Doctoral Degrees (Medical Virology)
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- ItemIdentification and characterisation of paramyxoviruses in species-rich small mammals from South Africa(Stellenbosch : Stellenbosch University, 2022-12) Kleinhans, Bronwyn; Preiser, Wolfgang; Ithete, Ndapewa Laudika; Drexler, Jan Felix; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: Paramyxoviruses are negative-sense RNA viruses and include a substantial collection of ubiquitous viruses, comprising one of the most important viral groups with numerous pathogens historically impacting on public and veterinary health. The Paramyxoviridae family consists of four subfamilies including Avulavirinae (genera: Metaavulavirus, Orthoavulavirus and Paraavulavirus), Metaparamyxovirinae (genus: Synodonvirus), Orthoparamyxovirinae (genera: Aquaparamyxovirus, Ferlavirus, Henipavirus, Jeilongvirus, Morbillivirus, Narmovirus, Respirovirus and Salemvirus) and Rubulavirinae (genera: Orthorubulavirus and Pararubulavirus). Over the past three decades, an increasing number of novel paramyxoviruses of especially the Orthoparamyxovirinae and Rubulavirinae subfamilies have emerged from small mammal reservoir hosts, some of which, especially the deadly henipaviruses, Hendra- and Nipah virus, have demonstrated a propensity to spillover from their natural reservoir hosts into human and domestic animal populations. Although some small mammals have been implicated as potential hosts for novel paramyxoviruses within southern Africa, little to no data exists on such discovery in insectivorous bats, rodents and particularly shrews and sengis in South Africa. This study identified an additional 23 previously unimplicated species representing four different orders (Chiroptera, Eulipotyphla, Macroscelidea and Rodentia), greatly expanding on the known host and geographic range of these putative paramyxoviruses, reiterating this group of virus’ ubiquitous nature and high diversity. The presumptive paramyxoviruses discovered in this study demonstrated phylogenetic relatedness to at least five of the known genera including: Henipavirus, Jeilongvirus, Morbillivirus, Narmovirus and Rubulavirus as well as to previously discovered viral sequences clustering within the Orthoparamyxovirinae subfamily but that could not be assigned to any of the currently known genera. Unique to this study and of particular value in the South African context was the discovery that: Cape horseshoe bats (Rhinolophus capensis) harbour different variants / strains of a paramyxovirus displaying multiple nonsynonymous mutations resulting in amino acid changes, raising concerns over these putative paramyxoviruses’ zoonotic potential; the widely distributed and populous Rhabdomys (R. bechuanae, R. dilectus, R. intermedius and R. pumilio), endemic to southern Africa, harbour diverse and abundant (overall prevalence of 19.41% [106/546]) paramyxoviruses, displaying diversity on the individual, species and population levels; at least four different shrew species commonly found in South Africa were implicated as hosts for putative henipaviruses; the sustained presence of one of these novel viruses in greater red musk shrews (Crocidura flavescens) implicated this particular species as the reservoir host thereof, of which the near full-length genome sequence further revealed a close phylogenetic relationship to the rat-borne henipavirus, Mòjiāng virus. Given the abundance and diversity of novel potential paramyxoviruses discovered herein and the implication of a multitude of previously unimplicated species as potential reservoir hosts, this study reaffirms the importance and need for ongoing surveillance efforts of especially small mammals in South Africa. This study further highlights the importance of not only the identification of novel paramyxoviruses but also their characterisation, especially those demonstrating close relation to pathogenic members within the family. Further investigation into host-pathogen dynamics at the wildlife – domestic animal – human interface is however warranted to establish the potential for these viruses’ ability to spillover into humans and/or their domestic animals.
- ItemAnalysis of HIV-1 diversity and inflammatory markers in HIV-associated neurocognitive disorders (HAND).(Stellenbosch : Stellenbosch University, 2022-04) Ruhanya, Vurayai; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH SUMMARY: HIV-associated neurocognitive disorders (HAND), which involve impairment or disruption of neurocognitive functioning have become one of the most frequent complications in adult HIV-1 infections with global estimates ranging from 42% to 45%. The screening and diagnosis for HAND relies on multiple clinical and neuro-psychometric methods. However, these methods have a low reliability because they are not precise as most of possess inadequate psychometric properties and diagnostic accuracy. Therefore, this study aimed to describe and characterise viral and immunological determinants of HAND and evaluated their relationship with specific clinical, neuromedical and neuropsychological data to identify putative easy-to-measure biological markers for diagnosis of the condition. This study demonstrated that higher peripheral blood lymphocyte-derived HIV-1 proviral DNA is a predictor of global and domain-specific neurocognitive impairment among individuals infected with HIV-1 subtype C. The study also determined proviral load cut-off /threshold value for neurocognitive impairment and associated diagnostic accuracy. It also identified IP-10 and RANTES as a plasma chemokine bio-signature for HIV-associated neurocognitive impairment with diagnostic accuracy comparable to standard psychometric tests used to screen and describe severity of HAND. In addition, the study identified 3 viral genetic signatures for cognitive impairment, namely Lysine at codon 24, (24K) and Arginine at codon 29 (29R) on Tat protein and Tyrosine (Y) at position 45 (45Y) of Vpr. These three signature amino acids were related to classical markers for monitoring HIV infection. Finally, we identified 4 conserved fragment sequences, PEDQGPQREPYNEWTLE (5 to 21), LGQYIY (42 to 47), TYGDTW (49 to 54), PEDQGPQREPYNEW (5 to 18) on viral protein R, that were associated with higher plasma viral load and proviral load. The study has identified novel cytokine/chemokine and viral biomarker signatures for HIV associated neurocognitive impairment with low to moderate diagnostic accuracy. The findings demonstrated a need for interdisciplinary approach to elucidate possible molecular interactions between the peripheral blood immune markers, viral signatures and the CNS that are linked to observed clinical outcomes of neurodegradation in HIV infection. The identified biomarkers can be further investigated for use as screening tools and treatment endpoints for HAND.
- ItemA Longitudinal Perspective on the Impact of Immune Status on the HIV-1 Latent Reservoir and Neurocognitive Outcomes in Virologically Suppressed Children(Stellenbosch : Stellenbosch University, 2022-04) Naidoo, Shalena; Glashoff, Richard; De Beer, Corena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH SUMMARY: Background: Children remain the most vulnerable population affected by the Human Immunodeficiency Virus-1 (HIV-1) pandemic whose reliance on lifelong therapy is accompanied by several immune abnormalities. In the absence of an effective vaccine, there is currently a strong emphasis on HIV-1 “cure” and although cART leads to viral suppression the virus is still able to establish latent reservoirs within host cells and this is considered the major barrier to achieving cure. Immune factors are considered critically important for the establishment and maintenance of HIV-1 latent reservoirs, but these factors are not well characterised, particularly in children. Delineating and understanding the immunological mechanisms that drive HIV-1 persistence and other chronic diseases such as metabolic, cardiovascular and neurodegenerative diseases is important in children approaching adolescence. Longitudinal studies evaluating the relationship between immune status, the HIV-1 latent reservoir and neurocognitive outcomes are limited, and inadequately studied, specifically within the South African subtype C context. The aim of this study was to longitudinally investigate and characterise host immune status before and after early initiated, delayed and interrupted cART in relation to HIV-1 latent reservoir size and neurocognitive outcomes in perinatally HIV-1 infected children. Methods: Study participants originated from the well-characterised Children with HIV Early AntiRetroviral Therapy (CHER) randomised controlled trial. This was a descriptive study that employed both a longitudinal (birth to 8 years of age) and cross-sectional study design and analysed samples collected retrospectively and prospectively. For the extensive longitudinal evaluation of immunological biomarker (cytokine, chemokine and receptor antagonist) profiling, we utilised Luminex® Multiplex Assays as well as Enzyme Linked ImmunoSorbent Assays (ELISAs). Multiparameter flow cytometry was utilised for the analysis of monocyte subset distribution. The quantitative viral outgrowth assay (QVOA) was implemented on a subset of participants for measuring of the HIV-1 replication-competent viral reservoir in conjunction with a novel highly sensitive RT-qPCR single copy assay targeted at HIV-1 integrase (iSCA). In addition, HIV-1 cell-associated DNA, specific for integrase (iCAD), were measured longitudinally as a molecular biomarker of HIV-1 persistence and correlated to immune and neurocognitive parameters. Longitudinal neurocognitive assessments were completed independently and correlated to immune, virological and clinical parameters. Age and demographically matched HIV exposed uninfected (HEU) and HIV unexposed uninfected (HUU) were included at the last time point (8 years of age). Key Findings: HIV-1 infected (HIV+) children showed significantly higher levels of biomarkers associated with generalised chronic inflammation, particularly those associated with myeloid cell activation compared to HEU and HUU controls at 8 years of age. These included hsCRP (p=0.01), MIP-1β (p=0.03), IL-1α (p<0.001), INF-α (p<0.001), CD40L (p<0.001), sCD14, sCD163, IL-18, IL-17F (p<0.001) and PDGF-BB (p<0.00001). We also observed a significant elevation of soluble calcium binding alarmin protein involved in the regulation of the inflammatory process and immune response, s100A8/A9, in the HIV+ group compared to the two study control groups (p<0.001). Within the HIV+ group, significant elevation of biomarkers associated with gut epithelial damage was observed at 8 years of age. IL-18 was the only immune biomarker that significantly correlated with HIV-1 CAD at baseline (r = +0.35; p=0.04) and at the 8-year follow-up (r = +0.38; p=0.02) and associated to the innate IL-1 family of immune biomarkers including IL-1RA (r = +0.33; p<0.01), IL-1α (r = +0.22; p<0.01), IL-1β (r = +0.26; p<0.01), and IL-1RA (r = +0.32; p<0.01). IL-18 also significantly correlated with biomarkers of monocyte/macrophage activation and gut damage, including: sCD14 (r = +0.50; p<0.01), sCD163 (r = +0.40; p<0.01), LBP (r = +0.26; p<0.01) and MIP-1β (r = +0.19; p=0.02). IL-18 showed significant negative associations (p<0.01) with CD4 % at baseline, longitudinal CD4 count and CD4:CD8 ratio at 8 years (r = +0.35; p=0.04). For longitudinal cytokine analysis, principal component analysis indicated that about 36% of the soluble biomarkers measured including: IL-15, TNFβ, GCSF, IL-1RA, IL-5, IL-4, IL-8, IL-10 and RANTES contributed to the largest parameter change over time across all treatment groups. The key early clinical predictors of plasma biomarker expression over time include time to therapy initiation, time to viral suppression, longitudinal CD4% and absolute count and CD4 and CD8% and absolute counts at birth. Neurocognitive outcomes can be predicted by early immunological, virological and clinical parameters. Conclusion: By investigating and profiling participants from the CHER randomised control trial cohort, we were able to provide important insights into immunological mechanisms that contribute to driving HIV-1 persistence during long-term suppressive therapy. The findings reported in this thesis have highlighted some key features of immune abnormalities that persists after early initiated long-term ART in paediatric populations. The evaluation of the persistence of any of the key associations through and beyond adolescence will aid in building a lifelong profile of immune changes in the MTCT-infected children. This research also provides important knowledge to further exploring PHIV children as potential “cure” and remission candidates.
- ItemSeroprevalence and incidence of Toxoplasma gondii, rubella and cytomegalovirus among Namibian women of childbearing potential(Stellenbosch : Stellenbosch University, 2020-12) Van der Colf, Berta Elizabeth; Van Zyl, Gert U.; Reju, Sunday A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.Introduction: Data on the prevalence or incidence of congenital infections in Namibia are limited. Therefore, this study aimed to determine the prevalence and model the incidence of three important vertically transmitted infectious diseases: Toxoplasma gondii (T. gondii), rubella and cytomegalovirus (CMV) infections in women of childbearing potential in Namibia. Methodology: Three hundred and forty-four consenting women attending public antenatal care in Windhoek were included in the study. Clotted blood was collected, and a questionnaire included demographic data, immunization and obstetric history data as well as information on the exposure to risk factors. Seroprevalences of IgG against T. gondii, rubella and CMV and specific IgM antibodies against CMV were determined. T. gondii IgM and T. gondii and CMV IgG avidity were determined with ELISA. Statistics: Fisher’s exact test was used for categorical associations and Kruskal Wallis test for continuous variables. Results: Anti-T. gondii IgG was found in 9 (2.61%) pregnant women. There was no association of anti-T. gondii IgG with demographic characteristics or exposure to risk factors. Anti-T. gondii IgM was positive in 1 (0.3 %) woman while 3 (0.9 %) women had borderline positive results. Specific IgG avidity was equivocal and high in 33% and 67% of seropositive women. Seroprevalence of rubella did not increase with age and the overall seroprevalence of specific IgG was 95.9%. The majority of the participants had never been vaccinated against rubella infection. The percentage of women with IgG levels of <10 IU/ml, 10-14.9 IU/ml and > 15 IU/ml were 2.0%, 2.0% and 95.9% respectively. An overall anti-rubella IgG mean level of 164.5 IU/ml (95% CI 150.4-178.7) was found in five age groups, namely 15-20 (195.8; 159-232); 21-25 (167.8; 143-193); 26-30 (165.2; 136-195); 31-35 (147.6; 116-179) and 36-47 (150.4; 107-194). Demographic factors like maternal age, gestational age and immunization history did not show significant associations with anti-rubella IgG levels. Seroprevalence of anti-CMV IgG among pregnant women was 100%. Eleven participants (3.2%) had a positive or equivocal anti-CMV IgM result. Specific IgG avidity was high in all of these cases. Neither maternal age nor gestational age was associated with a positive or equivocal zone IgM result. Incidences of infections could not be modelled due to either a very high or very low prevalence across age ranges. Conclusion: Seroprevalence of anti-T. gondii IgG is much lower in central urban Namibia than in other developing countries. Investigation into specific IgM seropositivity and IgG avidity showed that pregnant women in the central region of Namibia are at low risk of vertical transmission and development of congenital toxoplasmosis. A high percentage of pregnant women in the study were immune to rubella virus despite no history of vaccination. This is likely due to a high rate of natural infection with rubella of children before reaching child-bearing potential. This was the first study to investigate seroprevalence of CMV in Namibia. The high seroprevalence of CMV suggests a risk of reinfection or reactivation rather than primary CMV infection in pregnancy. Further studies are needed to determine the prevalence of congenital CMV in Namibia.
- ItemThe role of clonally expanded HIV-1 infected cells in maintaining HIV reservoirs in adults and children on antiretroviral treatment(2020-12) Botha, Johannes Christiaan; van Zyl, Gert U; Engelbrecht, Susan; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology. Medical Virology.ENGLISH ABSTRACT: The HIV-1 pandemic remains a major public health dilemma. Treatment with combination antiretroviral therapy (cART) can, in the majority of patients, sufficiently suppress HIV viral replication. However, cART is not a cure as it does not affect long-lived reservoirs. HIV proviruses are harboured in the genome of long-lived CD4 helper cells that are maintained by having long half-lives and by being replenished through cellular proliferation. HIV infected cells that proliferate can be identified as clonal populations by all having the same integration site. A small proportion of proviruses are intact from where HIV rebounds after therapy interruption and is the barrier to achieving cure or remission. Therefore, many molecular assays have been designed to characterise HIV proviruses, with a focus on intact proviruses. From this the proviral landscape is estimated to consist of ~2% intact proviruses and up to 98% defective proviruses comprising deletions and hypermutations. However, highly deleted proviruses such as solo-long terminal repeats (LTRs) remain largely uncharacterised. Recently, it has become apparent that some proviral clones are able to express infectious HIV and cause clonal viraemia in patients on cART, which are not indicative of ongoing cycles of viral replication. This has further confirmed the importance of proviral clones in maintaining the HIV reservoir. Subsequently, aspects of proviral clones were investigated in this study. A novel assay capable of characterising severely deleted proviruses by targeting the unique integration sites was developed. The characterisation of proviral clones in paediatric patients with this novel assay revealed that severely deleted solo-LTR proviruses may be more prevalent than previously expected. Further investigation of these solo-LTR proviral clones was performed with another novel assay capable of quantifying proviruses based on unique integration sites. The longitudinal waxing and waning of solo-LTR proviral clones could be observed. Although solo-LTR proviruses do not contribute to the true reservoir, it suggests that current proviral landscape proportions require adjustment to account for all HIV integration events in cells. Clonal viraemia was studied in HIV positive adult patients on long term cART presenting with non-suppressible HIV viraemia. Three clusters of monotypic plasma viraemia and cell associated DNA HIV sequences were identified in one patient. These monotypic proviruses were shown to be replication competent by viral outgrowth. This provides evidence that clonally proliferated cells harbour at least a proportion of intact proviruses and therefore constitute an important component of the true HIV reservoir. Two additional patients were identified with uncommon cases of sustained viraemia. Firstly, a probable large cell clone harbouring non-infectious virus was found to leak proviral nucleic acid into plasma, resulting in apparent treatment failure. Secondly, possible clonal viremia was observed in a patient with very slow decaying viral load, despite objective evidence of adherence and initial undetectability of treatment-relevant drug resistance over a period of 2 years. We conclude that clonal viraemia may be underreported, and that cases like these show that clonal viraemia should be considered as an explanation of non-suppressed viraemia or apparent therapy failure in the management of HIV infected patients on cART.