M.Sc. / It is the widely accepted view that the human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS) and that South Africa harbors mainly HIV type 1 subtype C (HIV-1 C). Extensively characterized biological isolates (especially of HIV-1 subtype C) for use in HIV/AIDS vaccine and drug development are not readily available. This study evaluated three different protocols for the expansion of virus from infected PBMC's of 68 HIV/AIDS patients (designated HJ1 — 22 and INN1 — 97). Factors influencing the success of a protocol for the expansion of HIV-1 were 1) the amount and time of addition of IL-2 and PHA to the culture media; 2) the fact that freshly isolated clean PBMC's (treated with PHA prior to co-cultivation) was necessary while infected PBMC's could be used fresh or frozen; 3) whether the absence or presence of polybrene as a tissue culture additive had any effect. The I-11V-status of patients could be confirmed with rapid tests and/or NASBA assays, while successful expansion of the virus could be confirmed or refuted by determining p24 levels of sera or culture supematant (with values ranging from <7.8pg/ml to about 280pg/ml). Less sensitive assays like the reverse transcriptase (RT) and gpl 20 ELISA's give much lower absorbance values when compared to the p24 ELISA. Using expanded virus to infect PBMC's and T-cell lines (PM1, U87.CD4-CCR5, U87.CD4-CXCR4 and CEM.NKR-CCR5) and then measuring p24 levels showed that the final protocol chosen was capable of producing high titre, biologically active virus. To further test the biological activity of the isolates, the virus was used in assays evaluating the potential inhibitory ability of natural products and neutralizing antibodies. PCR using universal primers (SK22/SK38/SK39) was not consistently successful in amplifying out the correct sized region of gag (for SK22/SK39 a fragment of 600bp and a 115bp fragment for SK38/SK39 was obtained but not for all the samples). Primers (Cgag189(+/-)) were designed during this project to specifically amplify an 189bp region of gag from subtype C, which proved (in some instances) to be more successful. PCR amplification of the proviral DNA fragments was confirmed by sequencing of selected PCR products. Virus titre was determined by calculating TCID50 (login: 1.054). By modifying expansion and detection protocols it is possible to standardize the process to suit a particular isolate and/or circumstance. This production of large volumes of high titre, biologically active isolates has filled a desperate need for reagents to aid HIV researchers in the development of an effective vaccine or other drug therapy.

KwaZulu-Natal has the highest prevalence of HIV in South Africa. The prevalence of dual infection in a normal-risk population in this region is unknown. Dual HIV infection has important implications for diagnosis, treatment response and vaccine development. This cross-sectional study aimed to establish and optimize methods for subtyping and detection of dual infection in KZN. Samples were from chronically-infected patients on ARV treatment within the ARV Rollout Programme, from sites throughout KZN. Subtyping of the samples was performed using HMA. Four samples had indeterminate results by HMA and were then cloned and sequenced. Phylogenetic analysis showed that one of the four samples was a dual infection. This study showed 1/46(2%) samples to be dually infected which suggests that the prevalence of dual infection is low in the sample population. The low prevalence of dual infection reported could be due to the low-risk profile of the sample population. It was concluded that the low prevalence of dual infection is unlikely to have a considerable impact on HIV management. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2007.

Thesis (MMedSc)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Thesis (M.A.)--Ball State University, 2009. / Title from PDF t.p. (viewed on June 07, 2010). Includes bibliographical references (p. [47]-51).

This thesis provides an examination of how Papuan university students in eastern Indonesia react to Indonesian governance. Qualitative interviews investigate students' understandings of HIVIAIDS, an emerging threat in Papua around which the state makes moral claims and promotes development. Media discourse analysis reveals the way that "development" is used by the state for control, evaluation, regulation, and to make assertions about the quality and qualities of local people. Papuan students in Manado, Sulawesi are strongly influenced by development ideology. As they negotiate their way through state discourse, they show conformity and resistance to Indonesian development ideology, and by extension, governance.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Biochemistry." Includes bibliographical references (139-146).

Human immunodeficiency virus type 1 (HIV-1) infection is a life-long threat which cannot be prevented by vaccination or completely cured by antiretroviral (ARV) treatment. The establishment of genotypic resistant testing (GRT) greatly improved the infection management and provided further guidelines on ARV strategies. The current study aimed to utilize the accumulated GRT data to investigate the HIV-1 molecular epidemiology, trends of drug resistance mutations (DRMs) among local patients and intra-host viral evolution. In order to maximize the therapeutic options for HIV-1 patients, the genotypic study also extended to two relatively new ARV classes for determination of integrase polymorphisms and tropism prevalence. From 1994 through 2013, a total of 3,108 plasma samples were available from 2,475 HIV-1 positive patients for epidemiological and viral investigations. The predominant genotypes in Hong Kong were subtype B (42.1%) and CRF01_AE (39.7%). Other genotypes including subtypes A1, C, D, F1, G, CRF02_AG, CRF06_cpx, CRF07_BC, CRF08_BC and CRF12_BF were also identified. Though the phylogenetic studies of subtype B and CRF01_AE identified several sporadic distinct clusters, their transmissions had been widely established between all local risk groups. In contrast, most of the non-B and non-AE cases were infected outside Hong Kong or circulated within non-Chinese population. However, one of the clusters in CRF07_BC suggested that this genotype might have been established quite well among local Chinese men-who-have-sex-with-men community. Routine GRT also provided valuable longitudinal data to study intra-host viral evolution. The observed intra-host evolutionary rates in CRF01_AE variants (11.68 x 〖10〗^(-4), IQR: 8.87 – 20.54 x 〖10〗^(-4) substitutions per site per year) was significantly higher than subtype B variants (5.27 x 〖10〗^(-4), IQR: 3.32 – 8.01 x 〖10〗^(-4) substitutions per site per year). Of 2,157 treatment-naïve patients included in this study, 4.4% of them were found carrying surveillance protease (PR) or reverse transcriptase (RT) DRMs. More importantly, half of the GRT data from 407 treatment-failure patients were potentially resistant to PR or RT inhibitors. The introduction of integrase inhibitors and CCR5 antagonists provided new insights and era for treating HIV-1 patients who were resistant to PR or RT inhibitors. The integrase GRT results showed that there were no major DRMs observed in integrase inhibitors-naïve patients. The distribution of integrase polymorphic sites between subtype B and CRF01_AE was significantly differed. On the other hand, the prevalence of X4/Dual-Mixed tropism in CRF01_AE variants was always significantly higher than subtype B, regardless of the interpretation algorithms and treatment history. In conclusion, this study demonstrated the HIV-1 molecular epidemiology and intra-host viral evolution of Hong Kong in the last two decades by utilizing the GRT data obtained from the ARV surveillance program. The in-house integrase GRT and genotypic tropism test were as well evaluated. The high prevalence of X4/Dual-Mixed tropism in CRF01_AE isolates would definitely limit the salvage therapeutic options for CRF01_AE-infected patients. Summarizing these findings, it is possible to project that CRF01_AE virus has unique characteristics from subtype B and should be investigated further in terms of disease progression and replicative capacity. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Not available / text

When expressed in Spodoptera frugiperda cells using recombinant baculoviruses, the HIV - 1 gag gene product, p55, self assembles to form immature HIV core like particles that are secreted into the culture medium. Using this system, the gag open reading frame was progressively truncated from the carboxy terminus, and each deleted Gag protein examined for its ability to produce core like particles. Deletions that removed the distal region of the Gag nucleocapsid domain, including both Cys - His motifs thought to function as RNA capture signals, did not disrupt particle formation. Analysis of the truncation mutants by north - western blot with a radiolabelled HIV - 1 RNA encapsidation signal, confirmed that only precursors with the Cys - His motif present were able to bind the probe. It was concluded that HIV - 1 Gag particle formation per se does not require RNA encapsidation. This finding clarifies uncertainties proposed previously regarding the role of RNA encapsidation in particle assembly. Further deletion mutants led to the delineation of the carboxy terminal boundary of a Gag assembly domain. Properties of the SIV env encoded TM glycoprotein were also explored. Previous reports have found that the cytoplasmic tail of this membrane spanning protein was preferentially deleted in vivo when SIV was grown in a variety of cultured human cells. However, when propagated in cells of simian origin, the TM glycoprotein cytoplasmic tail was retained. Analysis of the function of this domain was addressed by expression of both the truncated and the full length proteins in insect cells using recombinant baculoviruses, coupled with a study of their biochemical characteristics. A role for the cytoplasmic tail was identified in the post - translational modification and localization of the glycoprotein. The use of the Gag particles in designing vaccines was investigated. Acting as non - infectious carriers of immunogenic antigens, the particles represent a safe and efficient means for presentation of epitopes for eliciting a protective immune response. Two possible approaches for loading the particles with foreign antigen were examined. In the first, the deleted portion of Gag was replaced with a sequence encoding the foreign antigen. The second method involved co - expression of Gag with another recombinant virus that expressed a foreign antigen on the surface of the cell. Both methods were found to be feasible, although limitations were identified when addition of the fusion protein promoted excess degradation of the Gag precursor.

Thesis (Ph. D.)--Universiteit van Amsterdam, 2004. / Title from ebook title screen (viewed Mar. 18, 2005.). Includes bibliographical references.