Doctoral Degrees (Medical Microbiology)
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Item Open Access Adaptive immune response in COVID-19 patients and innate immune modulation of SARS-CoV-2(University of the Free State, 2023) Litabe, Matefo Millicent; Burt, FelicityIn December 2019, a cluster of cases of atypical pneumonia were reported in Wuhan, China. All patients had a history of attending a large seafood market. Surveillance and detection methods established during the 2003 severe acute respiratory syndrome coronavirus (SARS-CoV)-1 outbreak contributed towards the identification of the virus as a novel coronavirus (CoV). The virus was later named SARS-CoV-2 and identified as the causative agent of Coronavirus Disease 2019 (COVID-19). Despite massive attempts to contain the virus in China, the cases rapidly increased and spread globally, and the World Health Organization (WHO) declared COVID-19 a Public Health Emergency of International Concern on 30 January 2020 and characterized the outbreak as a pandemic on 11 March 2020. Assessing immunoglobulin (Ig)-G and neutralizing antibodies is essential to comprehensively evaluate the efficacy and duration of immunity conferred by natural infection and the COVID-19 vaccines. Effectively determining SARS-CoV-2 seroprevalence within a population is important as it helps to improve our understanding of virus circulation dynamics, identify individuals at risk of infection, and the extent of virus exposure in the community. Therefore, this study investigated the duration and kinetics of adaptive immunity, particularly the persistence of IgG and neutralizing antibodies, in patients who recovered from SARS-CoV-2 in the Free State, South Africa. Commercial assays are expensive, and hence two anti-spike (S) inhouse assays, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), were developed and validated for detection of anti-S IgG. A total of 89 serum samples were collected from COVID-19 PCR-confirmed patients between 2-94 days post-symptom onset to validate the assay. A 100 prepandemic samples were used as a negative control panel to determine the cutoff of the assay. A cutoff value of 30% was considered accurate to differentiate between negative and positive samples using a two-graph receiver operating characteristic (TG-ROC). The assays exhibited a sensitivity of 100% for ELISA and 98.8% for IFA when testing samples collected more than one week after the onset of illness. The positive predictive values (ppv) were 92.1% for ELISA and 91.0% for IFA on PCR-confirmed positive samples. The assays were also compared to a commercially available SAPRHA-approved assay, where the ELISA showed a higher ppv of 95.8 %, while the Roche assay was 89.6 %, and the commercial lateral flow was 93.9 %. The two in-house assays also detected IgG antibodies in samples collected from waves in which new variants were circulating, showing that despite mutations of the SARS-CoV-2 S protein, the assay was still sensitive to detect IgG antibodies in circulating variants. This indicates that these assays could be used for surveillance of the South African population. To investigate the duration of anti-S IgG and neutralizing antibodies against SARS-CoV-2, 100 individuals with previously confirmed COVID-19 infection were recruited for the study. The cohort included 64/100 vaccinated and 36/100 unvaccinated individuals. Initial samples were collected between March 2021 and January 2022, with confirmed infection between June 2020 and December 2021. Follow-up samples were collected from 82/100 in 2022 and 62/100 in 2023 of the initial cohort. Samples were tested for anti-S IgG antibodies and neutralizing antibodies against Ancestral strain, Delta, and Omicron variants. A total of 95/100 baseline samples tested positive for anti-S IgG and 79/82 and 53/62 for follow-up samples. A total of 99/100, 78/100, and 72/100 baseline samples tested positive for neutralizing antibodies against the Ancestral strain, Delta, and Omicron variants. A total of 80/82, 63/82, 77/82 in 2022 and 53/62, 50/62, 56/62 in 2023 tested positive for neutralizing antibodies against the Ancestral, Delta, and Omicron variants, respectively. Samples were grouped based on the time (days) the samples were collected post-onset of illness. Results showed that IgG antibodies were significantly higher directly after infection, between 1-180 days, then gradually declined significantly with time. Neutralizing antibody titers against the Ancestral strain and Delta variant were significantly higher early after infection, between 1-180 days, remained relatively stable for an extended period, and then waned gradually before declining significantly. In contrast, although not significant, neutralizing antibodies against the Omicron variant increased with time, but this may be because samples were collected when the Omicron variant was still prevalent. Results also showed that vaccinated individuals had significantly higher antibody titers than unvaccinated, highlighting the importance of continued vaccination. The study shows the longevity of antibodies against SARS-CoV-2, as most individuals still had detectable titers at least 24 months post-acute infection, possibly boosted by vaccination or reinfection. The study also shows that the dynamics of antibodies vary among individuals; most individuals display declining antibody titers with time, and a smaller proportion maintain stable antibodies or a fluctuation of antibodies over time. Traditional medicinal plants have been proposed as promising, cost-effective treatments for SARS-CoV-2, with studies showing the potential to induce protection against different viral infections. The study also investigated the potential of Phela, a traditional medicine prepared from the extracts of four South African plants, and the individual components to modulate the release of cytokines in SARS-CoV-2 Omicron-infected mammalian cells and to investigate the influence of the plant extracts on viral replication. Cells were treated with the plant extracts before or after infection with SARS-CoV-2. Subsequently, cell culture media was collected at 12, 24, 48, and 72 hours post-infection and tested for virus replication and levels of Interleukin (IL)-1β, IL-2Rα, IL-6, IL-10, tumour necrosis factor (TNF)-α, and interferon (IFN)-γ cytokines. There was no statistically significant difference in viral load between infected cells treated with plant extracts compared to infected and untreated cells, showing that the plant extracts may have little to no effect on virus replication. Treatment with plant extracts resulted in significantly lower release of IL-1β, IL-2Rα, and TNF-α, with better response post-treatment than pretreatment, showing that the plant extracts may have the potential to manage cytokine storms and be a potential source of treatment for SARS-CoV-2.Item Open Access Investigating the role of viroplasm formation and calcium levels on the production of prostaglandin E₂ during rotavirus infection(University of the Free State, 2022) Sander, Willem Jacobus; O'Neill, H. G.; Pohl-Albertyn, C. H.Both in vitro and in vivo studies have shown that levels of prostaglandin E₂ (PGE₂), an immunomodulatory eicosanoid, are increased during rotavirus (RV) infection. Although it has been shown that inhibition of cyclooxygenase (COX) (PGE₂ biomes) has adverse effects on viral yield, the mechanism of PGE₂ induction during replication remains unknown. Viroplasms are viral factories that consist of several viral proteins, in particular NSP2 and NSP5, and cellular lipid droplets. Lipid droplets (LDs), with their high content of neutral lipids and the proximity of PGE₂ biosynthetic enzymes, are well known sites for PGE₂ biosynthesis. In addition, during replication, RV has been shown to increase the total lipid content of infected cells, while modulating specific lipid classes during infection. Inhibitors that prevent the formations of LDs severely limit the amount of viroplasms formed and subsequently decrease viral progeny production. Another viral protein critical for viral replication is the enterotoxin, NSP4. NSP4, contains a viroporin domain that selectively conducts calcium (Ca²⁺) from the endoplasmic reticulum to the cytosol, increasing free intracellular Ca²⁺. Intracellular Ca²⁺ levels are crucial for the activation and function of cytoplasmic phospholipase A₂, the rate-limiting enzyme in PGE₂ biosynthesis. Both LDs and phospholipase A₂ are also essential for PGE₂ biosynthesis. Therefore, the main objective of the study was to determine when and by which mechanism(s) RV induces/amplifies the production of prostaglandin E₂. During early infection, RV attaches to several cellular receptors and enters the cells by either clathrin-dependent or -independent endocytosis. Other viruses such as bovine ephemeral fever virus haven been shown to require the activation COX-2-mediated PGE₂/EP receptor signalling for enhanced clathrin-mediated endocytosis. To determine if PGE₂ exerts its proviral effects during internalisation we supplemented MA104 cells with γ-linolenic acid (GLA), a precursor of arachidonic acid. Infection of supplemented cells with RV SA11 led to increased production of PGE₂ as monitored by ELISA. Confocal microscopy demonstrated that PGE₂ co-localises with the viroplasm-forming proteins, NSP5 and NSP2. Due to the known association of viroplasms as well as PGE₂ with lipid droplets, our results indicate a possible role for viroplasms in the production of RV-induced PGE₂. Replication kinetics showed that inhibitors, targeting the biosynthesis of PGE₂, had negative effects on RV yield, especially during the early stages of infection. Using flow cytometry and PGE₂ addback experiments, we show that PGE₂ enhances the attachment and internalisation of rotavirus in MA104 cells, indicating a possible role for PGE₂ during clathrin-mediated RV entry. Due to the well-known association between viroplasms and LDs, and the fact that LDs are production centres for the PGE₂, we next explored the possible role if any, of viroplasm components in the induction of PGE₂ production during RV infection. Transfection of HEK293 cells with plasmids, encoding the ORFs for NSP2 and NSP5 or both NSP2 and NSP5, showed that neither protein on their own, nor the formation of viroplasm-like structures was able to induce PGE₂ production. A MA104 cell line, stably expressing NSP5, was used to generate and characterize several SA11-based rescued rotaviruses (rSA11_aNSP5 and rSA11_aNSP5) with mutations in the α-helix within the C-terminal of NSP5. We demonstrate that a rSA11 with replaced hydrophobic amino acids in the C-terminal appeared to from less viroplasms compared to rSA11. This led to reduced replication of both rSA11_aNSP5 and rSA11_pNSP5, confirming the pivotal role of the α-helix within the C-terminal during RV replication. These mutations also affected the production of PGE₂ in HEK293 infected cells, although this is more likely due to decreased viral replication. Furthermore, we investigated how the introduced mutations affect NSP5 co-sedimentation with LD-associated protein, perilipin 2, and showed that the NSP5 mutations decreasing the hydrophobicity or abolishment of the α-helix changed the sedimentation profile. Our results indicate that individual viroplasm components or the formation of VLS do not induce PGE₂ in transfected cells, but that mutations in the C-terminal of NSP5 decrease PGE₂, most probably due to decreased replication. After showing that viroplasms mainly indirectly induce the production of PGE₂, we switched our focus to the role of Calcium (Ca²⁺) as it is essential for several cellular signalling and physiological processes, including the activation of cytoplasmic phospholipase A₂ (cPLA₂). This enzyme plays a role in lipid droplet (LD) biogenesis and is the rate-limiting enzyme in prostaglandin E₂ (PGE₂) biosynthesis. During rotavirus (RV) replication, NSP4 modulates the levels of cytoplasmic Ca²⁺ (cyto[Ca²⁺]) by a viroporin domain, which selectively conducts Ca²⁺ from endoplasmic reticulum (ER) stores to the cytoplasm. This modulation of cyto[Ca²⁺] is crucial for several viral process, including entry and assembly. We, therefore, investigated the role of the viroporin domain of NSP4 in the induction of PGE₂ production during RV infection. We show that RV infection of HEK293 cells increases the activity of cPLA₂, and that the chelation of cyto[Ca²⁺] decreases the activity of cPLA₂, leading to decreases in viral progeny and RNA yield. Mutations within the viroporin domain, which decreases the conductively of Ca²⁺, decreased the activity of cPLA₂ and subsequently affected PGE₂ levels as well as viral progeny and RNA yield. Our results indicate that the viroporin domain of NSP4 plays a role in the induction of PGE₂ production by increasing the activity of cPLA₂ in a Ca²⁺-dependent manner. Taken together, the data shows that PGE₂ is most likely induced in a NSP4-cyto[Ca²⁺]-cPLA₂-dependent manner enhancing RV internalisation. This enhanced internalisation increases viral yield, which could contribute indirectly to PGE₂ production by increasing the numbers of LDs and thus sites of PGE₂ production.Item Open Access Drug resistance mutations in newly diagnosed HIV-infected infants and in children and adolescents with virological failure on ART(University of the Free State, 2020-12) Barakzai, Iqra; Goedhals, DominiqueIntroduction and aim: South Africa has never experienced an epidemic of the same extent and magnitude as the one with human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS).The roll out of antiretroviral therapy (ART) and other interventions, such as prevention of mother to child transmission (PMTCT), has greatly reduced the incidence of new HIV infections among children. However, early initiation of ART among children has increased their overall exposure to antiretrovirals (ARVs), resulting in an increased prevalence of acquired drug resistance (ADR) on treatment. Presence of drug resistant virus further complicates clinical management of HIV infection, resulting in fewer present and future treatment options. Therefore, drug resistance testing has been recommended in selecting appropriate treatment regimens among children and adolescents diagnosed with virological failure. The aim of the current study was to determine and characterise drug resistance mutations in newly diagnosed HIV-infected children and in children and adolescents with virological failure on ART. Methods: To detect drug resistance mutations, the present study developed and validated a Sanger sequencing assay using dried blood spot (DBS) samples. The Sanger assay was used to perform HIV drug resistance testing on DBS samples testing positive on the early infant diagnosis (EID) platform. Lastly, a retrospective analysis was performed of resistance data from samples of children and adolescents submitted to the routine diagnostic laboratory. Results: A high level of concordance was found among major mutations detected using the Sanger DBS and plasma assays. Since all the discordant mutations were either minor or existed as mixtures, the interpretation of resistance profiles was found to be identical. The DBS assay was then used to detect pre-treatment drug resistance (PDR) among samples from children on the EID platform. Overall, PDR was detected among 73% of the children, with the majority of the children showing resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) (72%). Dual class resistance was most prevalent to NNRTIs and NRTIs (11% [33/308]), followed by NNRTIs + protease inhibitors (PIs) (0.6% [2/308]). Triple class resistance (NNRTI + NRTI + PI) was very rare and was only detected in two children. Protease resistance was very rare among the children and none of the children displayed high-level resistance against any PI. Results from children (0 to 9 years) and adolescents (10 to 19 years) with virological failure while on ART indicated that most of the children were resistant to NRTIs (86% [79/92]), followed by NNRTIs (75% [69/92]) and PIs (28% [26/92]). Among adolescents, prevalence of resistance to NNRTIs (87% [146/167]) was the highest, followed by NRTIs (66% [111/167]) and PIs (30% [51/167]). Dual class resistance to NRTIs + NNRTIs was the most common among both children (44% [41/92]) and adolescents (38%, [63/167]). Triple class resistance (NNRTI+NRTI+PI) was detected in 18% of children (17/92) and 19% of adolescents (32/167). Conclusion: The prevalence of infants, children and adolescents experiencing treatment failure due to the presence of transmitted, acquired and PDR is increasing. While the implementation of virological monitoring and genotypic testing play an essential role in identifying and managing drug resistance among children and adolescents experiencing treatment failure, these services are limited in many developing countries. The current study supports the implementation of HIV drug resistance testing for national surveys and optimal clinical management of HIV-infected children and adolescents.Item Open Access Evaluation of constructed recombinant mengoviruses and other HIV vaccine candidates in murine and primate models(University of the Free State, 2001-03) Van der Ryst, Elna; Smith, M. S.; Borman, A. M.; Botha, P. L.The development of an effective vaccine against HIV is a formidable challenge. The overall objective of this work was to evaluate different HIV -1 vaccine approaches in primate and murine models. In a first approach recombinant Mengoviruses expressing several HIV -1 and SIV gene products were evaluated for their immunogenicity in mice and macaques. Results indicated that Mengovirus recombinants expressing HIV -1 Nef or SIV CTL epitopes are weak immunogens. This was disappointing in light of the promising results previously obtained using other Mengovirus recombinants and indicated that the nature of the insert might play an important role in the immunogenicity of Mengovirus recombinants. As a second approach, protection of chimpanzees from intravenous and vaginal challenge by immunisation with a recombinant canarypox virus expressing the HIV-ll1lB/LAI gp 120rrM, gag and protease genes was evaluated. In animals challenged by the iv route protection from homologous challenge was seen in one of two animals and this correlated with the neutralising antibody levels. One of five females resisted a total of 3 vaginal challenges, while two further animals resisted 2 challenges. However, only low levels of HIV-l-specific neutralising antibodies were present at time of challenge. This suggests that neutralising antibodies may have little importance for protection from mucosal infection in chimpanzees, in contrast with what was seen for iv challenge. Finally, macaques were immunised with a primary isolate of HIV -1 in order to evaluate the breadth of the immune response induced by HIV-1 in its "native" state. The animals developed moderate to high titers of total anti-HIV -1 antibodies as measured by EIA, which was mainly Gag directed. However, no antibodies capable of neutral ising HIV -1BX08 were demonstrated, and sera From the animals induced strong facilitation of HIV -1 replication in PBMC, raising the concern that whole virus based HIV vaccines might induce facilitating antibodies that can result in Facilitation of transmission and/or evolution of disease.Item Open Access Immunogenicity and serological applications of flavivirus ED III proteins and multiplex RT-PCR for detecting novel Southern African viruses(University of the Free State, 2015-01) Mathengtheng, Lehlohonolo; Burt, Felicity; University of the Free State, Grow Our Own Timber FellowshipEnglish: West Nile virus (WNV) is endemic to southern Africa but the true burden of disease associated with WNV infection remains unknown in this region. The presence of the mosquito-borne Wesselsbron virus (WESSV) has also been established in southern Africa. Although not considered a serious human pathogen, WESSV has been associated with encephalitis in humans. No routine testing is performed for WESSV diagnosis in South African patients and hence, similar to WNV infections, the virus remains unreported and overlooked. The presence of tick-borne flaviviruses in southern Africa on the other hand, has not been established despite the presence of suitable vectors. A challenge associated with serological identification of flaviruses is the high level of cross-reactivity between members of flaviviruses and the impracticality of using neutralization assays. Serological assays using reagents that can be handled in a biosafety level 2, or lower facility, were developed and evaluated for the detection and differentiation of tick- and mosquito-borne flaviviruses in the Free State province of South Africa. A total of 2393 serum samples from a variety of species including humans, cattle and sheep were tested using Kunjin virus (KUNV) cell lysate antigen for the detection of anti-flavivirus antibodies in an indirect IgG enzyme-linked immonosorbent assay (ELISA). To further differentiate positive reactors on KUNV assay for antibodies against tick- or mosquito-borne flaviviruses, recombinant envelope domain III (r-EDIII) proteins of Langat virus (LGTV), WNV and WESSV were expressed in a bacterial expression system and used in ELISA. A total of 722 samples were positive on the KUNV assay of which 71, 457 and 431 were positive on the r-LGTVEDIII, r-WNVEDIII and r-WESSVEDIII assays, respectively. A total of 70 samples were reactive on the KUNV assay but not on any of the other assays, suggesting that there are other flaviviruses circulating in the Free State province for which specific r-EDIII assays were not available. Collectively, the results suggest a strong presence of flaviviruses co-circulating in the Free State province with an abundance of mosquito-borne flaviviruses. There is evidence suggesting the presence of tick-borne flaviviruses but it has yet to be confirmed. The EDIII protein is a useful tool that can be utilized in the detection and differentiation of flaviviruses in resource-limited laboratories. Vertebrate hosts play a role in the maintenance and circulation of flaviviruses and, although not involved in the direct transmission of tick- and mosquito-borne flaviviruses, form a link for virus transmission between vectors. In addition to rodent involvement in maintenance of flaviviruses, rodents have also been implicated in the transmission of other medically significant viruses such as arenaviruses, lyssaviruses and hantaviruses. Arboviruses and viral heamorrhagic fevers are among the most pathogenic and devastating disease agents in many parts of the world. It is therefore important for surveillance of such pathogens to be conducted as they may result in considerable public health implications. Molecular assays were developed for the detection of a selected number of arboviruses and viral heamorrhagic fevers, specifically Crimean-Congo haemorrhgaic fever virus (CCHFV), mosquito-borne and tick-borne flaviviruses, as well as hantaviruses. To date, the presence of hantaviruses have not been confirmed in southern Africa despite their emergence in the western and eastern parts of Africa in recent years. In our study, serum samples of patients presenting with a tick-bite and febrile illness without diagnosis were screened for hantavirus IgG antibodies using commercial assays that represent the American and Eurasian hantavirus species. The overall seropositivity rate obtained was 10% and 6% for assays representing the Eurasia and America hantavirus species, respectively. The emergence of hantaviruses in Africa and their seroprevalence in the Cape region of South Africa as well as in our study warranted the development of a molecular assay to further investigate the presence of these viruses in southern Africa. In order to achieve this, a real-time RT-PCR was designed and optimized. The assay was designed by identifying in-house primers targeting the partial region of the S-segment of hantaviruses and hydrolysis probes targeting the inner region of the amplicon. The probes were based on nucleotide sequences targeting the Murinae-associated hantaviruses for the HNLS probe, Sigmodontinae- and Arvicolinae-associated hantaviruses for the ASPRB probe, as well as the SANGV probe for the African hantavirus Sangassou virus. The flavivirus RT-PCR targeted the NS5 region with a probe shown to successfully detect RNA samples that represent eight different flavivirus species. The hantavirus primers and probes were evaluated using RNA transcribed from synthetic genes representing the different hantaviral genotypes and subsequently reverse transcribed cDNA. The limit of detection was determined to range from ~160 to ~17 copies of DNA for the various hantaviral probes and flavivirus probe. In addition, a conventional multiplex PCR assay aimed at detecting CCHFV and flavivirus RNA in samples collected from undiagnosed patients presenting with a tick-bite and febrile illness was developed by using nested primers targeting the partial region of the genome of the S-segment of CCHFV and hemi-nested primers targeting the partial region of the NS5 gene of flaviviruses. When clinical samples from patients with known tick-bites, mild disease and no diagnosis were screened, a patient was restrospectively diagnosesd as having a CCHFV infection. This result highlights the need for awareness to arboviruses and viral hemorrhagic fevers in mild cases that may easy be overlooked but constitute a significant public health risk. Similarly, there needs to be an increase in awareness for travelers to South Africa at risk of returning to their country with an exotic viral haemorrhagic fever, highlighting the need for increased awareness and increased diagnostic capacity for arboviruses. Finally the current lack of registered human vaccines warrants continued investigation of the immunogenicity of selected viral proteins. The recombinant antigens developed for serological purposes were further employed in this study to determine the immunogenicity of the envelope domain III proteins of WNV and LGTV in a mouse model. Small molecule antigens or weakly immunogenic antigens frequently require an adjuvant to stimulate a stronger immune response. In addition, adjuvants can shift an immune response towards a Th1 or Th2 response as required based on immune correlates of protection. Groups of mice were immunized with purified r-WNVEDIII or r-LGTVEDIII protein alone, r-WNVEDIII or r-LGTVEDIII protein in combination with one of three adjuvants, including saponin, Titermax® gold and Alhydrogel® or one of the three adjuvants without a flavivirus protein. In the absence of any adjuvant the results from WNV protein alone were inconclusive whereas a strong IgG1 response was induced by LGTV EDIII. Briefly, protein alone or mixed with alum elicited a predominantly Th2 response whereas protein in combination with saponin or Titermax® gold induced a mixed Th1 and Th2 response. Mice immunized with r-WNVEDIII reacted against KUNV native antigen indicating that the protein was expressed in conformation exposing epitopes that are required to induce a detectable antibody response. The formulation of the WNV and LGTV proteins with different adjuvants produced similar results with a shift in response depending on the adjuvant. Despite an absence of being able to assess cell mediated responses using antigen stimulated splenocytes and profiling cytokine production as initially planned, the results do confirm that r-WNVEDIII and r-LGTVEDIII proteins are immunogenic in the absence of complete E protein, with ability to induce detactable antibody when formulated with adjuvant and that different adjuvants are able to have an immunomodulatory influence on the type of response induced.Item Open Access Immune responses to Crimean-Congo haemorrhagic fever virus and molecular characterization of viral isolates(University of the Free State, 2014-02) Goedhals, Dominique; Burt, F. J.; Paweska, J.Crimean-Congo haemorrhagic fever virus (CCHFV) is a tick-borne virus belonging to the family Bunyaviridae, genus Nairovirus. The distribution of the virus correlates with that of the principal vector, ticks belonging to the genus Hyalomma. This includes areas in Africa, Asia, Eastern Europe and the Middle East, with recent emergence in Turkey, Greece and India. CCHFV is associated with haemorrhagic fever in humans, with a case fatality rate of up to 30%. Current patient management relies on supportive therapy and administration of ribavirin, but the efficacy of this antiviral drug is controversial. Although an inactivated vaccine has been used in Eastern Europe and the former Soviet Union, it has not been accepted for widespread use. An understanding of immune correlates is therefore needed to guide further development of therapeutic and preventative interventions. This study aimed to investigate immune responses in survivors of CCHF in South Africa, focusing on the presence of detectable memory T lymphocyte responses and the identification of epitopic regions within the nucleoprotein and glycoproteins. In order to ensure applicability of identified epitopes to geographically distinct isolates, viral sequence diversity was also investigated by means of next generation sequencing and phylogenetic studies. A synthetic overlapping peptide library was used to screen for interferon gamma production by peripheral blood mononuclear cells from survivors of CCHFV infection in ELISPOT assays. Ten potential epitopic regions were identified, the majority of which were located on the nucleoprotein with only two regions identified on the glycoprotein GC in a single patient. Long-lived memory CD8+ T cell responses were detected in survivors of CCHF up to 13 years after infection. These findings indicate the presence of effective long term cellular immune responses which could be modulated through vaccination and gives an indication of epitopic regions that should be considered in candidate vaccines and testing vaccine immunogenicity. The presence of detectable memory responses in the absence of reexposure or chronic infection will allow future studies to fully characterize T cell responses in survivors. With an expanding area of CCHFV endemicity, safe, sensitive and specific serological assays are required for diagnostic and serosurveillance purposes. As the biosafety level 4 facilities required to culture the virus are lacking in many endemic areas, alternative means of producing reagents for diagnostic assays are needed which will not pose a safety risk to laboratory workers. The use of synthetic peptides in serological assays is one such alternative approach. In addition, identification of immunodominant epitopic regions may have application in vaccine development if they induce protective immunity. The peptide library was used to screen for antibodies recognizing human defined linear B cell epitopic regions in survivors of CCHFV infection by means of an enzyme-linked immunosorbent assay (ELISA). Two potential epitopic regions were identified on the GC glycoprotein with reactivity in 13 – 14 of 15 patients tested. Further investigation will be required to determine whether these epitopic regions also correlate with immune protection and to identify non-contiguous B cell epitopes which are likely to play an important role in antibody induction during natural infection with CCHFV. With new foci of CCHFV infections emerging in recent years, it is important to ascertain whether genomic variation will influence applicability of vaccine candidates and diagnostic assays in distinct geographic areas. Next generation sequencing techniques were used to obtain complete genome sequences for ten southern African CCHFV isolates. This is the first application of next generation sequencing technology to CCHFV isolates and proved to be a rapid and cost effective alternative to standard Sanger sequencing which can be effectively applied to the approximately 20kb CCHFV genome. The phylogenetic results confirmed that although there is extensive variability among geographically distinct CCHFV isolates at a genomic level, conserved areas are present which could be targeted for vaccine development and diagnostic purposes. The genetic variability seen results from point mutations and segment reassortment, which was shown to occur commonly in southern African CCHFV isolates. Despite the extensive variation in primary sequence, at a protein level, the motifs involved in protein function are well conserved. Prediction software analysis confirmed the presence of conserved OTU-like cysteine protease and RNA dependent RNA polymerase (RdRp) domains in the L segment of diverse southern African CCHV isolates. The RdRp is essential for viral replication while the OTU-like protease likely plays a role in immune evasion and therefore affects viral pathogenicity. Analysis of the M segment showed conservation of the basic protein coding strategy, with two structural and three non-structural glycoproteins. However, amino acid variation was notable across all predicted proteins but particularly in the variable mucin-like domain which is thought to play a role in viral pathogenicity. This study identifies targets for further investigation of viral pathogenicity which may include in vivo studies in animal models and mutagenicity assays.