A significant barrier to curing Acquired Immunodeficiency Syndrome (AIDS) is the presence of latent reservoirs of cells infected with an integrated but transcriptionally silent provirus that is unaffected by the immune response or by antiretroviral drugs. Therefore, while antiretroviral therapy is able to suppress patient viral loads to clinically undetectable levels, upon cessation of treatment, viral loads rebound rapidly as virus is released from the latent reservoir. An understanding of the Human Immunodeficiency Virus (HIV) transcriptional regulatory events that contribute to viral latency is an important step towards eradicating the latent reservoir. Of particular interest are the mechanisms early in HIV infection that direct the cell towards productive or latent infection. The current study aims to determine whether the strength of T cell receptor (TCR) signaling at the time of HIV infection is correlated with the level of HIV transcription and to characterize the T cell receptor signaling pathways that regulate HIV transcription. We hypothesize that the strength of signaling at the time of infection determines the magnitude of early HIV transcription. Simultaneous infection and stimulation of Jurkat E6.1 T cells expressing the C6.5 chimeric antigen receptor shows a 1.5-fold increase in transcription compared to unstimulated controls while Jurkat E6.1 T cells expressing the B1D2 chimeric antigen receptor shows a nearly 3-fold increase in transcription compared to unstimulated controls. These results suggest that the strength of signaling through the TCR at the time of infection determines the magnitude of HIV transcription early in infection and may contribute to the establishment of productive or latent infection within the cell. Simultaneous infection and stimulation of Jurkat E6.1 T cells expressing the C6.5 or B1D2 chimeric antigen receptors in the presence of select inhibitors of T cell receptor signaling molecules indicate that TCR signaling through PI3 kinase and protein kinase C pathways may negatively and positively regulate HIV transcription, respectively. Understanding the specific TCR signaling pathways that lead to the initial establishment of latency within newly infected cells may lead to the discovery of novel therapeutic targets aimed at eliminating the latent reservoir.

Over the past few decades, the human immunodeficiency virus and its progression to acquired immunodeficiency syndrome has become one of the most prominent global health issues. As the number of infected persons continues to grow, it is increasingly important to develop a protective vaccine to stop HIV transmission, and a cure for those already infected. Although current combination antiretroviral therapy can help patients maintain undetectable levels of the virus throughout their bodies, once the treatment is stopped, the virus will rebound. In this project, the effects of a vaccine therapy that targets the protease cleavage sites (PCS) of the HIV protease were evaluated in 16 Cynomolgus macaques. Preliminary results of the study show that in the vaccine group (n=11), a disruption to one or more of the HIV protease cleavage sites leads to a better maintenance of CD4+ T cells versus that in the control group (n=5). Furthermore, a correlation between the percentage of PCS mutations and viral load was also observed. Upon closer analysis, it was determined that the most common sites of mutation occur at PCS2 and PCS12. To assess the impact of these PCS mutations on viral fitness, we used site directed mutagenesis to introduce single amino acid mutations into a fully infectious SIV clone (SIVmac239). Ongoing studies include producing virus stocks of the SIVmac239 mutants (with multiple PCS mutations) and evaluating the viral fitness of the SIVmac239 clones in cell lines using growth competition assays. The data from this study and future studies will help provide information in the areas of vaccine and therapy development for HIV.

Thesis (M.S. - Microbiology and Medical Technology)--University of Arizona. / Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1963. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 57-64).

Zika virus (ZIKV) had remained a relatively obscure flavivirus until an unexpected series of epidemics that began in Micronesia brought the virus into the forefront of global public health consciousness. Unlike its closely related flaviviruses, such as dengue, West Nile, and Japanese encephalitis viruses, that cause severe diseases, ZIKV causes asymptomatic or mild febrile infections that are dengue-like in infected individuals. However, ZIKV has exhibited teratogenic effects and infection of pregnant women can lead to microcephaly, a neurodevelopmental disorder in newborns. The teratogenic effects of ZIKV was most clearly highlighted during the epidemic in Brazil, due to the alarming 20-fold increase in microcephaly incidence experienced by the northeastern states where the first outbreak of the Western Hemisphere began, and quickly brought this virus into the spotlight. Although public health officials predicted that other Latin American countries would also experience an increase in microcephaly numbers of comparable scale, such an increase was not observed. This thesis seeks to understand the mechanisms underlying what appears to be a relatively out-of-proportion increase in microcephaly numbers in Brazil compared to other ZIKV-affected countries. The role that differences in vector survival and control, women’s behavior, interactions between ZIKV and dengue virus, and timing of the outbreak may have played in causing the different magnitudes of microcephaly in Brazil, Colombia, and the U.S., in particular, is discussed. Understanding the factors that may have caused the abnormally large outbreak of ZIKV and microcephaly in Brazil versus other regions may assist countries that have not yet been affected by ZIKV develop effective programs to prevent future outbreaks and lessen the impact of a potential outbreak on microcephaly numbers. Experiments to elucidate the mechanism by which ZIKV infects the fetus to cause central nervous system damage will provide an understanding to develop safe and effective vaccines that may assist in efforts to prevent future rise in ZIKV-associated microcephaly cases.

abstract: Vaccinia virus (VACV) is the current vaccine for the highly infectious smallpox disease. Since the eradication of smallpox, VACV has been developed extensively as a heterologous vaccine vector for several pathogens. However, due to the complications associated with this replication competent virus, the safety and efficacy of VACV vaccine vector has been reevaluated. To evaluate the safety and efficacy of VACV, we study the interactions between VACV and the host innate immune system, especially the type I interferon (IFN) signaling pathways. In this work, we evaluated the role of protein kinase R (PKR) and Adenosine Deaminase Acting on RNA 1(ADAR1), which are induced by IFN, in VACV infection. We found that PKR is necessary but is not sufficient to activate interferon regulatory factor 3 (IRF3) in the induction of type I IFN; and the activation of the stress-activated protein kinase/ c-Jun NH2-terminal kinase is required for the PKR-dependent activation of IRF3 during VACV infection. Even though PKR was found to have an antiviral effect in VACV, ADAR1 was found to have a pro-viral effect by destabilizing double stranded RNA (dsRNA), rescuing VACVΔE3L, VACV deleted of the virulence factor E3L, when provided in trans. With the lessons we learned from VACV and host cells interaction, we have developed and evaluated a safe replication-competent VACV vaccine vector for HIV. Our preliminary results indicate that our VACV vaccine vector can still induce the IFN pathway while maintaining the ability to replicate and to express the HIV antigen efficiently. This suggests that this VACV vector can be used as a safe and efficient vaccine vector for HIV. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2013

Introduction: HIV viral load testing is the preferred monitoring approach for HIV infected patients on combination antiretroviral therapy (cART) as it is more sensitive than CD4 count and clinical monitoring. In resource limited settings, timely plasma separation and transportation to testing laboratories is a major barrier to the access of HIV viral load testing. The 2015 World Health Organisation guidelines recommend that cART should be initiated in all adults and children living with HIV regardless of disease stage or CD4 count, thereby escalating the demand for HIV viral load testing. Potential solutions to expand implementation and scale up of viral load testing in low and middle income countries are whole blood testing through point of care (POC) viral load assays or dried blood spots (DBS) collected at the health facility. Utilization of whole blood instead of plasma would simplify sample collection, storage and transportation requirements and be cost effective. However, the paucity of studies comparing whole blood HIV viral load across different test platforms, especially in the correct classification of virological failure, has resulted in the lack of a standardised programmatic approach to whole blood viral load testing. Methods: We evaluated four HIV whole blood viral load test methods namely Alere q HIV-1/2 POC, Abbott RealTime HIV-1 DBS original and updated protocols, and Roche CAP/CTM DBS free virus elution (FVE) protocol, against the standard of care, plasma viral load, on 299 samples across the viral load spectrum from South African patients on cART. Virological failure was defined at >1000 copies/ml. Proportions of correct classification of virological failure and overall correlation with plasma were used for evaluating each method's performance. Results: Alere q, Abbott original and updated, and Roche FVE correctly classified virological failure in 61%, 89%, 87% and 76% of all samples tested respectively. The performance varied across plasma viral load categories. Alere q showed good correlation above plasma viral load of 1000 copies/ml, with correct classification of virological failure in 100% of samples. However, below the plasma threshold of 1000 copies/ml, Alere q demonstrated significant over-quantification, resulting in reduced specificity and upward misclassification of virological failure in 39% of all samples tested. Abbott original and updated also had good sensitivity of 98% and 91% respectively and the best overall correlation with plasma (r² = 0.76 and 0.72 respectively), but there was upward misclassification in 10% and 8% of samples tested respectively. Roche FVE had the best specificity of 99% but with significantly reduced sensitivity of 53%, especially between 1000–10,000 copies/ml of plasma, resulting in downward misclassification in 24% of all samples tested. Greatest variability between the different testing methods was seen when plasma viral load was 40-1000 copies/ml. Correlation was best for all whole blood viral load assays at >10,000 copies/ml. Conclusion: The key finding highlighted by this study is the great variability between the different whole blood test methods. Various factors influence the ability to quantify whole blood HIV viral load such as input volume used in each assay vary, sample treatment/processing (DBS versus fresh blood samples versus FVE), extraction (RNA selective, total nucleic acid extraction), amplification target and detection methods are different for each of the platforms tested. Based on our study, Alere q and Abbott DBS need to raise their whole blood threshold for virological failure in order to reduce upward misclassification and Roche FVE needs to achieve better sensitivity around its limit of detection. Receiver operating characteristic curve analysis can be used to determine the optimum threshold of virological failure for each assay.

HIV represents a global health problem. The phenomenon of HIV latency presents challenges in treating and curing HIV infection. Understanding the mechanisms behind HIV latency may provide the route to cure HIV. In a subset of elite controllers, elevated levels of p21 provide resistance to HIV replication by inhibiting transcription. I attempted to understand the mechanisms by overexpressing p21 in HEK 293T cells.

Bibliography: pages 139-150. / The diversity of Subgroup I geminiviruses causing streak disease in maize, sugarcane, and indigenous wild grasses was investigated. The virus. isolates studied originated from maize (several southern African isolates), two sugarcane cultivars (from Natal province, South Africa, and from Mauritius), wheat, and three grasses (Panicum, Setaria, and Eleusine spp. from South Africa). The following methods were used: analysis of restriction fragment length polymorphisms (RFLPs) between viral genomes in individual infected plants; DNA cross-hybridization between virus isolates; restriction endonuclease mapping of whole virus genomes; and nucleic acid sequencing. The complete genome of the Natal sugarcane streak virus isolate was sequenced. Partial sequences were obtained for other isolates, either by sequencing the ends of cloned viral genomes, or by sequencing a 250 base pair fragment of a highly conserved open reading frame that had been amplified using the polymerase chain reaction technique. The viruses being studied were compared both among themselves and with other Subgroup I geminiviruses of known DNA sequence, on the basis of sequence (nucleotide and amino acid) and restriction map data. Distance matrix methods were used to infer phylogenetic relationships between Subgroup I geminiviruses from restriction map and sequence data. Phylogenies deduced from sequence data were considered to be more accurate than those deduced from map data. Regardless of the method of analysis used, however, the relationships between the Subgroup I geminiviruses studied here remained constant. Thus, three strains of MSV (maize, Setaria, and Eleusine strains) were distinguished. Streak viruses distinct from MSV were also identified: panicum streak virus (PanSV), and two distantly related strains (Natal and Mauritius) of sugarcane streak virus (SSV). Restriction mapping of different geographical isolates of the maize strain of MSV demonstrated that variation existed within a single strain of virus. RFLP analysis indicated that minor variation existed between virus genomes within single diseased plants. Methods used to. type Subgroup I geminiviruses were evaluated, and discrepancies in the serological typing of geminiviruses from Subgroups I and III were pointed out. A unified scheme was proposed for distinguishing between distinct Subgroup I geminiviruses and strains of geminiviruses. The origins of maize and sugarcane streak viruses were speculated upon.

Human Immunodeficiency Virus (HIV) is a global disease that has yet to be cured. Its most popular outbreak was in the 1980s. Current treatment involves holding the virus in its dormant phase using Antiretroviral Therapy (ART). By keeping the virus dormant, patients are able to live their lives normally without worry of the disease progressing. Although their quality of life and length improve, they are still limited by the daily medications they are required to take. Mishaps in following the medical plan can result in the virus spreading and gaining tolerance to the medication, deeming it ineffective. Genuine elimination of the virus will remedy this issue and truly give patients a better quality and length of life. In order to accomplish this goal, current research has been investigating ways to target the latent population of HIV. This study was aimed at understanding the role of BCL-2 in HIV latent immune cells, specifically CD4+ T cells. This was mainly accomplished by isolating Rhesus Macaque monkey peripheral blood mononuclear cells (PBMCs). Once the cells were isolated, they were stained with the appropriate antibodies to help in identifying specific subpopulations. These subpopulations were Monocytes, B cells, and T cells. BCL-2. OX40 and p27 levels of expression were analyzed and measured on these subsets. OX40 and p27 were also of interest because of the similar roles they play when viruses infect healthy cells. The results of this study showed high correlation between all three proteins (BCL-2, OX40, and p27) on latent infected CD4+ T cells. These correlations were transient through all subpopulations of PBMCs, as well as, Simian Immunodeficiency Virus (SIV) infected and non-infected specimens. Although no statistical test to prove significance was completed, these preliminary findings were promising for further studies.