Retroviral vectors are used as mighty tools for an introduction of recombinant genes into the recipient genome in gene therapy trials. In the vector design, great emphasis is put on safety and efficiency. In spite of a great progress in retroviral vector design with the purpose to stabilize its expression, e.g. introduction of protective elements into the viral regulatory sequences, the current approaches are still not sufficiently effective and the majority of proviruses is transcriptionally silenced. The understanding of the silencing mechanism is of special importance to the optimization of the vector design and handling. In this master thesis, I have designed and constructed an expression system for study of the mechanism involved in the silencing of retroviruses integrated inside gene bodies. This artificial system will be utilized for testing of hypothesis that retroviruses integrated into gene bodies are silenced by DNMT-dependent mechanism and this process is triggered by transcriptional read-through of the provirus from nearby host promoter. I have obtained preliminary results suggesting the validity of the suggested hypothesis; however the verification of general validity of this hypothesis for various retroviruses and elements will be a matter of further studies in our laboratory. Powered by...

Production of infectious retrovirus particles is a complex and poorly-understood process with multiple steps that are often linked to one another. Our aim in this study was to gain better understanding of the path the murine leukemia virus (MLV) structural protein Gag follows to assemble into immature capsid structures, the process of which is central to retroviral assembly and release. Extensive studies of human immunodeficiency virus type 1 (HIV-1) assembly have led to the development of a model proposing that the assembly of immature HIV-1 capsids proceeds sequentially through multiple intermediates, in association with an RNA granule containing some well-conserved cellular factors, such as ATP-binding cassette subfamily E member 1 (ABCE1) and DEAD-box helicase 6 (DDX6). In this work, we provided evidence suggesting that MLV Gag associates with endogenous ABCE1 in human cells expressing assembly-competent MLV, and can be found in at least three high-molecular weight complexes with sedimentation properties highly resembling the HIV-1 assembly intermediates. Furthermore, we assessed the Gag proteins of select assembly-defective MLV mutants in terms of their expression levels, ability to form viral particles, involvement in intracellular complexes, membrane association, and ABCE1 interaction. Our findings were not only consistent with a model of MLV assembly through host-mediated intermediates, but also provided novel information about the effects of various MLV Gag mutations that are associated with defects in particle production.

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Jaagsiekte is a contagious cancer affecting the lungs of sheep. Although the etiological agent is Jaagsiekte retrovirus (JSRV), two other retroviruses viz South African maedi visna virus ( SA - OMVV) and a novel Bovine retrovirus (BRV) have been associated with or implicated in the jaagsiekte disease complex. JSRV was sufficiently purified from lung rinse material using a Freon extraction, Percoll density gradient centrifugation and chranatography on a Sephacryl column, its polypeptide composition was studied by gel electrophoresis and its morphology observed electron microscopically. Monoclonal antibodies were made against purified preparations of the virus. Two hybridomas were isolated that produced MAbs which appear to be tumour cell specific. A third hybridoma, called 4A1O, produces antibodies considered to be viral specific. These MAbs have been used in the development of JS specific immunoassays. A cross reaction between JSRV and a polyclonal serum against Mason Pfizer monkey virus (MPMV) was confirmed and used in a Western blot technique to identify, monitor and differentiate JSRV from other viruses. During the study of JSRV it became apparent that another retrovirus was often present in JS infected lungs. This virus, referred to as SA - OM1V I, is a novel South African isolate of maedi visna virus (MVV). As SA - OM1V I has physicochemical characteristics similar to JSRV, it was often found in purified JSRV preparations. Being a retrovirus it is also detected by the reverse transcriptase assay which was the only method used to assay and monitor for JSRV during the early stages of our work. Using a Westen blot technique and sera against MVV and MPMV it was possible to simultaneously detect and differentiate JSRV from SA - OMVV I. A method was also developed whereby the two viruses could be separated from each other during purification. The information gained and techniques developed whilst studyiing JSRV were also used to isolate and characterize BRV. This novel virus originated from bovine cells that had been co-cultivated with white blood cells from an ox suffering from malignant catarrhal fever. Three out of four sheep inoculated with BRV developed JS. It therefore had to be· ascertained whether this virus was related to JSRV or not. The comparative study revealed that BRV was biochemically and morphologically quite different fran JSRV. Interestingly, it was shown that serum against MPMV cross reacted with a 32 kd protein of BRV indicating a serological relationship between JSRV, MPMV and BRV. The possible role of BRV in the etiology of jaagsiekte remains to be elucidated. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1987.

Human immunodeficiency virus type 1 (HIV-1) isolates can be differentiated based on their ability to use particular coreceptors – R5 viruses use CCR5, X4 viruses use CXCR4 and R5X4 (dual tropic) viruses use both CCR5 and CXCR4. It is widely reported that HIV-1 subtype C (HIV-1C) has a unique viral coreceptor evolution pattern in that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for this subtype compared to other subtypes. However, dual tropic HIV-1C isolates have occasionally been described. Furthermore, it has been reported that certain highly active antiretroviral drugs (HAART) may select for X4 viral variants. Therefore, this thesis study was undertaken to better understand the functional and genotypic characteristics of dual tropic HIV-1C isolates, and to characterize drug resistance and coreceptor usage patterns in HAART-naïve versus HAART-failing HIV-1C infected patients. Thirty-five functional HIV-1 env clones derived from seven dual tropic HIV-1C strains were generated and their coreceptor usage characterized in transformed cell lines. All 35 env clones efficiently infected transformed cells expressing CXCR4. Twenty of 35 clones (57%) also utilized the CCR5 receptor. No R5-only clones were detected. Functional coreceptor usage data was correlated to env gene sequence data. The ability of the HIV-1C env clones to facilitate infection of primary lymphocytes and monocyte-derived macrophages was next investigated. The majority of clones characterized as X4 or R5X4 on cell lines used either CXCR4 alone or CXCR4 and CCR5, respectively, in primary cells. A few viruses displayed comparable CCR5 and CXCR4 usage and clones from one virus preferred CCR5 usage in macrophages. Thus in a few cases coreceptor phenotyping in transformed cell lines does not predict usage in primary cells. Genetic determinants for coreceptor usage in primary cells require further investigation. Finally the patterns of drug resistance mutations were studied and coreceptor usage among 45 HAART-naïve and 45 HAART-failing HIV-1C infected patients analyzed. Ninety-five percent of HAART-failing patients had viruses with at least one drug resistance mutation. Thymidine analog resistance mutations (TAMs) were present in 55% of patients. HAART-failing patients had significantly higher prevalence (59%) of X4/R5X4-utilizing viruses compared to HAART-naïve patients (30%) (p<0.02) using the Trofile Co-receptor Tropism Assay while 41% of HAART-failing patients used CCR5 and 70% of HAART-naïve patients used CCR5. Functional results correlated with predictive algorithm methods. This study enhances our understanding of HIV-1 pathogenesis and the results have important implications for the use of coreceptor antagonists for the clinical management of HIV-1C infection. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2011.

The study of retroviruses has been of pivotal significance to the field of biomedical science, where it has provided fundamental insights into the processes underlying both viral and non-viral carcinogenesis. Ovine pulmonary adenocarcinoma (OP A), a contagious lung cancer of sheep and goats, has emerged over the past three decades as an invaluable model of human epithelial cancers. It is one of the very few animal models of retrovirus induced neoplasia of epithelial tissues, whereas most other such animal models of human cancers pertain to the haematopoietic system. OP A represents a unique, naturally occurring, inducible, outbred animal model of peripheral lung carcinomas, and is caused by a betaretrovirus - jaagsiekte sheep retrovirus (JSRV) - that is receiving increasing attention in the fields of retrovirology and lung cancer research. JSRV exists in two highly homologous, yet molecularly distinct forms. The first is an exogenous form of the virus that is transmitted horizontally from one animal to another. This form is infectious and the direct cause of OP A. The other is an endogenous form, 15 to 20 proviral copies of which reside benignly in the genome of sheep and are transmitted vertically from one generation to the next. At the time this study commenced, no knowledge existed regarding the underlying pathogenic mechanism by which JSRV causes OPA. Even though the nucleotide sequence of exogenous JSRV had been elucidated seven years earlier, only limited sequence information was available on endogenous JSRVs. With a view towards identifying genetic regions or elements within exogenous JSRV that could potentially be implicated in its pathogenic function, this study began with the cloning of the first three full-length endogenous JSRV loci ever isolated from sheep. The DNA sequences of these full-length endogenous JSRV loci were determined and comprehensively analysed. Comparison with exogenous JSRV isolates revealed that the two forms of the virus are highly homologous, yet can be consistently distinguished in three short regions within the coding genes. Two of these reside in the gag gene, and one at the end of the env gene. These regions were named the variable regions (VRs) of sheep betaretroviruses. The JSRV VR3 in env was linked by our collaborators to the virus's ability to transform cells in tissue culture. The effects and biological significance of VRI and VR2 in gag are subtler and more difficult to determine. After identifying these regions, it became the objective of this study to develop relevant molecular tools that could be used to discern the significance of these variable regions in vivo, and to characterise these tools in vitro to assess their suitability for in vivo studies. The development of these tools entailed the design of a novel strategy that was implemented to precisely substitute the endogenous VRI and VR2 (individually and in combination) into an infectious molecular clone of exogenous JSRV. These chimeric constructs were shown to support retroviral particle release into the supernatant of transiently transfected cells in tissue culture. These particles were confirmed by independent experiments to have arisen specifically from transfection with the chimeric clones. Finally, the particles were shown to be capable of infecting cultured cells and of productively integrating their genomes into those of their host cells, rendering these particles fully competent retroviruses that can be used in the context of in vivo studies to determine the biological significance of VRI and VR2. This study has made a significant contribution to the further development of the OP A / JSRV model system of human epithelial lung cancers. It has also led to the design of a molecular substitution strategy that can be adapted to introduce any genetic region into a cloned DNA construct, regardless of the degree - or lack of interrelation - of the two DNA sequences, thereby creating a highly versatile molecular biological tool. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.

Background Human T cell Lymphotropic Virus Type I (HTLV-I) associated infective dermatitis, first described by Sweet in Jamaican children, is a pattern of eczema characterized by exudation, crusting around the nostrils, ears and scalp with eventual appearance of a generalized fine papular rash. More recently LeGranade and co-workers have proposed major and minor criteria in establishing the diagnosis of HTLV-I associated infective dermatitis (HAID). HTLV-I has been aetiologically linked to Adult T cell leukaemia/lymphoma (ATLL) and tropical spastic paraparesis (TSP). HAID is not only a marker of childhood infection with HTLV-I but may be a harbinger of more serious HTLV-I associated diseases later on in life such as ATLL or TSP. The pathogenesis of HAID is poorly understood so are the histopathological features of this entity. The effects of co-infection with human immunodeficiency virus- 1 (HIV-1) are inconclusive. HAID is described in Sub Saharan Africa, Senegal but no data is published on this entity in Southern Africa, characterizing the clinical, laboratory features and the histopathology of this entity. Aims and Objectives 1) To describe the clinical and histological features of HTLV-I associated infective dermatitis in KZN, South Africa 2) To determine the virological characteristics of HTLV-I in KZN, South Africa 3) To assess for HTLV-I / HIV co-infection Methods This was a prospective study of all patients with HAID who presented to King Edward VIII hospital (KEH), outpatient department over a period of 42 months. These were patients who fulfilled the clinical criteria of HAID. Enrolled patients were subjected to a confirmatory HTLV-I serology testing. Demographic data was obtained from all HTLV-I seropositive patients. Their clinical examination included dermatological, neurological and pathological examination. A blood count, immunoglobulin levels, serum protein electrophoresis measuring albumin levels and globulin fractions were measured. For bacteriological assessment skin swabs were taken from the affected sites with stool samples examined for parasites, ova and cysts. The HIV-1 status together with HIV-1 viral load were determined on those enrolled. The CD4 count, CD8 counts and CD4/CD8 ratio were also calculated. Skin biopsies were taken for histological examination. PCR for HTLV subtyping was performed on a subset of the cohort. Results Demography Of the 60 patients recruited, 33 fulfilled criteria for HAID. The majority of patients fell between age categories of 6 to lOyears. The male to female ratio was 1:1. There were more females in the adult group than there were within the childhood group. All of the patients in our cohort were African. Clinical features The lesions were erythematous, scaly, exudative, and crusted in all cases. The distribution of lesions was as follows: scalp (77.4%), retroauricular areas (71%), the axilla (65%) and paranasal areas (58%) were the sites more commonly affected. Nasal crusting was not a significant feature in this series. Bacteriology Culture was positive for Staphylococcus aureus (S. aureus) in 90%, with streptococcal group of organisms found in 68% of the skin swabs taken from the lesional skin. Haematological Our patients were mildly anaemic as has been shown in previous studies. They had a mean Hb of 11.5g/dl. In 12 of the 14 patients tested, the erythrocyte sedimentation rate (ESR) was elevated. Serum protein electrophoresis and levels of Immunoglobulin A, G and M were raised. The mean CD4 count in the entire group was elevated at 1730 cells/fil, CD8 was 1299 cells/ul Histopathology The major histological findings were as follows: 38% demonstrated a superficial and deep perivascular inflammatory infiltrate, 28% had a superficial and deep perivascular inflammatory infiltrate together with a lichenoid dermatitis, 12.9% had features of superficial and deep inflammatory infiltrate with an interface dermatitis, 6.4% revealed features of seborrhoeic dermatitis. Genotyping Our patients were infected with the strains belonging to the Cosmopolitan, A Subtype (HTLV-Ia). Complications Complications were low in this series with the commonest being scabies in 6(18.1%), corneal opacities in 3(8.6%), 2(6 %) with HAM/TSP. No parasitic worm infestations were isolated. HIV/HTLV-I co-infection Of the 33 patients, 9 (30 %) were co-infected with HIV. The mean viral load in this group was 52 000 copies/ml. Their mean CD4 count was also elevated at 1505cells/^il with a CD8 of 1704 cells/Mi and a CD4/CD8 ratio of 1.15. Discussion Thirty three of the 60 patients enrolled met the diagnosis for HAID according to the established criteria. The mean age in this series was 17 years (range: 8 months-46 years)however; almost a third (30.3%) were children under 12 years, reinforcing the entity as a childhood infective condition. There was an equal male female distribution in the childhood group and a female predominance in the adult group. Clinically patients presented with infected erythematous, scaly lesions mainly on the scalp, neck and post- auricular area. The clinical features were in keeping with other series worldwide. The complication rate was low in our cohort. S. aureus was the predominant organism in both anterior nares and lesional skin. The most common histological pattern was superficial and deep perivascular inflammatory infiltrate. The subtype in our series was the Cosmopolitan Subtype A (HTLV-Ia) as opposed to subtype B in Japan. We share with Brazil a common subtype. A subset of our patients (30%) was co-infected with HIV. The CD4 cell count in this subgroup was lower than the entire group but this was not statistically significant. The histological patterns found in this subgroup infected with HIV were similar to the rest of the group except for a more intense eosinophilic infiltrate in these skin biopsy specimens. Conclusion HTLV-I associated infective dermatitis is distinct entity which affects the African population of KwaZulu Natal, South Africa. It is predominantly a disease of childhood with an equal female to male ratio in children. The clinical features are an exudative, erythematous scaly rash most commonly found involving the scalp, axillae, paranasal and retroauricular areas. HTLV-I positivity is essential for the diagnosis; the Cosmopolitan Subtype A is commonest in South Africa. The commonest histological pattern is a superficial and deep perivascular infiltrate in 38%. A subset, 30%, was co-infected with HIV. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2008.

Lavendamycin, an aminoquinolinedione antibiotic is similar to streptonigrin, an antibiotic with known antiretroviral activity. Their applicability as drugs is limited due to their high toxicity to mammalian cells. A series of novel analogs of lavendamycin has been recently synthesized. In initial screening, three of the analogs showed significant inhibitory activity toward the reverse transcriptase (RT) of the avian myeloblastosis virus (AMV) and exhibited little or no animal toxicity and relatively low cellular cytotoxicity.In this study, we determined the anti-retroviral activity of nine analogs by assessing their anti-reverse transcriptase(anti-RT) activity in comparison to streptonigrin. Using both the human immunodeficiency virus (HIV) and AMV reverse transcriptase in vitro we found that the analogs exhibited significant anti-RT activity. The inhibitory activity of the analogs was dose dependent, and they had different effects on the two enzymes. At 30 µM seven of the analogs inhibited HIV-RT activity by 50% or more. At this concentration, two of the analogs were significantly more effective than streptonigrin. AMV-RT was more sensitive toward both streptonigrin and several of the analogs than was HIV-RT. Furthermore, combination of azidothymidine (AZT)-triphosphate (TP) and several of analogs showed synergistic inhibitory effects at low doses. / Department of Biology

Endoplasmic reticulum (ER) stress is a homeostatic mechanism, which is utilized by cells to adapt to inter- and intra-cellular changes. There is a burgeoning literature showing that the human endogenous retroviral envelope glycoprotein, Syncyin-1, oxidative stress and reactive oxygen species participate in the pathogenesis of multiple sclerosis (MS). I investigated the contribution of Syncytin-1-induced ER stress in MS and its animal model, experiment autoimmune encephalomyelitis (EAE). The prototypic ER stress biomarker, XBP-1 spliced variant (XBP-1/S), was increased in cerebral white matter of MS patients compared to non-MS controls and was correlated with Syncytin-1 expression. Syncytin-1 over-expression caused glia cytotoxicity but was mitigated by the ROS scavenger, crocin. Treatment with crocin on day 7 post-EAE induction ameliorated EAE disease severity in mice by reducing EAE pathology. Herein, I demonstrate that crocin attenuates Syncytin-1-induced ER stress in astrocytes while also diminishing disease severity in EAE in conjunction with suppression of neuroinflammation.

<p>Nevirapine (NVP) is a cheap anti-retroviral drug used in poor countries worldwide, administered to pregnant women at the onset of labour to inhibit HIV enzyme reverse transcriptase. Viruses which may get transmitted to newborns are deficient in this enzyme, and HIV-1 infection cannot be established, thereby preventing mother to child transmission (MTCT). In some cases, babies get infected and positive selection for viruses resistant to nevirapine may be inferred. Positive selection can be inferred from sequence data, when the rate of nonsynonymous substitutions is significantly greater than the rate of synonymous substitutions.</p> <p>Unfortunately, it is found that available positive selection methods should not be used to analyse before- and after- NVP treatment sequence pairs associated with MTCT. Methods which use phylogenetic trees to infer positive selection trace synonymous and nonsynonymous substitutions further back in time than the short time duration during which selection for NVP occurred. The other group of methods for inferring positive selection, the pairwise methods, do not have appreciable power, because they average susbtituion rates over all codons in a sequence pair and not just at single codons. We introduce a simple counting method which we call the Pairwise Homologous Codons (PHoCs) method with which we have inferred positive selection resulting from NVP treatment in longitudinal HIV-1 reverse transcriptase sequences. The PHoCs method estimates rates of substitutions between before- and after- NVP treatment codons, using a simple pairwise method.</p>