Despite the high prevalence of the human immunodeficiency virus (HIV) in South African women of reproductive age, the South African (SA) Prevention of Mother-to-Child Transmission (PMTCT) programme has significantly reduced the incidence of new HIV infections in infants from >20% in 2004 to <2% and overall MTCT approximately >5%. The PMTCT programme, however, faces challenges in terms of early infant diagnosis (EID) because of HIV polymerase chain reaction (PCR) indeterminate results as well as HIV drug resistance (HIVDR) secondary to antiretroviral therapy (ART) exposure of mothers and infants. The National Health and Laboratory Services (NHLS) uses the Roche COBAS®AmpliPrep (CAP)/COBAS®TaqMan® (CTM) HIV-1 Qualitative Test (Roche Molecular Systems, Pleasanton, CA) (CAP/CTM) platform as part of EID. Recently, the Roche cobas® 6800/8800 System has been introduced to test HIV viral load and HIV DNA PCR for EID. The platform is already processing samples for HIV viral load; however, verification for HIV DNA PCR for EID with dried blood samples (DBS) is needed, especially for CAP/CTM HIV PCR indeterminate results (Cycle threshold [Ct]>33 with any relative fluorescent intensity [RFI] value or Ct≤33 and RFI <5 on the CAP/CTM, Ct>38 on the Roche cobas® 6800/8800 System). In addition, HIVDR in newly diagnosed infants significantly limits treatment options. Therefore, the current study verified the Roche cobas® 6800/8800 System against the CAP/CTM system for the detection of HIV in EID and determined the HIVDR prevalence and profiles in infants <6 months, and how this affects current SA PMTCT and EID guidelines. The study comprised 642 DBS samples (235 HIV PCR positive, 193 HIV PCR negative and 214 HIV PCR indeterminate) previously tested on the CAP/CTM assay. Overall, 99.6% (234/235) CAP/CTM HIV PCR positive samples remained positive, while 99.5% (192/193) HIV PCR negative samples remained negative with the Roche cobas® 6800/8800 System. The HIV PCR indeterminate results as detected by the CAP/CTM decreased from 100% (214/214) to 8.4% (18/214) with the Roche cobas® 6800/8800 System. The Roche cobas® 6800/8800 System had a specificity of 99.5% and a sensitivity of 99.6%, but this decreased to 96.3% and 90.8% when HIV PCR indeterminate results were included. The kappa value increased from 0.5, which signifies moderate agreement, to 0.9, which is excellent agreement, when RFI from the CAP/CTM was excluded for result determination. The overall agreement between the two assays, taking only cycle threshold values into account, was 93.8%. As for HIVDR, mutations were detected in 42.9% (24/56) of infants <6 months. The most common non-nucleoside reverse transcriptase inhibitor (NNRTI) mutation causing high-level resistance was K103N (21.4% [12/56]), followed by Y181C and the NRTI mutation, M184V, both in 8.9% (5/56) of infants. Also, major protease inhibitor (PI) mutations, M46L and V82A were detected in one case each (1.8%). In conclusion, the performance of the Roche cobas®6800/8800 System was comparable to the CAP/CTM; however, it detected fewer HIV PCR indeterminate results, thus potentially offering conclusive results in a larger proportion of infants. The detection of high levels of the NNRTI mutation, K103N, emphasises the need for constant surveillance since nevirapine is included as part of the SA PMTCT guidelines and the World Health Organization recommends that NNRTIs should be phased-out of as part of PMTCT once the resistance prevalence exceeds 10%. / Dissertation (MSc (Medical Virology))--University of Pretoria, 2021. / National Health Laboratory Services Trust and RDP UP / Medical Virology / MSc (Medical Virology) / Restricted

Rotavirus gastroenteritis is a preventable public health concern, and diarrhoeal deaths persist in low-income settings. Rotavirus is prevalent in children under five years, despite widespread vaccination. Rotavirus P[8] vaccines display lowered efficacy (30-50%) in low-income settings compared to high-income (~80%). Disparities in vaccine efficacy are multifactorial but may be influenced by host factors. Understanding the interplay between the host, pathogen infection, and gut microbes may allow for targeted interventions against diarrhoeal disease. The FUT2 secretor gene encodes the availability of human histo-blood group antigens (HBGAs) in the gut, and the VP4 spike of rotavirus displayed binding affinity in a strain-specific manner to HBGAs. Secretor status may, therefore, influence rotavirus susceptibility, strain distribution, and partially contribute to varied vaccine efficacy. Furthermore, gut microbiomes may be shaped by HBGAs, influencing gut health and the subsequent risk of diarrhoeal disease. The aim of this study was to investigate secretor status, rotavirus VP4 genotypes, and gut microbiomes in South African children to elucidate the interplay between these factors. Methods for determining secretor status were evaluated since unique strengths and limitations exist that should be tailored to study design and population. Genotyping of the FUT2 gene targeting a known non-secretor mutation proved to be limited in genetically diverse participants. Phenotyping methods inaccurately classified children potentially due to an underdeveloped secretor phenotype that is masked by maternal antibodies introduced via breastmilk. Both methods yielded comparable yet distinct information about the cohort, and the study aim is essential to consider prior to method choice. In this study, South African secretors were significantly more susceptible to rotavirus infection, while non-secretors displayed a natural resistance. Rotavirus susceptibility was also strain-specific, where secretors were susceptible to predominantly circulating P[8] and P[4] strains, while less prevalent rotavirus P[6] strains were more common in non-secretors. Our findings indicate that secretor status is an important mediator of VP4 strain-specific rotavirus susceptibility in the South African population. Non-secretors in a population could modulate the circulation of wild-type rotavirus and contribute to lowered vaccine efficacy. In our study, non-secretors made up ~30% of the population. Higher frequencies of non-secretors in a population could reduce P[8] and P[4] rotavirus circulation, causing increased prevalence of P[6] strains with phylogenetically different VP4 genes. P[6] rotavirus strains are relatively common in South Africa, which contrasts to regions where non-secretors and P[6] strains are uncommon. Non-secretors may also respond poorly to rotavirus P[8] vaccines, and although their resistance to wild-type P[8] strains negates the need for a strong P[8] response, it may be important in areas where rare rotavirus strains are prevalent. Investigating the gut microbiome of healthy children did not show that microbial composition was influenced by secretor status. However, this pilot study provided valuable insights into microbiome methods and analysis pipelines. The gut microbiome is an emerging field of study, with advancements in sequencing technologies making the field more accessible to young scientists. The fight against diarrhoeal disease will require comprehensive insight into the gut environment. / Dissertation (MSc)--University of Pretoria, 2020. / The thesis/dissertation is under embargo until September 2022. / Rotavirus Surveillance grant (GSK E-Track 200238) / Poliomyelitis Research Foundation (Grant 19/48 and 19/18) / Medical Virology / MSc (Medical Virology) / Restricted

Tuberculosis (TB) is a global pandemic which resulted in 5.8 million disease cases and approximately 1.5 million deaths worldwide in 2020. TB disease is the leading cause of death worldwide from a single infectious agent, Mycobacterium tuberculosis (M.tb). It is estimated that a one fourth of the world's population is latently infected with TB and of these 5-10% will go on to develop active TB in their lifetime. Human immunodeficiency virus (HIV) infection is the greatest risk factor for developing active TB, with people living with HIV (PLWH) up to 19 times more likely to develop active TB compared to HIV uninfected individuals. Epidemiological studies show that there is increased risk of developing active TB (either reactivation of latent TB or new M.tb infection) throughout the course HIV infection, with the highest risk observed within the first year of infection, even when CD4+ T cell counts are still high. Previous studies have demonstrated that M.tb-specific CD4+ T cell responses are depleted early after HIV infection, within 3-12 months. This early defect in M.tb-specific CD4+ T cells may explain the elevated risk of TB even within the first year of HIV infection, prior to substantial immunosuppression. Antiretroviral therapy (ART) is an important strategy in preventing TB in PLWH. Earlier studies done in sub-Saharan Africa show that ART is associated with a 70-90% reduction in TB risk and a 52% decrease in TB related mortality. Several studies have also shown the importance of early ART initiation for HIV pathogenesis and transmission. However, despite ART intervention studies in sub-Saharan Africa, as well as Europe and North America, show that in these regions rates of TB remain high in PLWH. These high TB rates are most likely linked to the incomplete restoration of TB immune responses during ART. Overall, this thesis focused on characterising M.tb-specific CD4+ T cell responses during the course of HIV infection, from the acute phase of infection to post ART initiation. In Chapter 3, we aimed to confirm and extend previous findings that show there is a rapid depletion of M.tb-specific CD4+ T cells soon after HIV infection. We examined the kinetics of M.tb-specific CD4+ T cell responses over the course of HIV infection in two cohorts, a cross sectional cohort (n=58) and a longitudinal cohort (n=17). Consistent with previous findings, our results showed that there is a significant decrease in the frequency of M.tb responders 3 months after HIV infection. However, not all participants experienced M.tb-specific CD4+ T cell loss after HIV infection, with half the cohort losing 50% or more of their responses and the other half maintaining their responses. In the group that maintained their M.tb responses after HIV infection, the majority had very little fluctuations in their responses during the acute and chronic phase of infection. When comparing the clinical characteristics and the function and phenotype of M.tb-specific CD4+ T cells between individuals who maintained their M.tb-specific response and those who don't, no significant differences were found. In Chapter 4, to investigate the impact of early ART on M.tb immunity, we characterised M.tb-specific CD4+ T cell responses in individuals who initiated ART at an early stage of HIV infection (median 7.5 months post infection, n=16) in comparison to those who started ART during the chronic phase of HIV infection (median 66 months post infection, n=22). In this study, we examined multiple parameters between the two groups including their clinical characteristics, as well as the magnitude, function and phenotype of their M.tb-specific CD4+ T cells. We show that 2 years after ART initiation (irrespective of its timing) induced a significant immune reconstitution, marked by an increase in CD4 T cell count and restoration of the C4/CD8 ratio, but had no significant impact on the magnitude, function or phenotype of M.tb-specific CD4+ T cells. In Chapter 5, we sought to characterise M.tb-specific T cell responses in a small (n=13) cohort of participants who developed active TB during the CAPRISA 002 study. We performed a descriptive study examining the magnitude and phenotype of M.tbspecific T cells longitudinally over the course of TB treatment: before TB treatment/diagnosis, during treatment and after successful treatment. Despite the limited number of participants in this sub-study, we showed that after TB treatment there was a decreasing trend of activated M.tb-specific CD4+ T cells coincident with an increase in IFN-+ IL-2+ dual functional cells. Overall, our data confirms previous findings that there is an early depletion of M.tbspecific CD4+ T cells within a year HIV infection. However, in our study we found that not all HIV infected individuals lose their M.tb-specific responses. Instead, there was a variation in M.tb-specific responses after HIV infection, with some individuals maintaining their responses and others completely losing them. Results of our study also showed there were no major differences between participants who initiated ART early and those who initiated later during chronic HIV infection. To our knowledge this is the first study which looks at the impact of ART timing on M.tb-specific immunity in PLWH. The study therefore provides further insight, for future research, into whether early ART preserves TB immunity and therefore reduce the early, elevated risk of TB in HIV infected individuals.

No description available.

Mosquito-borne arboviruses are of considerable public health importance as they cause some of the most important emerging and re-emerging infectious diseases affecting humans and animals in many parts of the world including southern Africa. The threat of large epidemics of mosquito-borne arboviruses are often associated with climatic conditions, global warming, animal migrations, surface water, wind, topography, harbourage, vegetation, food supply and abundance of competent mosquito vectors. The goal of this project is to provide an in depth understanding of mosquito community dynamics and the importance of mosquito vector populations in the maintenance and transmission of mosquito-borne diseases in southern Africa. Firstly, a review of past and current literature was conducted to highlight: (a) the current state of knowledge regarding the most important mosquito-borne viruses of medical significance in southern Africa (b) lesser known mosquito-borne arboviruses with the potential of causing zoonotic health threats for humans in southern Africa. (c) key aspects of the ecology of mosquito vectors of medically significant mosquito-borne viruses in southern Africa. d) gaps in knowledge regarding southern African arbovirus mosquito vectors. Most of the studies on mosquito-borne viruses in southern Africa can be clustered into specific programmes led by Kokernot and Smithburn in the 1950s, McIntosh in the 1970s and 1980s, Swanepoel in the 1970s, Venter and others in more recent years, and have largely been restricted to South Africa, Mozambique and Zimbabwe. Twenty-six (26) arboviruses have been isolated from mosquitoes in southern Africa. Of these, Chikungunya (CHIK), Sindbis (SIN), West Nile (WN), Wesselsbron (WES), Spondweni (SPO), Banzi (BAN), Dengue (DEN), Bunyamwera (BUN), Germiston (GER) and Rift Valley fever (RVF) viruses are known to cause human illness. Middelburg (MID) and Shuni (SHUN) viruses are also important, causing neurological symptoms in animals with zoonotic potential for humans in South Africa. There are eight mosquito-borne arboviral infections most likely to impact humans in southern Africa (CHIK, MID, SIN, DEN, WES, WN, SHUN and RVF viruses). Mosquitoes in the subfamily Culicinae (mostly Aedes and Culex mosquitoes) are the most frequently associated with arbovirus transmission (115 and 105 types of arbovirus, respectively). Understanding the role of mosquito vector species in arbovirus transmission is vital for the development of new strategies to control the spread of arboviral diseases. In southern Africa, a few species in the genera Anopheles, Coquillettidia and Mansonia have also been implicated as vectors of arboviruses. Surveys over multiple decades across southern Africa have provided an insight regarding which species of mosquitoes are involved in the transmission of at least the most common of the mosquito-borne zoonotic arboviruses. These cluster within the genera Aedes and Culex, each representing a different transmission strategy. Aedes-borne viruses such as CHIK, DEN and WES tend to have primate or human reservoir hosts (McIntosh, 1986), while Culex-borne viruses often use birds as reservoir hosts, and these factors influence the distribution and epidemiology of the diseases they cause in humans and animals. Aedes and Culex have different breeding strategies and preferences which also represent fundamental differences. These mosquitoes are Aedes aegypti, Aedes furcifer/cordellieri, Aedes circumluteolus, Aedes unidentatus, Aedes mcintoshi, Aedes caballus, Aedes juppi, Culex theileri, Culex zombaensis, Culex univittatus, Culex neavei and Culex rubinotus. To determine mosquito community dynamics and mosquito vector distributions, sampling mosquito vectors at six sentinel sites in three provinces in the northern part of South Africa where recent cases had been detected in animals. Adult mosquitoes were collected from two horse properties in Gauteng Province; two wildlife reserves in Limpopo Province and at Orpen Gate in Kruger National Park and Mnisi Area in Mpumalanga Province between 2014–2017, using carbon dioxide-baited light and tent traps. Culex poicilipes, was the most abundant species caught during the study period. Highest diversity and species richness were found at Lapalala Wilderness Reserve, while the lowest diversity and abundances were at Orpen in Kruger National Park. Aedes aegypti, Ae. mcintoshi, Ae. metallicus, Ae. vittatus, Cx. pipiens sensu lato, Cx. theileri and Cx. univittatus, which are potential arbovirus vectors, had the widest geographical distribution in northern South Africa. Also collected were Anopheles arabiensis and An. vaneedeni, both known malaria vectors in South Africa. Therefore, arbovirus surveillance and vector control programs should be augmented in peri-urban and mixed rural settings where there is greater risk for arbovirus transmission to humans and domestic stock. Since climate has reportedly been associated with disease transmission, it’s important to understand the extent of its influence on mosquito abundance and distribution in northern South Africa. Thus, population composition, abundance and diversity of mosquitoes collected over a three-year period were determined and correlated to diverse climatic conditions during those years in order to determine seasonal trends in occurrence, abundance and distribution. Marked differences in the temporal distribution and seasonal abundances of the seven medically important mosquito vectors encountered from the two distinct geographic regions and climates. Statistical models have shown that climatic factors play a crucial role in shaping the population dynamics of Ae. mcintoshi, Ae. vittatus, An. arabiensis, Cx. pipiens s.l., Cx. poicilipes, Cx. theileri and Cx. univittatus both in Highveld Grassland and Middleveld Bushveld regions of northern South Africa. High summer temperatures and rainfall lead to increased vector density which might trigger outbreaks of RVF, SIN and WN viruses on the inland plateau of South Africa. This study also showed that abundances of RVF and WN virus vectors are related to elevation. These findings will be important in predicting the timing of onset and spread of future epidemics such as WN and RVF viruses, in southern Africa and other geographical settings with similar climates. / Thesis (PhD)--University of Pretoria, 2020. / University of Pretoria US Centers for Disease Control and Prevention / Medical Virology / PhD / Unrestricted

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Sudden Unexpected Death in Infancy (SUDI) refers to any such sudden demise in a child. If the child dies while asleep within the first year of life, and if no conclusive cause of death can be ascertained by means of complete autopsy and investigation into the circumstances surrounding death, including visit of the death scene, such a case is classified as one of Sudden Infant Death Syndrome (SIDS). By South African law, a full medico-legal autopsy is mandated in cases where the cause of death is not evident – including cases of possible SIDS. There can be little doubt that viral infection can be a cause of death in cases of supposed SUDI. At the Tygerberg medico-legal (forensic) laboratory, the evaluation of lung tissue for the presence of fatal viral lung infections forms part of the institutional protocol for the examination of SUDI cases. Lung samples of these SUDI cases are routinely tested for the presence of Cytomegalovirus (CMV), adenovirus and respiratory syncytial virus (RSV) by means of shell vial cultures. In a retrospective pilot study of 366 SUDI case files from Tygerberg Hospital, Western Cape, from 2004 – 2006, it was evident that in only 13.9% of possible SIDS cases, positive results for one or more of the aforementioned viruses were obtained. We hypothesise that the current method of virus detection, together with other factors such as the interval between death and post mortem examination, transport time of the specimens to the laboratory etc. might not be optimal to give a realistic picture of death in infancy caused by viral pulmonary infection. As other test modalities exist for the diagnosis of pulmonary viral infections, these methods were compared in terms of positive yield and association with viral pneumonitis, keeping the cost and time needed for each assay in mind. A total of 82 samples were collected over an 8 month period and routine shell vial cultures were done, followed by real-time Polymerase Chain Reaction (PCR) and immunohistochemical (IHC) staining of the lung sections with consensus pathology opinion. As expected, the real-time PCR method was much more better suited for identifying positive samples than shell vials (35% vs. 3.7% respectively). IHC staining also aided the pathologist in diagnosing viral infections microscopically. We expect the findings to be instrumental in streamlining not only our institutional SIDS investigation protocol, but also the development of a standardised national SIDS investigation protocol. / AFRIKAANSE OPSOMMING: “Sudden Unexpected Death in Infancy” (SUDI) verwys na enige skielike sterfte van ‘n kind. Indien die kind sterf tydens sy/haar slaap periode en geen oortuigende oorsaak van dood bepaal kan word deur middel van ’n volledige nadoodse ondersoek en ondersoek na die omstandighede tydens die dood, insluitend ’n besoek aan die doodstoneel nie, word so ’n geval as Wiegiedood (SIDS) geklassifiseer. SuidAfrikaanse wetgewing vereis ’n volledige medies-geregtelike nadoodse ondersoek in gevalle waar die oorsaak van dood onbekend is – insluitend gevalle van moontlike Wiegiedood. Daar is min twyfel dat virusinfeksie ‘n oorsaak van, of bydraende faktor tot dood kan wees in gevalle van moontlike SUDI. By die Tygerberg forensiese laboratorium vorm die evaluasie van long weefsel vir die teenwoordigheid van dodelike virusinfeksies deel van die institusionele protokol vir die ondersoek van SUDI gevalle. Long monsters van hierdie SUDI gevalle ondergaan roetine toetse vir die teenwoordigheid van sitomegaalvirus, respiratoriese sinsitialevirus en adenovirus deur middel van selkulture (“shell vial cultures”). In ‘n retrospektiewe steekproef van 366 SUDI gevalle by Tygerberg Hospitaal, Wes-Kaap van 2004 – 2006, is bevind dat in slegs 13.9% van moontlike SUDI gevalle die teenwoordigheid van een of meer van bogenoemde virusse bevestig kon word. Ons hipotese is dat hierdie metode van virus deteksie, tesame met ander faktore soos die tydsinterval tussen dood en nadoodse ondersoek, tyd om monsters na die laboratorium te vervoer ens. moontlik nie optimaal is om ‘n realistiese beeld van dood in babas as gevolg van pulmonale virusinfeksie te gee nie. Aangesien ander toets modaliteite bestaan vir die diagnose van pulmonale virusinfeksies, is hierdie metodes vergelyk in terme van positiewe opbrengs en assosiasie met virale pneumonitis, teen ’n agtergrond van die koste en tyd benodig per toets. ’n Totaal van 82 monsters is oor ‘n 8 maande periode versamel en roetine selkulture is gedoen, gevolg deur “real-time” Polimerase Ketting Reaksie (PKR), asook immunohistochemiese (IHC) kleuring van long snitte met patologiese verslae. Soos vermoed, is gevind dat die real-time PKR metode baie meer akkuraat is om positiewe monsters te identifiseer as roetine selkulture (35% vs 3.7% onderskeidelik). IHC kleuring het ook mikroskopiese diagnose van virale infeksies deur die patoloog vergemaklik. Ons verwag dat hierdie bevindinge grootliks kan bydra in die vaartbelyning van ons institusionele SIDS ondersoek protokol, asook in die ontwikkeling van ’n gestandaardiseerde nasionale SIDS ondersoek protokol.

Thesis (MScMedSc)--Stellenbosch University, 2013. / The diversity of HIV-1 sequences derived from patients in Bushbuckridge, Mpumalanga, was investigated. The gag p24, pol p10 and p66/p51, pol p31 and env gp41 gene fragments from 51 patients were amplified and sequenced. Quality control on the sequences was carried out using the LANL QC online tool. HIV-1 subtype was assigned using the LANL QC (RIP), REGA and jpHMM online tools. Subtype for the pol gene fragment was further designated using the SCUEAL online tool. Most of the sequences, that is 89%, belonged to HIV-1 subtype C. LANL QC (RIP), REGA, jpHMM also detected recombinants in 11% of the sequences. One of the isolates could only have the env gp41 gene fragment amplified and sequenced, which was determined to be HIV-1 subtype B. Phylogenetic analysis using the Neighbor-Joining and Maximum Likelihood methods from MEGA v 5 showed that, except for the env gp41 designated as a subtype B, all sequences in the study clustered with HIV-1 subtype C. Significantly, phylogenetic analysis showed that not only are the Bushbuckridge, Mpumalanga sequences related to HIV-1 subtype C sequences from southern Africa, India, Ethiopia and Brazil, but it is possible there has been multiple introductions of HIV-1 in the province. SDRMs were observed in two samples.

Thesis (MScMedSc) –Stellenbosch University, 2014. / ENGLISH ABSTRACT: HIV-infection is characterized by depletion of CD4+ T-cells from the gut-associated lymphoid tissue (GALT) which causes irreparable gastrointestinal tract damage and subsequent microbial translocation of bacterial products such as lipopolysaccharide (LPS), a component of Gram-negative bacteria, into systemic circulation. HIV infection also affects the functions and relative population sizes of various immune cells, such as monocytes. Monocytes are important innate immune cells as they are often the first cells recruited to sites of infection and inflammation. They then either promote inflammatory processes; elicit adaptive immune responses, through their antigen presenting ability; aid in pathogen and debris clearance or aid in damage repair. This cross-sectional study investigated functional changes to monocytes and monocyte subsets (CD14+CD16- and CD14+CD16+) in HIV+, treatment naïve individuals and healthy uninfected controls, using whole blood assays and isolated monocytes. A number of chemokine receptors associated with function and homing, and specific gut-homing receptors, were investigated. Monocyte activation, apoptotic potential and intracellular monocyte cytokine production were also investigated. All markers were evaluated using multi-parameter flow cytometry. Monocyte responsiveness to in vitro LPS stimulation and expression of the afore-mentioned chemokine receptors to viral load, CD4+ count and CD38/8 T-cell expression was also assessed. During HIV-infection monocytes appeared primed to exit systemic circulation and migrate towards the gut, as seen through elevated CD62-L (p < 0.005) and CCR7 (p < 0.005), whereas the CD14+CD16+ subset was increased (p = 0.0461) and exhibited a higher activation status through increased CD69 expression (p < 0.005) compared to the CD14+CD16- subset. An interesting observation was the significantly increased IL-10 production by the CD14+CD16+ subset (p < 0.005). An elevated CCR5 expression in total monocytes (p < 0.005) was also seen. After LPS stimulation, the HIV+ group displayed unique and significant percentage increases in the total monocyte population. The findings of the current study suggest that monocyte functionality may be retained during HIV-infection and that CD14+CD16+ monocytes play a vital role during HIV-infection evidenced by their preferential expansion and priming for GALT migration. The production of IL-10 by this subset further highlights their importance and emphasizes the need for future studies on the role of these cells in chronic stable HIV-1 infection and whilst disease progresses. / AFRIKAANSE OPSOMMING: MIV-infeksie word gekenmerk deur die uitputting van CD4+ T-selle, veral uit die derm-verwante limfweefsel (GALT). Dit veroorsaak onherstelbare skade aan die spysverteringskanaal en die daaropvolgende mikrobiese translokasie van bakteriële produkte soos LPS, „n komponent van Gram-negatiewe bakterieë, wat gaan binne sistemiese sirkulasie. MIV-infeksie beinvloed die funksies en relatiewe bevolkingsgrootte van verskeie immuun selle, insluitend monosiete. Monosiete is belangrike ingebore immuun selle en is dikwels die eerste selle wat gewerf word na areas van infeksie en inflammasie. Monosiete kan inflammatoriese prosesse bevorder of aanpabare immuunstelsel reaksies ontlok deur middel van hul antigeen aanbiedings vermoë of help met patogeen en puin klaring en skade herstel. In hierdie deursnee-studie het ons veranderinge aan monosiete (CD14+CD16+ en CD14+CD16-) ondersoek in MIV+ behandelde naïef individue en gesonde onbesmette kontroles, deur die gebruik van hele bloed toetse en geïsoleerde monosiete. 'n Aantal chemokine reseptore, wat verband hou met homing en funksie was ondersoek in toevoeging tot spesifieke derm-homing reseptore. Monosiet aktivering, apoptese potensiaal en intrasellulêre monosiet sitokien produksie was ook ondersoek. Alle merkers is ondersoek deur multi-parameter vloeisitometrie. Die beoordeel reaksies van monosiete na in vitro LPS stimulasie en die uitdrukking van die merkers met merkers van algemene immuun aktivering en MIV-siekte patogenese was ook ondersoek. CD14+CD16+ monosiete was gedurende MIV-infeksie verhoog (p-waarde = 0.0461). Daar was 'n hoër algehele monosiet uitdrukking van verskeie chemokine merkers soos CD69 (p-waarde < 0.005) uitdrukking; CD62-L (p-waarde < 0.005), en CCR7 (p-waarde < 0.005) uitdrukking in die CD14+CD16+ subgroep. Daar was ook „n toename in IL-10 produksie, veral in die CD14+CD16+ subgroep (p-waarde < 0.005). Hoewel baie funksionele merker reaksies dieselfde was, het die MIV+ groep „n unieke en beduidende persentasie verhooging in die totale monosiet bevolking getoon. Ons algehele bevindinge dui op 'n voorkeur uitbreiding van CD14+CD16+ monosiete tydens MIV-infeksie. Die CD14+CD16+ monosiet subgroep blyk ook bevoordeel word met betrekking tot voorbereiding vir migrasie na limfknope en die GALT. Die toename in geaktiveer de CD14+CD16+ monosiete op siekte webwerwe is waarskynlik 'n groot bydraende faktor tot aanhoudende immuun aktivering wat op sy beurt virale replikasie bevorder. Hierdie resultate beklemtoon die behoefte om die rol van hierdie selle en in veral die CD14+CD16+ subgroep, in kroniese stabiele MIV-1 infeksie verder te studeer en terwyl siekte bevorder.

Thesis (PhD (Pathology. Medical Virology))--Stellenbosch University, 2006. / Host susceptibility to human immunodeficiency virus-1 (HIV-1) infection and disease progression to acquired immunodeficiency syndrome (AIDS) varies widely amongst individuals. This observation led to the identification of host genetic factors playing a vital role in HIV-1 pathogenesis. Previous studies mainly focusing on Caucasian-based populations have indicated possible associations between genetic variants and host susceptibility to HIV-1/AIDS. The limited studies performed on African-based populations have emphasised the need for extensive investigation of both previously reported and particularly novel genetic variants within the older and genetically diverse Sub-Saharan African populations. In this study, the case-control samples were represented by African individuals of Xhosa descent, all residing in the Western Cape Province of South Africa. This included 257 HIV-1 seropositive patients and 110 population-matched HIV-1 seronegative controls. Mutational screening was performed in a subset of individuals for the entire coding regions of the CC chemokine receptor 5 (CCR5) and CC chemokine receptor 2 (CCR2) genes, and the 3’ untranslated region of the CXC chemokine ligand (CXCL12) gene, as previously reported (Petersen, 2002). Further analysis of these genes in a larger study sample involved the genotyping of previously identified mutations and single nucleotide polymorphisms (SNPs), which forms part of the present study. In addition, mutational screening was performed for the entire coding region of the CXC chemokine receptor 4 (CXCR4) gene, partial coding region of the mannose binding lectin (MBL) gene, and the promoter regions of interleukin 4 (IL4), interleukin 10 (IL10) and the solute carrier 11A1 (SLC11A1) genes. This was followed by genotyping of SNPs occurring in CCR5, CCR2, CXCL12, MBL, IL4, IL10, CX3C chemokine receptor 1 (CX3CR1), CC chemokine ligand 5 (CCL5) and tumour necrosis factor alpha (TNFα) genes. Significant associations were observed with HIV-1 susceptibility in the Xhosa population of South Africa. These included the CCR5-2733A>G, CX3CR1V249I, IL10-819C>T and IL10-592C>A SNPs being associated with a reduced risk for HIV-1 infection, while the CCR5-2135C>T and SDF1-3’G>A (CXCL12-3’G>A) SNPs were associated with increased susceptibility to HIV-1 infection. Furthermore, certain haplotypes for IL4 and IL10 showed association with reduced risk for HIV-1 infection. This included the identification of a novel IL4 haplotype restricted to the HIV-1 seronegative control group. This study emphasises the importance of considering genetic diversity across all populations, as certain HIV-1/AIDS associations appear to be restricted to specific ethnic groups. These findings have also provided an understanding for further elucidating the functional roles of genetic variants in determining HIV-1/AIDS susceptibility. Ultimately, such genetic association studies will contribute to establishing HIV-1/AIDS risk profiles for African-based populations from pandemic-stricken Sub-Saharan Africa.

Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010. / Bibliography / ENGLISH ABSTRACT: Throughout history disease entities have been described which match the description of diseases now known to be caused by hantaviruses; however these viruses were first identified as the aetiologic agent in 1976, the first species named Hantaan virus after the river near which its natural host, the rodent species Apodemus agrarius, was captured. Since then numerous species in the Hantavirus genus, family Bunyaviridae, have been found, with today more than 30 species worldwide being known. Hantaviruses are hosted by rodents from the Muridae and Cricetidae families and by shrews (insectivores) in the Soricidae family. There are two types of hantavirus disease, Haemorrhagic fever with renal syndrome (HFRS) in the Old World and Hantavirus cardiopulmonary syndrome (HCPS) in the New World. The first two African hantaviruses were identified in 2006 in Guinea, West Africa; Sangassou virus (SANGV) in a rodent, the African wood mouse (Hylomyscus simus), and Tanganya virus (TGNV) in Therese’s shrew (Crocidura theresae). In this study, rodents and shrews were trapped at localities in the Western Cape and Northern Cape provinces of South Africa, and in the southern regions of Namibia. RNA was extracted from their lungs and screened for hantavirus sequences by RTPCR, using degenerate primers designed to detect all members of the Hantavirus genus. In addition, an in-house IgG ELISA assay was set up, based on recombinant N antigen from Dobrava virus, DOB-rN, and Puumala virus, PUU-rN. The assay was used to screen patient sera collected in an anonymous convenience serological survey using residual serum samples left over from routine testing at NHLS laboratories in the Western Cape for hantavirus-specific antibodies. RNA from 576 animal specimens was screened by RT-PCR; no hantavirus genome was detected in any of the specimens. Sera from 161 patients were screened for hantavirus antibodies; 11.18% of the sera were reactive to DOB-rN, 4.97% against PUU-rN and 2.48% against both antigens. v Though no virus was detected in the animals screened, this does not necessarily mean that there are no hantaviruses present in Southern Africa. A previous seroepidemiological survey conducted in South Africa reported on the presence of hantavirus specific antibodies by IFA in two species of rodents trapped in the Western Cape and Northern Cape Aethomys namquensis and Tatera leucogaster. Our was the second known study in South Africa conducted that determined and proved the presence of hantavirus specific antibodies in humans. / AFRIKAANSE OPSOMMING: Dwarsdeur die geskiedenis was daar beskrywings van siektes wat ooreenstem met die beskrywing van hantavirus simptome, maar die eerste etiologiese oorsaak van die siekte is eers in 1976 geïdentifiseer en Hantaan virus genoem, vernoem na die rivier waar naby die gasheer, Apodemus agrarius, gevang is. Van daar af het die soektog na nuwe hantavirusse intensief gevorder en vandag is daar meer as 30 spesies wêreldwyd wat aan die Hantavirus genus, ’n lid van die Bunyaviridae familie, behoort. Knaagdiere van die Muridae en Cricetidae families, sowel as spitsmuise (insekvreters) in die Soricidae familie is gasheer vir hantavirusse. Twee tipes hantavirus siekte is bekend, hemorragische koors met nier sindroom (HFRS) in die Ou Wêreld en hantavirus kardiopulmonale sindroom in die Nuwe Wêreld. Die eerste twee Afrika hantavirusse is in 2006 in Guinee Wes-Afrika geïdentifiseer; Sangassou virus (SANGV) in ’n knaagdier, die Afrika hout muis (Hylomyscus simus) en Tanganya virus (TGNV) in Therese se spitsmuis (Crocidura theresae). In hierdie studie is knaagdiere en spitsmuise op verskeie plekke in die Wes- en Noord-Kaap provinsies, asook die Suide van Namibië, gevang. RNS is onttrek vanuit die longe en hantavirus volgordes is gesoek deur middel RT-PKR deur gebruik te maak van Pan-Hanta primers wat ontwerp is om alle lede van die Hantavirus genus op te spoor. ’n Self-ontwerpde IgG ELISA, gebasseer op rekombinante N antigeen van Dobrava virus, DOB-rN en Puumala virus, PUU rN, is opgestel en gebruik om pasiënt serum, verkry in ’n anonieme serologiese opname, te toets; oorblywende serum, na toetse uitgevoer is deur NHLS laboratoriums in die Wes-Kaap, is verkry en getoets vir hantavirus spesifieke teenliggaampies. RNS van 576 dier monsters is getoets deur middel van RT-PKR en geen hantavirus is in enige van die monsters geïdentifiseer nie. Serum van 161 pasiënte is getoets vir hantavirus teenliggaampies; 11.18% van die serum was reaktief teen DOB-rN, 4.97% teen PUU-rN en 2.48% teen albei antigene. Alhoewel geen virus in die diere geïdentifiseer is nie, beteken dit nie noodwendig dat geen hantavirusse in Suidelike-Afrika voorkom nie. ‘n Vorige sero-epidemiologiese opname wat in Suid-Afrika gedoen is het die teenwoordigheid van hantavirus spesifieke teenliggaampies in twee knaagdier spesies, Aethomys namquensis en Tatera leucogaster gevang in die Wes-en Noord-Kaap, gevind. Ons studie is die tweede studie bekend in Suid-Afrika uitgevoer, wat die teenwoordigheid van hantavirus spesifieke teenliggaampies bevind en bewys het.