The development and optimization of anti-viral screening methods are essential to develop newer more effective, treatments against HIV. The XTT method is a widely described method for antiviral screening. Both continuous HIVinfected cells and experimentally infected T-cells have been used in the XTT assay. We compared these methods to screen several plant-derived extracts for cytotoxicity. Several considerations were taken into account when performing these tests (effect of media, solvents and plant enymes). Experiments were performed to investigate these effects. In addition, p24 and viral load quantification were compared as antiviral screening methods. The study showed that several modifications were necessary when performing the XTT assay on plant extracts, due to the effect of media, solvents and plant enymes. The XTT assays and p24 assays performed using experimentally infected cells are far more specific than those using chronically infected cells. The use of viral loads as an antiviral screening method consistently demonstrated the expected efficacy of AZT. / Thesis(MMed.)-University of KwaZulu-Natal, 2009.

Recently a new Flavivirus, GB Virus C also referred to as Hepatitis G virus (GBV-C/HGV) was identified in humans with indeterminate hepatitis . Whilst in non-African countries this discovery led to an enormous enthusiasm to elucidate an association with liver disease, very little was known about the prevalence and pathogenicity of GBV-C/HGV infection in KwaZulu Natal, South Africa, where Hepatitis B Virus (HBV) infection is endemic and infection with the Human immunodeficiency virus (HIV) is a catastropic health problem. Sera from patients with liver disease (chronic liver disease [n = 98]; alcoholic liver disease [n = 50]); high risk groups (haemodialysis patients [n = 70]; HIV positive mothers and their babies [n = 75]) and control groups (alcoholics without liver disease [n = 35] and blood donors from the four racial groups [n = 232]) were screened for GBV-C/HGV RNA and Anti-E2 antibodies by reverse transcription polymerase chain reaction (RT-PCR) and an enzyme linked immunosorbent assay (ELISA), respectively. Overall 43.9% (43/98) of patients with chronic liver disease; 60 % (30/50) of patients with alcoholic liver disease; 47.1% (33/70) of haemodialysis patients; 60% (21/35) of alcoholics without liver disease and 31.9% (74/232) of blood donors (Africans] 44/76; 5.9%); Asians (5/52; 9.6%); Whites (15/49; 30.6%) and "Coloureds" [mixed origin] (9/54; 16.6%)]) were exposed to GBV-C/HGV infection as determined by the detection of Anti-E2 &/or RNA in serum. There was a significant difference in the prevalence of GBV-C/HGV infection (RNA &/or anti E2) between African blood donors and the other racial groups (p < 0.001), between blood donors and haemodialysis patients (p = 0.02) and or patients with chronic liver disease (p =0.04). There was no significant difference in the prevalence of GBV-C/HGV between African blood donors (45/76, 59.2%) and alcoholics with and without liver disease (30/50, 60% and 21/35, 60%, respectively). Anti-E2 antibodies and GBV-C/HGV RNA were almost mutually exclusive. GBV-C/HGV infected dialysis patients tended to have had more transfusions (p = 0.03) and had a longer duration of dialysis than non infected patients, indicating that the majority of patients on maintenance haemodialysis acquire their GBV-C/HGV infection through the transfusions they receive. There was no evidence for in utero and/or intrapartum transmission of GBV-C/HGY. However, there is some mother-to-infant transmission of GBV-C/HGV, though it is very probable that in KZN GBV-C/HGV is transmitted by as yet undefined non-parenteral routes. Sequence and phylogenetic analysis of the 5' non-coding region (5' NCR) and E2 gene segments of the GBV-C/HGV genome identified an additional "genotype" (Group 5) of GBV-C/HGV that is distinct from all other known GBV-C/HGV sequences (Groups 1-4). Although there is a high prevalence of Group 5 GBV-C/HGV isolates in KZN, there was no significant difference in liver biochemistry between GBV-C/HGV infected and noninfected patients with liver disease or between blood donors in each of the four racial groups. There was no significant differences in CD4 (461.12 ± 163.28 vs 478.42 ± 181.22) and CD8 (680.83 ± 320.36 vs 862.52 ± 354.48) absolute cell counts between HIV positive patients co-infected with GBV-C/HGV and those not infected with GBV-C/HGV, respectively. However, significantly higher relative CD3 [80.0 ± 4.17% vs 70.99 ± 19.79%] (p = 0.015), gamma delta T cells (yLT) [3.22± 1.30% vs 2.15 ± 29.12%] (p = 0.052) and lower CD 30 [35.45 ± 17.86% vs 50.59 ± 9.20%] (p = 0.041) status were observed in GBV-C/HGV positive compared to GBV-C/HGV negative HIV infected patients, respectively. Although there is a high prevalence of novel Group isolates of GBV-C/HGV in KZN, the lack of elevated liver enzymes and clinical hepatitis in blood donors and haemodialysis patients suggests that GBV-C/HGV is not associated with liver disease. HBV and not GBV-C/HGV modifies the course of alcoholic liver disease. The relatively higher number of CD3 cells and increased yLT expression, together with a decrease in CD 30 cells tends to suggest an association with protection and or delayed progression of HIV disease in GBV-C/HGV infected patients. Whilst GBV-C/HGV is not associated with liver disease, it may be an important commensal in HIV infected patients. / Thesis (Ph.D.)-University of Natal, 2003.

Viruses are common causes of both endemic and epidemic gastroenteritis, infecting millions of people per year, with norovirus, rotavirus and adenovirus-F as the main causative agents, and sapovirus and astrovirus as contributing viruses. These viruses are highly infectious and most severe in the very young, old, or individuals who are immunocompromised. The viral infection usually causes self-limited gastroenteritis, although chronic infection has been observed in highly immunocompromised patients. African and South-East Asian regions are disproportionally affected by diarrhoeal disease. These regions (especially South Africa) are also more severely affected by human immunodeficiency virus (HIV) infections. It has been suggested that immunocompromised individuals may form part of a reservoir for novel virus variants and recombinants. It should be taken into account that not every person is equally susceptible to infection after pathogen exposure and that not all infected persons develop clinical symptoms (Ramani and Giri, 2019). One host genetic factor that can influence susceptibility to enteric infection is the expression of histo-blood group antigens (HBGAs). Histo-blood group antigens are a major group of complex carbohydrates and are determinants of both human and animal ABO blood groups and the Lewis blood group systems, which are distributed in abundance on the mucosal epithelia of the gastrointestinal tract. Histo-blood group antigens have been proven to influence susceptibility to rotavirus and norovirus infections. Saliva, blood and stool specimens (n=205) have previously been collected from children (≤ 5 years of age) hospitalised with gastroenteritis at Kalafong Provincial Tertiary Hospital from June 2016 to December 2017. Follow up stool specimens were then collected six weeks after enrolment when possible. A descriptive questionnaire was completed by each child’s guardian, giving information on age, residential area, HIV status etc. of the participating child. The stool specimens were screened for six gastroenteritis causing viruses (norovirus GI and –GII, rotavirus, sapovirus, astrovirus and adenovirus) by multiplex PCR. Forty-seven percent (96/205) of specimens tested positive for at least one gastroenteritis causing virus. Rotavirus predominated (46/205), followed by norovirus (32/205), adenovirus (15/205), sapovirus (9/205) and astrovirus (3/205). A total of 27/32 norovirus (GI.3, GII.2, GII.3, GII.4, GII.7, GII.12 and GII.21), 44/46 rotavirus (G1P[8], G2P[4], G2P[6], G3P[4], G3P[8], G8P[4], G8P[6], G9P[6] and G9P[8]) and 8/9 sapovirus (GI.1, GI.2, GII.1, GII.4 and GII.8) strains have been genotyped, of which norovirus GII.4 and rotavirus G3P[4] predominated. A total of 46/205 children submitted a follow up stool specimen to be tested. Of the 46 children, 9 tested positive for norovirus infection with initial stool specimen testing. Follow up screening resulted in 13/46 (28%) specimens testing positive for either norovirus GI or GII, with all patients presenting as asymptomatic. After genotyping it was observed that only one of the follow up specimens were identical to the original sequence genotyped, indicating prolonged shedding. FUT2 genotyping of 205/205 children showed a 71%:29% ratio between secretors and non-secretors. Eighty percent (77/96) of the virus-infected children were secretors whereas only 20% (19/96) were non-secretors. Rotavirus (p<0.01) and norovirus GII.4 (p<0.05) specifically were found to be more prevalent in secretors. In this study, no statistical significance was observed in terms of severity of and susceptibility to gastroenteritis viruses between HIV-infected, HIV-exposed uninfected or HIV-uninfected individuals. Histo-blood group phenotyping has resulted in various combinations, with Le(b) being the most prevalent antigen found. Next generation sequencing was unsuccessful. In future, fresh specimens should be considered for testing, with more funding and time for optimisation of this process and to give adequate results. In summary, gastroenteritis is still a leading cause of childhood morbidity and mortality, with all advancements in understanding the disease helping to decrease the impact of it. This study again reinforced the importance of these viruses, as they are circulating in such high abundance. It also reinforced the concept that susceptibility to noro- and rotavirus infection is affected by the secretor status of a person. This could in future help with better understanding the viral infection mechanisms and in turn help with vaccine development and treatment / Dissertation (MSc (Medical Virology))--University of Pretoria, 2020. / Reese Mushrooms / Discovery grant / PRF / Medical Virology / Msc / Unrestricted

Bibliography / Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In just 25 years since the first reported cases in 1981, the number of Human Immunodeficiency virus (HIV) infected people has risen to 65 million, and over 25 million have died of acquired immunodeficiency syndrome (AIDS). Sub-Saharan Africa accounts for 67% of all people living with HIV and 72% of deaths in this region were AIDS related. Tuberculosis (TB) is one of the most common opportunistic infections in AIDS patients, particularly in developing countries, where 60 - 70% of TB cases occur in HIV-1-infected persons. HIV-1 is a high risk factor for the development of TB, the reactivation of a latent Mycobacterium tuberculosis infection and also progressive TB. CD8+ Cytotoxic T Lymphocytes (CTL) are pivotal in the host immune response to HIV infection. CTL are associated with resolution of acute infection and with reduction in viral load. Studies in macaques and humans indicate the importance of CTL in the control of HIV infection, where reduction in CD8+ T cell number has been correlated with progression to AIDS. The current study was a cross-sectional descriptive study of CD8+ T cells of HIV+ adult South Africans with and without TB co-infection (TB disease). The cohort consisted of anti-retroviral therapy (ART) naive patients and all CTL analyses were carried out on peripheral blood mononuclear cells (PBMCs). A total of 60 South African adults from the Western Cape were utilized in this study, including 15 healthy controls; 30 HIV+TB-individuals and 15 HIV+TB+ individuals. Expression of phenotypic, activation and functional markers were investigated by flow cytometry with the use of fluorochomeconjugated antibodies. The markers examined included the novel activation marker CD137, the CTL associated markers Perforin, Granzyme A, CD107a/b, Fas (CD95), and FasL (CD95L), intracellular cytokines IFN-y and TNF-a and the chronic HIV CTL dysfunction marker PD-1. HIV infection alone was associated with increased baseline expression of TNF-a, Perforin, Granzyme A, PD-1, Fas (CD95), and FasL (CD95L), but not CD137(4-1BB) or IFN-y as compared to uninfected controls. TB co-infection resulted in further increased baseline expression of TNF-a, perforin, PD-1, FasL (CD95L), as well as increased IFN-y. HIV-1 antigen (gag)-specific stimulation in vitro indicated that in HIV infection was associated with antigen-specific upregulation of activation and cytotoxicity markers CD137, IFN-y, TNF-a, Fas, FasL and CD107a/b. In TB co-infection a reduction in antigen-specific degranulation (CD107a/b up-regulation) and also Fas and FasL expression was observed. TB co-infection (in the form of active pulmonary TB) reduced antigen-specific CTL functional activity, but simultaneously there was an association with increased baseline PD-1 expression and also cytolytic marker expression (Fas, FasL, TNF-a). These cytolytic markers could be involved in non-antigen-specific bystander target cell death. The expression of the co-stimulatory molecule CD137 appeared to correlate with interferon-y production and levels of degranulation, confirming its usefulness as a putative surrogate marker of functional responsiveness. These data indicate that in addition to impacting on CD4 T cell function, TB co-infection leads to higher baseline expression of CTL-associated markers, but to dysfunctional antigen-specific CTL responses. / AFRIKAANSE OPSOMMING: Slegs vyf en twintig jaar na die eerste berigte van die menslike immuniteitsgebrekvirus (MIV) in 1981, het die getal MIV-geinfekteerde individue gestyg tot 65 miljoen en het meer as 25 miljoen mense alreeds gesterf aan die verworwe immuniteitsgebrek sindroom (VIGS). Sub Sahara Afrika maak 67% uit van alle HIV gevalle en het `n MIVverwante doodsyfer van 72%. Een van die algemeenste opportunistiese infeksies in VIGS pasiente is Tuberkulose (TB). In ontwikkelende lande, veral, kom 60-70% van TB gevalle voor in MIV-1 geinfekteerde individue. MIV-1 is `n hoe risiko faktor vir die ontwikkeling van TB, die heraktivering van latente Mycobacterium tuberculosis infeksie en progressiewe TB. Die CD8+ sitotoksiese T Limfosiete (STL) se immuun reaksie teen `n MIV infeksie is noodsaaklik en word geassosieer met `n resolusie van die akute infeksie en `n afname in viruslading. Studies in die mens en macaque het getoon dat sitotoksiese T limfosiete belangrik is vir die beheer van MIV infeksies aangesien die afname in CD8+ sel getalle korreleer met die verloop tot VIGS. Hierdie deursnit-beskrywende studie het die CD8+ T selle van MIV+ volwasse Suid-Afrikaners, met of sonder`n TB mede-infeksie, ondersoek. STL analise is gedoen op die perifere bloed mono-nuklere selle (PBMS) van pasiente wat geen teen-retrovirale terapie (TRT) ontvang het nie. `n Totaal van sestig Suid-Afrikaanse volwassenes van die Wes-Kaap het deelgeneem aan die studie wat 15 gesonde kontroles; 30 MIV+TBen 15 MIV+TB+ individue ingesluit het. Die uitdrukking van fenotipiese, aktiverings en funksionele merkers is ondersoek deur middel van vloeisitometrie en fluorochroomgekonjugeerde teenliggaampies. Laasgenoemde het ingesluit die nuwe aktiversingsmerker CD 137, die STL geassosieerde merkers Perforien en Gransiem A, CD 107a/b, Fas (CD95) en FasL (CD95L), intrasellulere sitokiene IFN-y en TNF-a en PD-1, die merker vir chroniese MIV CTL disfunksie. Daar is gevind dat `n TB mede-infeksie (in die vorm van aktiewe pulmonere TB) die antigeen-spesifieke STL funksie verlaag en terselftertyd `n verhoging in die uitdrukking van PD-1 en sitolitiese merkers (Fas, FasL, TNF-a) bewerkstellig. Hierdie sitolitiese basislyn merkers is moontlik betrokke by die dood van nie-antigeen-spesifieke omstander teiken selle. Die uitdrukking van die mede-stimulatoriese molekule CD 137 blyk om te korreleer met die produksie van STL IFN-y en die vlakke van degranulasie. Dit bevestig die merker se bruikbaarheid as `n gewaande surrogaat merker vir funksionele reaksies. Die data toon verder dat `n TB mede-infeksie nie net `n effek het op die CD4 T sel funksie nie, dit lei ook tot `n verhoogde basislyn uitdrukking van STLgeassosieerde merkers, maar met disfunksionele antigeen-spesifieke STL reaksies. Hierdie studie het bepaal dat `n MIV infeksie verbind word met `n toename in die basislyn uitdrukking van TNF-a, Perforien, Gransiem A, PD-1, Fas (CD95) en FasL (CD95L). Dit is egter nie die geval wanneer die uitdrukking van CD 137 (4-1BB) of IFN-y vergelyk word met nie-geinfekteerde kontroles. `n TB mede-infeksie het `n verdere toename in die uitdrukking van TNF-a, Perforien, PD-1, FasL (CD95L) getoon, asook `n verhoging in IFN-y vanaf die basislyn. In vitro MIV-1 antigeen (gag)-spesifieke stimulasies het aangedui dat `n MIV infeksie met die antigeen-spesifieke op-regulasie van aktiverings en sitotoksiese merkers CD137, IFN-y, TNF-a, Fas, FasL en CD107a/b geassosieer word. In `n TB mede-infeksie, is `n verlaging van antigeen-spesifieke degranulasie (CD 107a/b op-regulasie) asook die uitdrukking van Fas en FasL waargeneem. / The Poliomyelitis Research Foundation / The National Health Laboratory Service

Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: The emergence and re-emergence of viral human pathogens from wildlife sources in the recent past has led to increased studies and surveillance of wildlife for potentially zoonotic agents in order to gain a better understanding of the pathogens, their sources as well as events that may lead to viral emergence. Of the >1407 known human pathogens, 13% are classified as emerging or re-emerging, and 58% as zoonotic; 37% of the (re-)emerging and 19% of the zoonotic pathogens are RNA viruses, accounting for the majority of recently emerged infectious diseases with a zoonotic origin, such as HIV, Ebola, Hendra, Nipah, Influenza and SARS. This study focusses on potentially zoonotic viruses hosted by rodents (Muridae family), shrews (order previously known as Insectivora/Soricomorpha, now reclassified as Eulipotyphla) and bats (order Chiroptera). Rodents and bats represent the largest (~40%) and second largest (~25%) mammalian orders and both occur on every continent except Antarctica. Together, the three mammalian orders investigated represent the most relevant potential sources of new zoonoses. In this study I investigated the occurrence of astroviruses, arenaviruses, coronaviruses and hantaviruses in South African small mammal species belonging to the orders mentioned above. These viruses have either been implicated in recent emerging zoonotic events or are considered to have the potential to cause cross-species transmissions resulting in a zoonotic event. In the first part of the study specimens collected from various bat, rodent and shrew species were screened for viral sequences by broadly reactive PCRs; positive samples were characterised by sequencing and sequence analysis. A separate part of the study focussed on hantavirus disease in humans: a seroprevalance survey was conducted to determine the presence of hantavirus antibodies in the local population. Additionally, acutely ill patients with potential hantavirus disease were tested in an attempt to identify possible acute infections and define clinical hantavirus disease in South Africa. Screening of rodent and shrew specimens resulted in the identification of eight novel arenavirus sequences. Seven of the sequences are related to Merino Walk virus, a recently identified South African arenavirus, and the eighth sequence represents a novel lineage of Old World arenaviruses. Screening of bat specimens resulted in the identification of highly diverse novel astrovirus and coronavirus sequences in various South African bat species, including the identification of a viral sequence closely related to the recently emerged Middle East Respiratory Syndrome coronavirus. While the study did not identify hantavirus infections in any of the acutely ill patients, it found seroprevalences similar to those observed in Europe and West Africa. The results obtained highlight the importance of small mammals in the emergence of potential zoonoses and further reinforce the importance of viral surveillance of relevant wildlife species. Further in-depth studies of naturally infected reservoir host populations are required in order to gain a better understanding of virus-host dynamics and the events that lead to virus emergence. / German Research Foundation (DFG) (project number: KR1293/9-1/13-1) / The Polio Research Foundation and the NHLS Research / Harry Crossley Foundation, the Polio Research Foundation and Stellenbosch University for granting scholarships and bursaries for PhD.

Thesis (MScMedSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Background: The conventional sequence analysis is the most common method used for the detection of drug-resistant mutants. Due to its sensitivity limitations, it is unable to detect these mutants when comprising less than 20% (minor populations) of the total virus population in a sample. However, real-time PCR-based assays offer a rapid, sensitive, specific and easy detection and quantification of such mutants. The HIV-1 variants harbouring the K103N mutation are associated with resistance to nevirapine (NVP) and efavirenz (EFV). The persisting drug-resistant mutants decay slowly to low levels, and therefore they are called minor drug-resistant mutants. Consequently, they affect subsequent treatment with the drugs of the relevant class. Objectives: The objective of this study was to design two TaqMan real-time PCR-based assays called selective-polymerase chain reaction (SPCR), namely the total viral copy SPCR assay and the K103N-SPCR assay. The former detects HIV-1 of subtype C reverse transcriptase sequences, whereas the latter detects K103N drug-resistant variants in these sequences. Design and Methods: In developing the SPCR assays, sets of appropriate primers and probes for the HIV-1 subtype C reverse transcriptase (RT) were developed to use in the K103N-specific reaction and the total copy reaction. Twelve DNA plasmid standards with sequence diversity were constructed for the assay from two HIV-1subtype C samples known to harbour the K103N mutation (AAC or AAT) in our Department‟s Resistance Databank. Their RT regions were amplified, cloned and verified with sequencing. Site-directed mutagenesis was used to induce mutations at 103 amino acid position in some of these clones to generate more standards with either one of the three codons (AAA, AAC and AAT). The two assays were optimized and validated, and a standard curve was generated for each assay using 10-fold serial dilution (5x107-5x100 DNA copy/μL) of a K103N-mutant plasmid standard. The optimized and validated SPCR assays were used to screen 40 nested PCR products of previously genotyped patient samples for minor K103N variants. Results: Two sensitive and reproducible selective real-time PCR (SPCR) assays, with cut-offs of 8.23 and 10.33 and a detection limit of 0.01% for the K103N resistance variants, were successfully developed. The assays detected a prevalence of 25.64-46.15% for the K103N resistance mutation in 39 patient samples. The genotyping (population sequencing) missed 40-53.85% of these variants. Conclusion: In conclusion, sensitive and reliable selective real-time PCR assays to detect and quantify minor K103N variants of HIV-1 in nested PCR products were successfully developed. The assay had a lower detection limit of 0.01%. / AFRIKAANSE OPSOMMING: Agtergrond: Konvensionele volgorde bepaling analise is die mees algemeenste metode wat gebruik word vir die opsporing van middel-weerstandige mutasies, maar weens beperkte sensitiwiteit is dit nie moontlik om hierdie mutante op te spoor wanneer dit minder as 20% (minderheids populasie) van die totale viruspopulasie in `n monster uitmaak nie. Nietemin, kwalitatiewe PKR-gebaseerd toetse bied vinnige, sensitiewe, spesifieke en makliker opsporings en kwantifisering van sulke mutante aan. MIV-1 variante wat die K103N mutasie bevat word geassosieer met weerstand teen nevirapine (NVP) and efavirenz (EFV). Volhoudende middel-weerstandige mutasies vergaan stadig na laer vlakke en word daarom na minderheids middel weerstandige mutasies verwys. Gevolglik affekteer dit opvolgende behandeling met die middel van die relevante klas. Doelwitte: Die doel van die studie was om twee TaqMan kwantifiserende PKR gebaseerde selektiewe polymerase ketting reaksies (SPKR), naamlik totale virale kopie SPKR en K103N-SPKR te ontwikkel. Die voormalige toets het die MIV-1 subtipe C omgekeerde transkriptase volgorde bepaal, waar K103N die middel-weerstand variante in hierdie volgorde opspoor. Ontwerp en Metodes: `n Geskikte stel inleiers en peiler was ontwikkel vir die MIV-1 subtipe C omgekeerde transkriptase (OT) vir gebruik in die K103N-spesifieke en die totaal kopie reaksie. Twaalf DNS plasmied standaarde met volgorde diversiteit was saamgestel vir die toets vanaf twee MIV-1 subtipe C monsters wat volgens ons Departement se weerstand databasis geklassifeer is vir die besit van die K103N mutasie (AAC of AAT). Die OT streke was geamplifiseer, gekloneer en geverifieer deur volgorde bepaling. Punt-gerigte mutagenese is gebruik om `n mutasie by die amino suur posisie 103 van sekere klone te induseer om meer standaarde te genereer wat een van die drie kodons (AAA, AAC en AAT) bevat. Die twee toetse is geoptimiseer en gevalideer en `n standard kurwe is genereer vir elk van die toetse deur die gebruik van tienvoud serie verdunnings (107-1 DNS kopie/μL) van `n algemene K103N-mutante plasmied standard. Die geoptimiseerde en gevalideerde SPKR toets was gebruik om vir die minderheids K103N variante in 40 “nested” PKR produkte van voorheen gegenotipeerde pasiënt te soek. Resultate: Twee sensitiewe en herproduseerbare selektiewe kwantitiewe PKR toetse met `n ΔCt afsnypunt van 8.23 en `n deteksie limiet van 0.006% was ontwikkel vir die K103N weerstand variant. Die toets het `n voorkomsyfer van 25.6 % vir die K103N weerstand mutasie in 40 pasiënt monsters bepaal, waar genotipering (populasie volgorde ) 40% van hierdie variante nie opgespoor het nie. Gevolgtrekking: `n Sensitiewe en betroubare selektiewe kwantitatiewe PKR toets vir die opspoor en kwantifisering van die minderheids K103N variante van MIV-1 in PKR produkte was ontwikkel. Hierdie toets het `n laer opsporings limiet van 0.01%. / Poliomyelitis Research Foundation (PRF) / National Research Fund (NRF) / National Health Laboratory Service Research Trust (NHLS RT)

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: Reactivation of human cytomegalovirus (HCMV) infection in individuals infected with human immunodeficiency virus (HIV) may lead to life-threatening end-organ diseases (EOD). The EOD becomes clinically apparent when a critical number of cells in the affected organs become damaged as a consequence of HCMV-infection. Treatment of the HCMV-associated disease at this point may not be effective. Therefore, early detection of HCMV reactivation may be useful to guide pre-emptive therapy. Aim: The aim of this study was to determine whether there is a point at which the HCMV-specific cellular immune response breaks down, as determined by the interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay, and HCMV reactivation occurs in HIV-positive, antiretroviral therapy (ART)-naïve individuals in a South African setting. This was done in relation to the CD4+ T cell count and the HCMV viral load as determined by real-time polymerase chain reaction (qPCR). Materials and methods: Fifty-two (52) HIV-infected, ART-naïve subjects were recruited from primary healthcare centres that they attended for the management of their HIV infection. Heparinised blood samples were collected to quantify the HCMV-specific cellular immune response using the IFN-γ-ELISPOT assay and to determine the HCMV IgG serostatus. Ethylenediaminetetraacetic acid (EDTA) blood samples were collected for the determination of the CD4+ T cell counts and the HCMV viral loads. Results: All 52 subjects recruited were confirmed to be HIV-HCMV co-infected based on their HCMV IgG serostatus. The results of 34 subjects with completed data sets were analysed. The CD4+ T cell counts of these subjects ranged from 10 to 784 cells/μl. Twenty-two (22) (65%) subjects had positive HCMV IFN-γ-ELISPOT results with 94% having no detectable HCMV viral loads. All subjects (28) with a CD4+ T cell count above 100 cells/μl had undetectable HCMV viral loads. Two of the six subjects with CD4+ T cell counts <100 cells/μl had detectable HCMV viral loads. There was no statistically significant association between the CD4+ T cell counts and the HCMV IFN-γ-ELISPOT results. Conclusion: No specific point could be determined when there is loss of integrity of the HCMV-specific cellular immune response in HIV-positive individuals. Low CD4+ T cell counts did not correlate with HCMV IFN-γ-ELISPOT results suggesting that the HCMV-specific cellular immunity did not necessarily break down at low CD4+ T cell counts. Nevertheless, a CD4+ T cell count above 100 cells/μl appeared to be protective against viraemia as determined by the HCMV viral load qPCR. The IFN-γ-ELISPOT assay was employed as a tool to determine the integrity of the HCMV-specific cellular immune response in HIV-positive individuals. However, the IFN-γ-ELISPOT assay should be used in conjunction with the CD4+ T cell count and the HCMV viral load qPCR to determine when there is loss of integrity of the HCMV-specific cellular immune response and HCMV reactivation occurs. This may assist clinicians in their choice of management and appropriate pre-emptive treatment in the HIV-HCMV co-infected individual at a risk for HCMV reactivation. / AFRIKAANSE OPSOMMING: Inleiding: Heraktivering van menslike sitomegaalvirus (MSMV) in menslike immuniteitsgebreksvirus (MIV)-MSMV ko-geïnfekteerde individue kan lei tot dodelike end-orgaan siektes (EOS). Die EOS word klinies duidelik wanneer 'n kritieke aantal selle in die organe beskadig raak as gevolg van die MSMV-infeksie. Behandeling van die MSMV-verwante siekte op hierdie punt mag moontlik nie meer effektief wees nie. Daarom kan die vroeë opsporing van MSMV heraktivering nuttig wees in die gebruik van voorkomende terapie. Doel: Die doel van hierdie studie is om die punt te bepaal wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word met behulp van die interferon gamma (IFN-γ) ensiem-gekoppelde immunospot (ELISPOT) toets en MSMV heraktivering voorkom in MIV-positiewe, antiretrovirale terapie (ART)-naïewe individue in' n Suid-Afrikaanse instelling. Dit word gedoen in verhouding met die CD4+ T sel telling en die MSMV virale lading. Materiale en metodes: Twee-en-vyftig (52) MIV-geïnfekteerde, ART-naïewe pasiënte is vanaf primêre gesondheidsentrums, wat hul bywoon vir die behandeling van hul MIV infeksie, genader. Gehepariniseerde bloedmonsters is gebruik om die MSMV-spesifieke sellulêre immuun reaksie met behulp van die IFN-γ-ELISPOT toets en die MSMV IgG serostatus te bepaal. Etileendiamientetra-asynsuur (EDTA) bloed monsters is versamel vir die bepaling van hul CD4+ T sel telling en hul MSMV virale lading met behulp van die ―real-time‖ polimerase kettingreaksie (qPKR) waardes. Resultate: Al 52 pasiënte is bevestigde MIV-MSMV ko-infeksies, gebasseer op hul serologiese status. Die resultate van 34 pasiënte met voltooide data is ontleed. Die CD4+ T sel tellings van hierdie pasiënte het gewissel 10-784 selle/μl. Twee-en-twintig (22) (65%) pasiënte het positiewe MSMV IFN-γ-ELISPOT resultate met 94% wat ‗n negatiewe qPKR resultate. Alle pasiënte (28) met 'n CD4+ T-seltelling bo 100 selle/μl het' n negatiewe qPKR resultate. Twee van die ses pasiënte met 'n CD4+ T-seltelling <100 selle/μl het waarneembare MSMV virale ladings oor die qPKR. Daar was geen statisties beduidende assosiasie tussen die CD4+ T sel tellings en die MSMV IFN-γ-ELISPOT resultate nie. Gevolgtrekking: Geen spesifieke punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word kon in MIV-positiewe individue bepaal word nie. Lae CD4+ T sel tellings het nie ooreengestem met die MSMV IFN-γ-ELISPOT resultate nie en dui daarop dat die MSMV-spesifieke sellulêre immuniteit nie noodwendig afgebreek word teen 'n lae CD4+ T sel tellings nie. Tog blyk 'n CD4+ T-seltelling bo 100 selle/μl om beskerming teen viremie te bied. Die meriete van die gebruik van die IFN-γ-ELISPOT toets die integriteit van die MSMV-spesifieke sellulêre immuun response in MIV-positiewe individue te bepaal, is waargeneem in die opgehoopte data. Tog kan die gebruik van die IFN-γ-ELISPOT toets in samewerking met die CD4+ T sel telling en die MSMV virale lading meer voordelig in die bepaling van 'n punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afbreek en herstel plaasvind. Dit kan help om klinici in hul keuse van bestuur en gepaste voorkomende behandeling in die MIV-MSMV mede-geïnfekteerde individu op 'n risiko vir herstel.

Thesis (MPH.) --Univesity of Limpopo, 2014 / Background: The main goal of HAART is to achieve maximal viral suppression. However, with poor adherence to therapy the chances of achieving and maintaining successful viral suppression are decreased, leading to virological failure. And virological failure has been recognized by WHO as one of the early warning signs of drug resistance. This operational research sought to explore virological failure as a treatment outcome to evaluate program performance at a facility level. Methods: Purposive sampling as per inclusion and exclusion criteria was used to retrospectively review clinical records of the first 700 adult HIV positive patients (350 males and 350 females) who initiated antiretroviral treatment between April 2004 and December 2007 at this adult HIV clinic, were followed up for at least 3 years and treated according to the South African government’s National Department of Health 2004 HIV treatment guidelines for adults and adolescents. Major Results: 268 clinical records, 97 (27.71%) male and 171 (58.86%) female records were eligible for inclusion in the study. The proportion of females was higher (63.8%) than males (32.8%) with an average age of 38.95 years. 24 (8.9%) patients in the study sample experienced virological failure during the study period; 11 (11.3%) males and 13 (7.6%) females. Two-thirds (66.6%) of patients who failed to suppress at their first viral load measurement proceeded to develop virological failure. Overall, there was no association of statistical significance between age, sex, baseline CD4 cell count and baseline regimen, and virological failure at various intervals, p> 0.05. Conclusion: It was a challenge to keep patients in care but those that remained in care had good treatment outcomes with only 8.9% developing virological failure. Failure to suppress at first viral load preceded virological failure in the majority of patients.

Thesis (MScMedSc)--Stellenbosch University, 2013. / Bibliography / The efficiency in which HIV-1 can infect, spread and evade the attack of therapeutic agents can be attributed to a high mutation rate and frequent recombination events. These factors have collectively contributed to the diversity observed in HIV-1 and resulted in a multitude of subtypes, sub-subtypes, circulating recombinant forms (CRF’s) and unique recombinant forms (URF’s). The aim of this study was to investigate HIV-1 diversity in Cape Town using a small cohort of treatment naive patients being investigated for HIV Associated Neurocognitive Disorders (HAND). Four different genomic domains: gag, pol, accessory and gp41 genes were sequenced to subtype the virus. HIV-1 tat was further investigated because the dicysteine motif has been reported to play a role in HAND. Viral RNA and proviral DNA was extracted from 64 patients and used for the amplification and sequencing of the genes. Rega and jpHMM online tools were used to identify HIV-1 subtypes and recombinants while Neighbor-joining phylogenetic trees were constructed for phylogenetic analysis. The pol gene was further investigated using SCUEAL to detect possible intra-subtype recombination and was also screened for the presence of transmitted drug resistance. In addition tat sequence datasets retrieved from the Los Alamos sequence database were investigated and compared with the newly generated sequences for the detection of point mutations and amino acid signature patterns. Sequencing identified most of the samples as subtype C; however six inter-subtype recombinants (AE, A1G, A1CU and two BC) and 9 intra-subtype C recombinants were identified. In addition 13% of pol sequences were identified with resistance mutations. Signature pattern analysis identified a high level of variability in the tat sequences: 68% were identified with C30S31; 29% with the C30C31 mutation and a single sequence with a novel mutation C30A31. Functional analysis of these mutations indicated that all mutations investigated were capable of inducing apoptosis in cell culture. The C30C31 mutation generated the highest level of apoptosis, closely followed by the C30A31 mutation. However no statistical significance could be detected between tat mutations and the observed levels of apoptosis.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations. / AFRIKAANSE OPSOMMING: Agtergrond: Die virus protein R (Vpr) van Menslike Immuungebrek Virus tipe 1 (MIV-1) is ‘n bykomstige protein wat met ‘n aantal sellulêre proteine van die gasheer en ander virus proteine in wisselwerking tree. Vpr het 'n invloed op verskeie funksies onder andere die induksie van apoptose, die induksie van selsiklus G2 staking, modulering van geen uitdrukking en onderdrukking van immuun aktivering. Die funksionaliteit van subtipe C Vpr, en veral die van Suid-Afrikaanse stamme, is nie beskryf nie. Die doelwit van die studie was om die diversiteit van Suid Afrikaanse MIV-1 subtipe C vpr gene te beskryf en ook om selektiewe funksies van die Vpr proteine te ondersoek Metodiek: Die MIV-1 vpr streek van 58 stamme is vermeerder, die DNA volgordes is bepaal en die stamme is gesubtipeer deur filogenetiese analise. Fragmente met natuurlike mutasies is in ekspressie vektore gekloon. ‘n Konsensus subtipe C Vpr geen is ontwerp en mutasies in posisies waar geen natuurlike mutasies beskryf is nie, is ontwerp deur mutagenese. Die funksionaliteit van die konstrukte is met die wilde tipe subtype B vergelyk deur 293T sellyn te transfekteer en te ondersoek vir subsellulêre lokalisering, induksie van apoptose, en G2 selsiklus stilstand. Die modulering van geen uitdrukking in die induksie van apoptose is deur TLDA ondersoek. Resultate: Filogenetiese analise het 54 stamme as HIV-1 subtipe C geklassifiseer en 4 stamme as subtype B. Die Vpr aminosuur volgordes was konstant insluitend die FPRPWL en TYGDTW motiewe, maar die C-terminaal was meer variëerbaar. Deur mutagenese is die volgende mutasies ontwerp: P14I, W18C, Y47N, Q65H and Q88S. Subtipe B en al die natuurlike mutante van subtipe C het in die selkern gelokaliseer, maar die W18C mutasie het die lokalisasie versteur. Die G2 selsiklus stilstand van alle mutante en konsensus C was laer as die van subtype B. Al die natuurlike subtipe C mutante het G2 selsiklus tot stilstand gebring in 54.0-66.3% van die selle, terwyl subtype B selsiklus tot stilstand gebring het in 71.5% van die selle. Subtipe B en die natuurlike Vpr mutante het apoptose op ‘n soortgelyke wyse geinduseer, wat wissel tussen 95.3-98.6% van getransfekteerde selle. Die protein met die kunsmatig ontwerpte konsensus C volgorde het egter ‘n verlaagde vermoë gehad om apoptose te induseer. Die konsensus subtipe C het apoptose in 82.0% van getransfekteerde selle geinduseer en die kunsmatige mutante in 88.4 – 96.2% van die selle. Die induksie van die apoptose verwante geen ekspressie deur die mutante was soortgelyk as die van konsensus C en subtipe B Vpr wat ’n aangeduiding is dat apoptose effektief veroorsaak is deur die intrinsieke roete. Gevolgtrekking: Hierdie studie het aangetoon dat kern lokalisering en apoptose op ‘n soortgelyke wyse by beide MIV-1 subtipe B en C Vpr plaasvind. Die induksie van selsiklus G2 stilstand deur MIV-1 subtipe B Vpr is egter meer robuust as baie van die subtipe C Vpr proteïene. Natuurlike mutasies in MIV-1 Vpr kan nie as gebrekkig beskou word nie, aangesien beter funksionaliteit 'n aanduiding is vandie aanpasbare rol. Die verhoogde krag van die gemuteerde Vpr proteïen dui daarop dat Vpr die patogenisiteit van MIV-1 kan verbeter deur die aanpassing van mutasies.