Chikungunya virus (CHIKV) is classified as an alphavirus in the Togaviridae family. This virus is known to rely on Aedes arthropod vectors for its dissemination. Human infection is characterized by rash, high fever, and severe chronic polyarthritis that can last for years. Recently, efforts in developing animal models have been made in an attempt to better understand CHIKV pathogenesis. CHIKV infection starts with a 7 to 10 day long febrile acute phase, in which most of the symptoms occur (rash, fever, and incapacitating pain in joints and muscle). Once the immune system clears most of the viral infection, a chronic phase starts in as many as 70% of the infected patients. Long term virus-related polyarthralgia is the hallmark of the CHIKV chronic phase. It is believed that CHIKV-infected macrophages infiltrate the joints during the acute phase, and CHIKV infects joint tissue and persists in it. Research into the effects of CHIKV infection in human and murine macrophages revealed that CHIKV-infected human macrophages produce high amounts of virions as well as induce the production of pro-inflammatory cytokines and monocyte recruiting chemokines. This contrasts with murine macrophage infection where low quantities of the virus were detected as well as lower production of pro-inflammatory cytokines. This may contribute to the lack of polyarthritis in murine animal models. Current literature suggests that CHIKV’s viral proteins bind and interact with human host cell machinery promoting viral replication more efficiently in humans than in mice. CHIKV-related neuropathology is not the most common outcome of the disease. However, recent outbreaks suggest that this pathology is becoming more prevalent, affecting as many as 30% of confirmed patients. The role of adaptive and innate immunity in CHIKV disease amelioration has been extensively, yet separately, explored. A RAG2-/-γc-/- Balb/c mouse model was used to study the role of these immune pathways and their associated immune cells in CHIKV infection. The mice in this study developed local arthritis at the site of inoculation as well as showed signs of viral invasion in the brain. This study added to the hypothesis that both innate and adaptive immune responses are necessary to ameliorate the disease and that the lack of adequately matured lymphocytes and STAT6-activation deficient macrophages may result in more severe pathologies.

Both humans and mice display an age-related decline in immunity. Reduced generation of mature B cells may be a contributing factor due to reduced entry of mature B cells with novel B cell receptors and specificity for pathogens into the mature B cell pool. In aged mice the numbers of B cell precursors within the bone marrow are diminished; there is a severe reduction in numbers of pre-B cells and an increase in numbers of re-circulated mature B cells. Other defects in developing B cells include reduced expression of rag1 and rag2 when measured in total bone marrow precursor populations. In the pro-B cell stage of development rag expression is essential to the process of V(D)J recombination and the generation of pre-B cells. It was not known prior to this work if rag levels were lower in pro-B cells. In Chapter 2 I show that rag2 expression is reduced in pro-B cells of aged mice. The reduction in rag2 expression is correlated with a loss of V(D)J recombinase activity in pro-B cells and reduced numbers of pre-B cells. This suggests that in aged mice the reduction in rag2 expression is sufficient to result in reduced V(D)J recombinase activity and reduced generation of pre-B cells, thus contributing to fewer pre-B cells in aged mice. Furthermore, I have shown that the loss of rag2expression and recombinase activity in pro-B cells are the result of age-associated defects in the bone marrow-microenvironment as opposed to cell-intrinsic defects in developing precursors. In Chapter 3 of this thesis I examine genetic influences on age-related defects in murine B cell development and correlations between bone marrow B cell subsets and peripheral T cell subsets. It was known that longevity and age-related defects in T cell subsets are influenced by genetic differences between strains of inbred mice. The impact of genetic polymorphisms on age-related defects in B cell development had not been previously assessed. Nor was it known if these defects were correlated with age-related changes in peripheral T cell subsets. Here I present evidence that B cell subsets in the bone marrow are influenced by genetic polymorphisms between mice strains. Genetic polymorphisms on Chromosomes 15 and 19 were found to influence the frequency of re-circulated and pre-B cells in the bone marrow of aged mice. Frequencies of bone marrow B cell subsets were compared with peripheral T cell subsets. Interestingly, an association between the frequency of pre-B cells was not observed with either re-circulated B cells in the bone marrow nor peripheral T cell subsets. However the frequency of pre-B cells was inversely correlated with the frequency of B220intIgM+cells, a subset that was found to correlate with more advanced age-related T cell defects. In addition, frequencies of re-circulated B cells in the bone marrow were found to be associated with less advanced age-related defects in peripheral T cell subsets. These observations indicate that defects in B cell development, including reduced rag2 expression and V(D)J recombinase activity, are the result of changes in the aged murine bone marrow microenvironment. In addition, a genetic polymorphism located on Chromosome 19 influences the frequency of pre-B cells in aged mice. Furthermore the frequencies of B cell precursors in aged mice are not correlated with peripheral T cell subsets, but are correlated with frequencies of B220intIgM+ cells in the bone marrow. These observations advance our understanding of age-related defects in murine B cell development and may lead to identification of genes that influence B cell development in aged mice and humans as well as to help devise therapeutics aimed at restoring humoral immunity in aged individuals.

Human noroviruses (HuNoVs) are the leading cause of viral epidemic acute gastroenteritis and responsible for the deaths of over 200,000 children each year worldwide. HuNoV research has been hampered by the long absence of a readily reproducible cell culture system and a suitable small animal model, while gnotobiotic (Gn) pigs have been a unique animal model for understanding HuNoV pathogenesis and immunity, as well as evaluating vaccine and therapeutics. Recent reports of HuNoVs infection and replication in B cells supplemented with commensal bacteria Enterobacter cloacae and in Blab/c mice deficient in RAG/IL2RG have gained extensive attention, and my studies utilized the well-established Gn pig model to investigate the effects of these two interventions on HuNoV infection. Surprisingly, the colonization of E. cloacae inhibited HuNoV infectivity in Gn pigs, evidenced by the significantly reduced HuNoV shedding in feces and HuNoV titers in intestinal tissues and blood compared to control pigs. Moreover, HuNoV infection of enterocytes but not B cells was observed with or without E. cloacae colonization, indicating B cells were not a target cell type for HuNoV in Gn pigs. On the other hand, using RAG2/IL2RG deficient pigs generated by CRISPR/Cas9 system, with confirmed severe combined immunodeficiency, I evaluated the effects of host immune responses on HuNoV infection. Compared to wild-type Gn pigs, longer HuNoV shedding was observed in RAG2/IL2RG deficient pigs (16 versus 27 days), and higher HuNoV titers were detected in intestinal tissues and contents and in blood, indicating increased and prolonged HuNoV infection in RAG2/IL2RG deficient pigs. In addition, I evaluated dietary interventions including probiotics and rice bran using Gn pig model of HuNoV infection and diarrhea. While the colonization of probiotic bacteria Lactobacillus rhamnosus GG (LGG) and Escherichia coli Nissle 1917 (EcN) in Gn pigs completely inhibited HuNoV fecal shedding, the two cocktail regimens, in which rice bran feeding started either 7 days prior to or 1 day after viral inoculation in the LGG+EcN colonized Gn pigs, exhibited dramatic anti-HuNoV effects, including reduced incidence and shorter duration of diarrhea, as well as shorter duration of virus fecal shedding. The anti-HuNoV effects of the cocktail regimens were associated with the enhanced IFN-𝛾⁺ T cell responses, increased production of intestinal IgA and IgG, and longer villus length. Taken together, my dissertation work improves our understanding of HuNoV infection and immunity, and further supports for Gn pigs as a valuable model for future studies of human enteric virus infection, host immunity, and interventions. / Ph. D.

Human herpesvirus 6A (HHV-6A) has yet to be definitively linked to a specific disease. This is due in part to the ubiquitous nature of the virus. Humanized Rag2-/-γc-/- (Rag-hu) mice were tested to determine if these were a suitable animal model to study the virus. Both cell-free and cell-associated virus was used for infection and both were found to be efficient at infecting the mice. Viral DNA was found in the plasma and cellular blood fractions, bone marrow, lymph node, and thymus, indicating successful infection and propagation of the virus in vivo. The CD3+CD4- population was depleted, while the CD3-CD4+ was increased in infected animals. The CD3-CD4+CD8- and CD3+CD4+CD8- populations were depleted and the CD3+CD4+CD8+ population increased when analysis was gated upon CD4+ cells. The CD3-CD4+CD8+ population expanded and the CD3-CD4+CD8- population was reduced when analysis was gated on the CD3- population. Additional flow cytometry analysis revealed increases in CD4+CD8+ double positive cells in the peripheral blood of cell-free infected mice, which could indicate improper T cell selection and a premature departure of these cells from the thymus, possibly contributing to autoimmunity. Previous research has shown that HIV and HHV-6A may have a synergistic effect on one another and that HHV-6A may act as a cofactor in the progression to AIDS. After determining the Rag-hu mouse model was suitable for studying HHV-6A infection, a coinfection of HHV-6A and HIV-1 was performed. Coinfected mice had fewer thymocytes when compared with the HIV-1 only, mock-infected, and to a lesser extent HHV-6A only groups which could indicate increased cell death in the coinfected group as well as possible disruptions in migration of cells, either causing cells to be sequestered in the bone marrow and unable to migrate to the thymus, or causing premature egress of the cells in the thymus due in part to premature upregulation of CCR7, both of which would explain the smaller cellular populations found in the coinfected mouse thymi. Additional studies were performed to determine if a preferential targeting existed between HHV-6A and HIV-1 as these viruses are found simultaneously coinfecting the same cell. Preferential targeting was not observed by cell-associated migration assay, but increased migration of HHV-6A-infected cells was observed in a CCL21 dependent manner. These studies have provided useful information about HHV-6A and its relevance to HIV/AIDS as well as a possible mechanism of the involvement of HHV-6A in multiple sclerosis (MS) and other autoimmune diseases.

Viral protein r (Vpr) of Human immunodeficiency virus type 1 (HIV-1) plays an important role in the ability of the virus to infect cells and cause disease. Two polymorphisms to Vpr have been shown to result in differences in disease progression in infected individuals. R36W tends to result in rapid disease progression while R77Q results in long-term non-progression. In order to better understand how these polymorphisms result in these different disease phenotypes, our lab has recently shown that in cell culture, the R36W polymorphism results in increased viral replication and greater induction of cell death. On the other hand, infection with R77Q results in increased G2 cell cycle arrest and increased induction of apoptosis. In this thesis, we have attempted to study how these two polymorphisms affect the ability of HIV-1 to cause cell death in primary CD4+ cells. We show that infection by a Vpr knockout virus results in increased apoptosis while infection with R77Q and R36W result in decreased apoptosis. Additionally, R77Q infection results in increased p24 production. Further, we attempted create a Rag2-/- γc-/- humanized mouse model in order to better study roles of these polymorphisms in vivo. An additional goal of this thesis was to characterize twelve novel Bacillus anthracis bacteriophage. B. anthracis is gram positive, anaerobic, rod best known for being the causative agent of anthrax. Bacteriophage, viruses that infect bacteria, have been used to identify bacterial contamination and to treat infection. Herein, we report the isolation, sequencing, and characterization of twelve novel phages that infect B. anthracis. The genomes were annotated using DNA Master and BLASTp. Hypothetical proteins were analyzed with Phyre2 to predict possible functions based on protein structure, revealing over 100 new predicted functions. Dotplot generation showed that these phages group into four distinct clusters. By running the major portal protein of one representative of each cluster through BLASTp, we have identified the closest relatives to our novel phages and placed them into their respective genera and groups.

Animal models have greatly contributed to the understanding of different aspects of human biology, as well as a variety of human-related pathogens and diseases. In order to study them, humanized mice susceptible to pathogens that replicate in human immune cells have been developed (e.g., humanized Rag2-/-γc-/- mice). These animals are engrafted with human hematopoietic stem cells (HSCs), resulting in the de novo development and maturation of the major functional components of the human adaptive immune system and the production of a variety of human cell types. Primary and secondary lymphoid organs in the mouse are populated with human cells, and animals have long term engraftment. These features make humanized mice an excellent in vivo model to study pathogenesis of human-specific viruses in the context of a human antiviral immune response. In addition, humanized mice have been shown to be useful preclinical models for the development and validation of antiviral therapeutics. In the present study, we aimed to successfully re-establish the humanized Rag2-/-γc-/- mouse model using cord blood-derived HSCs in our laboratory. We have shown that these mice sustain long term engraftment and systemic expansion of human cells, including the major targets of Kaposi's sarcoma Herpesvirus (KSHV) and Human immunodeficiency virus type 1 (HIV-1), in peripheral blood and different lymphoid organs. Further, we have begun to evaluate the susceptibility of the humanized Rag2-/-γc-/- mouse model to infection with KSHV. We demonstrate that human lymphocytes differentiated in reconstituted Rag2-/-γc-/- mice are permissive to KSHV infection ex vivo. This finding was corroborated by detection of KSHV mRNA expression in the spleen of a humanized mouse at 6 months post infection. In a different study, we tested the in vivo antiviral efficacy of a novel HIV-1 fusion inhibitor (PIE-12-trimer) in humanized Rag2-/-γc-/- mice. We have determined the half life of PIE-12-trimer in mouse plasma. Furthermore, the administration of PIE-12-trimer to HIV-1 infected humanized Rag2-/-γc-/- mice prevents depletion of CD4+ T cells in blood, thus it may be useful to prevent AIDS in human patients.