs of chicken as early as day one post-hatch, following pre-hatch delivery of resiquimod. When the resiquimod pre-treated day-old chickens were infected with IBV, reduction in viral shedding via oral and fecal routes was observed at 3 days post-infection (dpi). Similarly, in CpG ODN pre-treated birds at 3 dpi, we found increased recruitment of macrophages, CD8α+ and CD4+ T lymphocytes in addition to up-regulation of IFN-γ and IL-1β mRNA concentrations in chicken lungs. However, only in ovo delivered CpG ODNs significantly reduced the morbidity and mortality associated with IBV infection. Overall, these studies bring us closer to understanding mechanisms behind CpG ODNs and resiquimod induced immune responses in chickens when used as stand-alone prophylactic agents in ovo.
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    Compartmental Hepatitis B Virus (HBV) Evolution, Replication and Infectivity in vivo and ex vivo
    (2017) Gao, Shan; Coffin, Carla S; Lee, Samuel S.; Duan, Zhongping; Van Marle, Guido; van der Meer, Frank
    Hepatitis B Virus (HBV) is classically considered a hepatotropic virus, however, the presence of different HBV genomic molecules in lymphoid cells and tissues support its lymphotropic nature. Our previous studies showed that HBV evolved in a compartment- and disease phase-specific fashion. However, the effect of nucleos/tide analogue (NA) therapy on HBV evolution and replication in different compartments (i.e., liver, plasma and peripheral blood mononuclear cell (PBMC)) is unknown, as well as the replicative competence and infectious capacity of PBMC-derived HBV. We hypothesize that HBV replicating in lymphoid cells is infectious, and the HBV evolves in PBMC and/or plasma in chronic hepatitis B (CHB) patients under the influence of NA therapy or host immune pressure (i.e., fulminant hepatitis B). The study on HBV replication and genetic evolution in PBMC, liver and plasma of CHB patients under NA therapy revealed the HBV evolution varied between three compartments both before and after treatment. NA had little effect on HBV cccDNA level and persistence of mRNA in PBMC. The study on HBV genetic features in CHB carriers with acute-on-chronic liver failure (ACLF) revealed the frequent immune escape mutations with clusters of surface gene variants possibly associated with development of fulminant hepatitis B. Mitogen stimulation in PBMC of CHB patients revealed presence of replicating HBV, which can be upregulated in PBMC and exhibited infectious capacity to HepaRG cells in vitro. In summary, our data suggests potent NAs have little effect on HBV cccDNA in liver and PBMC, which highlights the need for continued suppressive antiviral therapy. The HBV evolution varied between different compartments in CHB patients under NA therapy. Distinct variants in HBV surface gene were found to be associated with HBV fulminant hepatitis. Moreover, HBV residing in lymphoid cells is increased after mitogen stimulation of PBMC and infectious to a hepatocyte cell line. The study furthers our understanding of HBV lymphotropism, role on viral persistence and the pathogenesis of chronic hepatitis B.
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    Design of Novel Algorithms for Comparative Analysis of Complex Gene Families and their Application to Nematode Detoxification Pathways
    (2017) Curran, David Michael; Wasmuth, James; Gilleard, John; Van Marle, Guido; Chua, Gordon; Cribb, Alastair
    Parasitic nematodes present a current and devastating global problem, infecting billions of people, and causing huge production losses in both crops and livestock. There are a limited number of anthelmintic drugs available to treat these infections, and resistance has arisen quickly and spread across the globe. Xenobiotic metabolism is a well-known mechanism of drug resistance in insects, and evidence suggests it may also play a role in the development of drug resistance in parasitic nematodes. Identifying candidate enzymes in the free-living nematodes may help to understand or combat the rising resistance crisis in the parasites. However, identifying many of the protein sequences that may be involved in xenobiotic metabolism can prove challenging due to high sequence divergence and draft quality genome assemblies. This work focuses on novel software to detect hard-to-find genes, as well as methods of performing comparative phylogenetic analyses, both between species and within a population. In the absence of specific selective pressures, the phylogeny of a multi-species gene family will tend to agree with the underlying species tree. Conversely, adaptive evolution tends to manifest as incongruence in the gene tree as well as lineage-specific expansions and contractions. These properties, collectively termed phylogenetic instability, have been found to be good predictors of proteins that directly interact with the environment. I have developed an algorithm to quantify phylogenetic instability in a gene tree, and show that it correlates exceptionally well with the known substrate specificity of human cytochrome P450 enzymes. I then apply this technique to five detoxification gene families (cyp, fmo, sdr, ugt, gst) from five free-living nematode species (Caenorhabditis elegans, C. briggsae, C. brenneri, C. remanei, and Pristionchus pacificus). These gene families are known to act on both endogenous and xenobiotic molecules, and these new methods allow me to predict which are likely involved in xenobiotic metabolism. This will aid in the study of these enzymes, including their orthologs in the parasitic species.
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    Diverse Structures and Strategies of 5′ Exon Recognition in Group II Introns
    (2018-06-05) Jarding, Ashley Marie; Zimmerly, Steven; Muench, Douglas G.; Noskov, Sergei Yu; Van Marle, Guido; Cousineau, Benoit
    Group II introns are a family of mobile elements found in bacteria and bacteria-derived organelles, which are thought to have a direct evolutionary link to spliceosomal introns. These introns can self-splice in vitro thanks to their highly organized ribozyme structure. Studying the diverse structural features of group II introns illuminates the breadth of their structural diversity and resultant unique splicing and mobility capabilities. In the first of three projects presented in this dissertation, bioinformatic approaches were used to identify over 800 new introns in a large scale, systematic collection and analysis of group II introns. This allowed inference of class-specific mobility properties and the description of novel intron arrangements in genomes. Additionally, one new class and 15 unique, previously unclassified introns with novel structural features have been identified. The newly collected introns revealed a conserved structural feature for Class A introns: they appear to have two 5′ exon recognition motifs, whereas all other group II introns are reported to have one. The second project investigates the binary splicing hypothesis, which states that either of these two motifs can be used to recognize the exon. This was investigated and confirmed through both experimental and bioinformatic approaches. This strategy appears to expand the set of homing sites available to an intron by permitting splicing from one sequence and reverse-splicing into another. In the third project, the 5′ exon recognition mechanism for a IIC intron was investigated. IIC introns lack the canonical IBS2-EBS2 interactions found for other group II introns, but have a conserved transcriptional terminator in place of EBS2. In B.h.I1 a 5S rRNA upstream of this motif was shown to have an unexpected effect on the splicing reaction in vitro. Mutagenesis of 5S rRNA showed that the A-loop is critical for splicing. RNA protection experiments to characterize the interactions with the 5′ exon support the terminator stem interacting with Domain I while 5S has an auxiliary effect on exon ligation. In summary, this work has increased our understanding of the structural diversity of group II introns and corresponding specialized strategies used to recognize the 5′ exon during splicing.
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    Evaluation of the Bacterial Transferrin Receptor as a Vaccine Antigen
    (2016) Fegan, Jamie; Schryvers, Anthony; Armstrong, Glen; Van Marle, Guido
    The bacterial transferrin receptor has been considered a candidate vaccine antigen for multiple important pathogens, including Neisseria meningitidis. It was previously shown that both components, transferrin binding proteins A (TbpA) and B (TbpB) are required for bacterial survival within the host and both can elicit bactericidal antibodies after immunization. Here, cross reactivity of multiple TbpB variants has been evaluated in a high-throughput ELISA-based assay that was developed to ensure all regions of TbpB are accessible during evaluation. An engineered C-lobe scaffold of TbpB has been used to host surface-exposed regions of TbpA. Antibodies developed against the hybrid antigens were able to bind TbpA on whole bacteria and inhibit TbpA- dependent transferrin-based growth. Serum bactericidal activity was elicited by antisera to these ‘hybrid’ antigens both with and without TbpB present in the targeted strain, indicating individual TbpA loops induce complement-based killing. In N. meningitidis, asymptomatic carriage of the nasopharyngeal mucosa represents the only reservoir of disease. Conjugate capsular vaccines have been shown to reduce the burden of colonization of N. meningitidis, indicating that systemic immunization is able to effect mucosal carriage. The recent development of transgenic mice expressing human CEACAM1, a molecule necessary for bacterial adhesion, has led to the first mucosal colonization model of N. meningitidis. This model has been utilized to evaluate mucosal protection by TbpB and another prominent meningococcal vaccine antigen, factor H binding protein (FHbp). These data suggest that while TbpB is able to elicit protection from both invasive disease and mucosal colonization, FHbp is unable to prevent mucosal colonization while still eliciting systemic protection. TbpB was evaluated as a carrier antigen for the development of conjugate capsular vaccines to target a pathogen by both the polysaccharide capsule and a required protein antigen. TbpB from N. meningitidis was conjugated to the Haemophilus influenzae type B polysaccharide and resultant sera was bactericidal against both N. meningitidis and H. influenzae with no significant decrease in TbpB cross reactivity or meningococcal protection when compared to sera against native TbpB. These data provide a more comprehensive evaluation of the efficacy of transferrin receptor-based vaccines.
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    Investigating the Unique Immunological and Virological Characteristics of Hepatitis B in Special Populations
    (2024-07-04) Patel, Nishi Harishkumar; Coffin, Carla; Van Marle, Guido; Sycuro, Laura; Patel, Trushar
    The hepatitis B virus (HBV) causes acute and chronic hepatitis B (CHB) infection. Most immunocompetent adults develop self-limiting acute hepatitis B and serum HBV surface antigen (HBsAg) clearance due to robust innate and adaptive anti-viral immune responses. If acute hepatitis B does not resolve within six months, with persistence of HBsAg in serum, it is diagnosed as CHB infection. In both acute and CHB, HBV covalently closed circular (ccc) DNA and integrated viral genome in host chromosomes persist within the hepatocyte and increase the risk of occult hepatitis B infection (OBI). Rarely, an exacerbated host anti-viral immune response and a cytokine storm can lead to acute liver failure (ALF). CHB is associated with high levels of subviral particles (i.e., HBsAg), suppression of innate immunity, and peripheral tolerance marked by T cell exhaustion. A cure for CHB will reduce the liver disease burden and need for lifelong anti-viral therapy. A complete (sterilizing) cure requires eradication of the HBV cccDNA reservoir and integrated viral genomes but is difficult to achieve. Thus, a functional cure that enhances host anti-viral immune response and clearance of HBsAg is the goal of new therapeutic approaches. We prospectively recruited three unique patient populations to investigate viral and/or immune factors associated with varying immune control of HBV infection: (i.e., OBI and human immunodeficiency virus co-infection, CHB and metabolic-dysfunction associated steatotic liver disease, and HBV induced ALF).We assessed standard and novel viral biomarkers, viral variants by Sanger and deep sequencing, serum cytokines and/or peripheral HBV specific T cell response by enzyme-linked immunosorbent spot assay (ELISpot). Complementary studies were performed in a transgenic mouse model of HBV. We found unique HBV variants associated with host immune escape, risk of reactivation, liver fibrosis and hepatocellular carcinoma development, which varied between patient cohorts. Patients with greater hepatic steatosis and metabolic syndrome displayed heightened pro-inflammatory cytokines, HBV-specific T cell responses, and suppression of HBV replication. Increased serum angiogenic factors were associated with improved prognosis (i.e., reduced need for liver transplant) in individuals with ALF. In conclusion, unique HBV variants and dynamic host anti-viral immune response are associated with reduced viremia and positive clinical outcomes in individuals with hepatitis B infection.
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    Kinetics of Immune Cells and Neutralizing Antibody Titres Against Viral Pathogens of Bovine Respiratory Disease
    (2024-01-19) Choden, Tshering; van der Meer, Franciscus Johannes; Orsel, Karsina; Van Marle, Guido
    Bovine Respiratory Disease (BRD) arises from complex interactions between viral and bacterial pathogens, environmental stressors, and host factors. BRD significantly impact feedlots due to its high prevalence, economic impact, and animal welfare concerns. Therefore, implementing an effective preventive management strategy, such as preconditioning (PC) aims to reduce the incidence and health impact of BRD in feedlots compared to conventional auction-derived practice. To measure and compare the health impact and effectiveness of this strategy, we evaluated the proportion of immune cells (cell-mediated and humoral) and neutralising antibodies (NAb) using the flow cytometry technique and virus neutralisation assay, respectively. Peripheral blood mononuclear cells (n=8) and serum (n=20) samples from preconditioned (PC) and auction-derived (AD) cattle were evaluated at three different time points in the feedlot (day 0, day 20, and day 40) to determine the proportion of immune cells (CD21+ B cells, CD4+ Th cells, CD3+ CTLs, and WC1+ γδ T cells) and to determine the NAb titres (against BHV 1, BVDV, BRSV and BPIV 3). We observed a high B cell response, and high and persistent NAb titres against BRD viral pathogens in PC cattle during the first 40 days in the feedlot compared to the AD group. Furthermore, most of the lymphocytes among the CD3+ T cells were WC1+ γδ expressing cells, with a smaller proportion of CD4+ expressing T-helper and CD8+ expressing cytotoxic T-lymphocytes, however, the CD4+ Th cells showed major fluctuations in their proportions within the PBMCs. In conclusion, the result provided us with a strong understanding of the immune response in PC calves, particularly the humoral immunity, and specifically the number of NAbs induced following preconditioning. This will help with the prediction of BRD in young calves during their first 40 days in a feedlot, and to evaluate the efficacy of vaccination strategies in preconditioning programs.
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    Molecular Diagnostics and Epidemiology of Anthelmintic Efficacy in Hookworms and Whipworms
    (2023-05-10) Venkatesan, Abhinaya; Gilleard, John; Wasmuth, James; Mains, Paul; Van Marle, Guido
    Broad-spectrum anthelmintics are widely used for treating parasitic nematode infections in humans and animals. The frequent and extensive use of these drugs has resulted in widespread anthelmintic resistance in nematodes of livestock, which is increasingly well documented. In contrast, while anecdotal and clinical data often suggest poor efficacy, the extent and molecular basis of anthelmintic resistance in parasitic nematodes of humans and companion animals is still poorly understood. This thesis uses molecular genetic approaches to investigate two significant and contemporary examples of poor anthelmintic efficacy in parasitic nematodes. Firstly, a parasite of dogs, Ancylostoma caninum, for which there have been many clinical and anecdotal reports of poor efficacy for multiple drug classes for some time. Secondly, an important parasite of humans, Trichuris trichiura, where a population which is refractory to albendazole-ivermectin combination therapy has recently been identified. Ancylostoma caninum (canine hookworm) is an important gastrointestinal parasitic nematode of dogs. The parasite prevalence in the USA has seen a dramatic increase in the past decade with anecdotal reports of reduced drug efficacy and suspected cases of anthelmintic resistance. Chapter 2 of the thesis identifies and functionally characterizes a novel benzimidazole resistance mutation at codon 134 of the A. caninum isotype-1 β-tubulin gene. A deep amplicon sequencing approach was utilized for the large-scale screening of this novel and other previously described benzimidazole resistance mutations, revealing that benzimidazole resistance is now widespread in A. caninum from pet dogs across the USA. Chapter 3 examines the molecular epidemiology of the benzimidazole resistance alleles and the genetic diversity of A. caninum in pet dogs to investigate the emergence and spread of anthelmintic resistance in the parasite populations. The results support the hypothesis that at least some benzimidazole resistance alleles originated in racing greyhound kennels due to intense drug selection pressure and subsequently spread into pet dog populations via the rehoming of retired racing greyhounds. Chapter 4 focuses on the second parasitic nematode, Trichuris trichiura (human whipworm). This soil transmitted helminth (STH) species of global health importance has been particularly problematic due to its poor sensitivity to benzimidazoles, which are widely administered anthelmintics in Mass Drug Administration (MDA) programs. This thesis chapter investigates genetic differences in Trichuris populations from clinical efficacy trials in three different countries: Côte d’Ivoire, Lao PDR, and Tanzania. Genetic analysis suggests that the parasite populations in Côte d’Ivoire, which is non-responsive to albendazole-ivermectin treatment, is a separate Trichuris species, distinct from T. trichiura. The results from this thesis illustrate the power of molecular genetic approaches to elucidate the basis of poor anthelmintic efficacy and provide a framework for molecular epidemiology and surveillance studies.
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    Structural Variation in the Caenorhabditis elegans Genome: Challenges and Quality Assurance Strategies for Reliable Variant Calling
    (2023-08) Lesack, Kyle James; Wasmuth, James; Van Marle, Guido; MacCallum, Justin; Gilleard, John
    Obtaining an accurate and comprehensive representation of structural variation is crucial for understanding how large alterations in chromosome structure contribute to phenotype diversity and drive genome evolution. Despite continuous efforts into improving methods for identifying structural variation from whole genome sequencing data, accurate variant calling remains challenging. The barriers to progress in this area are complex and multifactorial but the technical limitations of short-read sequencing technologies and limited availability of suitable benchmarking resources for non-human species feature prominently. This thesis includes an in-depth evaluation of several commonly used tools for identifying structural variants from short- and long-read DNA sequencing data from natural Caenorhabditis elegans strains. The results of these comparisons revealed that popular tools yield considerably different results, which are described in detail in Chapter 2. A major aim of this project was to identify sources of error and variability that tool developers could address in the future. Surprisingly, the order of reads in PacBio FASTQ files were revealed to affect the predicted structural variants. Chapter 3 describes these results and demonstrates how alignment sorting algorithms contribute to the problem. In Chapter 4, an analysis of structural variation in 14 natural C. elegans strains is described. Importantly, this work demonstrates how long-read DNA sequencing data can be successfully used to identify structural variants at the population level.
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    The Effect of Leishmania Donovani Cathepsin B on Transforming Growth Factor Beta-1 Activation and Regulation of Inducible Nitric Oxide Synthase and Arginase Activity Inside the Macrophages
    (2016) Murtatha, Asel; Gedamu, Lashitew; Storey, Douglas; Wong, Sui-Lam; Van Marle, Guido
    Leishmania parasites contain high level of cathepsin B and cathepsin L like cysteine proteases. The anti-inflammatory immune response depends mainly on the interleukin-10 (IL-10) and transforming growth factor beta-1 (TGF-β1) production. This study elucidated the role of L.donovani cathepsin B on L donovani parasites survival and multiplication. Our data suggest that cathepsin B could play a role in activating latent TGF-β1 in vitro and inside the macrophages. Our data also suggest that L.donovani cathepsin B could facilitate the parasite survival inside the macrophages via inhibiting iNOS and enhancing arginase expression. Exosomes, which are secreted vesicles, are important in cell to cell communication. We found that cathepsin B could be exported through L.donovani exosomes and could also activate TGF-β1 in vitro and inside the macrophages. Moreover, we found that L.donovani exosomes could deactivate macrophages and enhance the parasites survival inside macrophages.
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    The Evolutionary Potential of Bovine Viral Diarrhea Virus: A Model ssRNA Virus
    (2016) Chernick, Adam Alexander; van der Meer, Franciscus Johannes; Orsel, Karsina; Ambagala, Aruna P. N.; Van Marle, Guido; Wasmuth, James
    Single stranded RNA viruses are a group of pathogens infecting all animals. They include human pathogens such as human immunodeficiency virus, hepatitis C virus and influenza. Their error-prone replication generates mutations in their genomes, producing a reservoir of viral genomes capable of surviving variable selective pressures. Under certain conditions this is called a quasispecies. Studying these viral populations in vivo is challenging and model systems help to better understand their behavior. Bovine viral diarrhea virus (BVDV) is a ssRNA virus infecting cattle worldwide. Persistent BVDV infections, where animals are born immunotolerant to the virus due to an in utero infection, present a unique opportunity to study a quasispecies in the relative absence of immune selection. Methods and results useful for understanding both BVDV and ssRNA viruses in general are presented here. Canadian BVDV isolates were used to infer the evolutionary history of BVDV. Significant variability in circulating isolates was observed and clustering below the sub-genotype level may be of antigenic significance. Within a single, persistently infected host, substantial clustering of viral genomes by tissue compartment was described. The variable regions of the viral genome were also conserved from animal to animal; whether a region is variable or not appears to primarily be a feature of the virus, not the host. Preliminary data using a high-throughput viral neutralization assay showed potential to rapidly phenotype viral isolates and directly compare their antigenic properties. Although additional work is needed to ensure the assay is reproducible and reliable, statistical analysis of the data in conjunction with viral genome sequences offers a method of correlating genotype with antigenic phenotype. These data can improve BVDV control programs, especially the design of new vaccines. More broadly, these methods and results are also applicable to other ssRNA viruses. Data describing the intrahost variation of BVDV can be applied to viruses that have persistent phases or depend on the variation generated in infected hosts to escape acquired immune responses in a host population. This thesis presents findings that both further our understanding of an important pathogen of cattle and an important group of viruses in general.