Masters Degrees (Medical Microbiology)

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Open Access
Characterization of T cell responses to the non-structural proteins of the M segment in survivors of Crimean-Congo haemorrhagic fever
(University of the Free State, 2019) Maotoana, Makgotso Golda; Goedhals, Dominique; Burt, Felicity Jane
Crimean-Congo haemorrhagic fever orthonairovirus (CCHFV) is one of the most widely distributed arboviruses globally. The disease caused by the virus, Crimean- Congo haemorrhagic fever (CCHF), continues to emerge and re-emerge across the globe. There are currently various vaccines under development for CCHF prevention. The non-structural M protein (NSM), GP38, highly variable mucin-like domain and N-terminus of GC regions in CCHFV have proved to be immunogenic in vaccine studies. Furthermore, both arms of the immune system have been found to be fundamental for protection in mice. However, there is limited information about immunity in patients following natural infection. The aim of the study was to characterize T cell immune responses against the NSM, GP38 and the highly variable mucin-like domain of CCHFV in survivors of CCHF. This was achieved by first identifying immunogenic peptides in the regions of interest and determining the amino acid conservation of the identified peptides. An overlapping peptide library spanning the NSM, GP38 and highly variable mucin-like domain was designed using the South African CCHFV isolate SPU 103/87. The secretion of interferon-gamma (IFN-γ) by peripheral blood mononuclear cells isolated from 12 participants was screened using 24 peptide pools in an IFN-γ enzyme linked immunospot (ELISpot) assay. IFN-γ secretion was detected in eight of the twelve participants. Two participants showed no detectable IFN-γ responses to any of the peptide pools, and another two were excluded from the analysis due to a high background in the negative controls indicating non-specific reactivity. Nine peptides were identified with the IFN-γ ELISpot, including five peptides in the GP38 region and four in the NSM region, thus confirming the immunogenic potential of these regions during natural infection. No immunogenic peptides were identified in the highly variable mucin-like domain, which is possibly because of the high genetic diversity in the region. The identified immunogenic peptides were used to stimulate T cells of participants and a flow cytometry assay was performed to characterize the immune responses, with the focus on detecting the presence of the T cell memory subsets, the expression of CD107a, which is a cytotoxic marker, and the secretion of IFN-γ and tumour necrosis factor-alpha (TNF-α), which are antiviral cytokines. Cytotoxic CD8+ T cells were detected in six participants in response to nine peptides. IFN-γ and TNF-α secretion within the CD4 and CD8 populations were comparable; thus, highlighting the ability of the CD8+ T cell population to secrete antiviral cytokines, even though the population is known to be predominantly cytotoxic. The secretion of IFN-γ was more frequent than TNF-α secretion in both the CD4 and CD8 populations. Polyfunctional T cells were detected with the phenotypes IFN- γ+CD107a+ and IFN-γ+ TNF-α+, in both the CD4 and CD8 populations. Therefore, the results indicate the possibility of efficient antiviral responses upon stimulation with viral epitopes in survivors of infection. Heterogeneous functionality of the T cell memory subsets was observed, however the terminally differentiated (TEMRA) subset was the most dominant and abundant, followed by the naïve (TN), effector memory (TEM), with the least abundant being the central memory (TCM) T cell memory subset. The IFN-γ secretion detected with the IFN-γ ELISpot and the flow cytometry assay was used as basis for comparing the sensitivity of the two techniques. The IFN-γ ELISpot proved to be comparable to the flow cytometry assay. The ELISpot is suited for screening purposes, while the flow cytometry allowed further characterization of the T cell responses. Therefore, it is recommended that these complementary assays be used in combination. In conclusion, T cell epitopes were identified in the NSM and GP38 regions of CCHFV. Polyfunctional T cells were found in both the CD4 and CD8 populations, thus suggesting the presence of effective long-term memory T cells responses in survivors of CCHF.
Open Access
Development and application of molecular assays for mosquito-borne alphaviruses in South Africa
(University of the Free State, 2021) Dimaculangan, Micah; Burt, Felicity Jane
Surveillance of mosquito-borne alphaviruses is critical for the prevention of diseases and the control of outbreaks caused by these viruses, especially with the absence of approved vaccines and antiviral treatments available. Hence, the continual development of rapid and reliable tools for the surveillance of alphaviruses is important. This will aid in the understanding of which viruses are currently circulating with the potential to cause outbreaks. Molecular nucleic acid amplification tests (NAATs), particularly conventional and real-time reverse transcription (RT)-polymerase chain reaction (PCR), are typically employed in epidemiological surveys. In this study, a conventional nested RT-PCR assay was developed to detect alphaviruses in South Africa. In addition, an isothermal amplification technique, specifically a RT-helicase dependent amplification (HDA) assay, which only requires a simple heating device, for instance a heating block, and lateral flow dipsticks/ cassettes for end point detection, was developed to detect alphaviruses currently circulating in South Africa, as an alternative to the RT-PCR assay for application in low resource settings or for field application. The conventional nested RT-PCR assay was able to detect ≥620 copies of RNA compared to the RT-HDA assay which had a minimum limit of detection of 4.8 x 105 copies of RNA. Both assays were tested for theoretical cross-reactivity with other alphaviruses, which include Sindbis virus (SINV) and chikungunya virus (CHIKV) isolates from other regions and genotypes, and isolates from alphaviruses such as Ross River virus (RRV), Barmah Forest virus (BFV), Mayaro virus (MAYV), eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis virus (VEEV) and western equine encephalitis virus (WEEV) that are endemic to other parts of world. Alignment of the primers with the sequences of these isolates shows that both assays in theory would be able to detect SINV isolates from northern Europe, taking into account the transcontinental transmission of the virus between South Africa and northern Europe by migratory birds. The conventional nested RT-PCR assay may be able to detect most alphaviruses due to minimal mismatches (0 – 1) detected between the primers and the partial nsP4 sequences of the alphavirus isolates, while the RT-HDA assay may not be well suited to detect other alphaviruses due to the many mismatches (>4) detected between the primers and the partial nsP4 sequences of the alphavirus isolates. Nevertheless, this shows that the RT-HDA is theoretically more specific that the conventional nested RT-PCR assay. The RT-HDA however failed to detect any alphaviruses in the 42 mosquito pools tested, which was not unexpected as the assay could only detect up to 4.8 x 105 copies of RNA. In contrast, the conventional nested RT-PCR assay was able to detect alphaviral RNA in five out of the 42 mosquito pools tested, and the nucleotide sequences were determined to identify the alphavirus species. SINV RNA was detected in three mosquito pools and Middelburg virus (MIDV) was detected in two pools. Phylogenetic analysis was subsequently performed to determine the genetic relationship of these isolates from the Free State with previously published/ reported SINV and MIDV isolates in South Africa, Africa, and around the world. The conventional nested RT-PCR assay developed in this study has shown to be a useful surveillance tool for the detection of mosquito-borne alphavirus infections. Given the low sensitivity determined for the RT-HDA assay, improvements, or alternative rapid and fieldable NAATs should be considered in the future for alphavirus surveillance applications in low resource settings.
Open Access
Genetic analysis of human papillomavirus type 11 isolates from patients with recurrent respiratory papillomatosis treated at Universitas Academic Hospital
(University of the Free State, 2021-07) Thuynsma, Corne; Burt, Felicity Jane; Seedat, Riaz
Human papillomavirus type 11 (HPV11) is a causative agent of recurrent respiratory papillomatosis (RRP), a common benign laryngeal neoplasm that presents mainly in children. The genome comprises three regions: the early region (E1, E2, E4, E5a/b, E6 and E7), the late region (L1 and L2), and the upper regulatory region (URR). A sequence-based classification system is primarily used to genotype HPV. The L1 is used for HPV type discrimination, and in combination with the URR, can be used to differentiate between various lineages. However, optimal sub-lineage classification requires whole genome sequencing (WGS). A recent study investigating the genomic diversity of globally circulating HPV11 isolates identified a novel lineage and two novel sub-lineages. It has been proposed that phylogenetic tree topologies using the sequences of concatenated E5a/b- L1-URR genes, a 208bp segment of the E2 gene, and the complete genome generates similar tree topologies. Also, there is currently no published data on the HPV11 intratypic variants circulating in the Free State region. Hence, this study investigated HPV11 intratypic variants circulating in patients with RRP at the Universitas Academic Hospital, and aimed to identify novel (sub)lineages through phylogenetic investigations. The study population included patients diagnosed with RRP caused by HPV11, and sequence data for geographically distinct HPV11 (sub)lineage representatives. The genetic variation of HPV11 isolated from patients with RRP was determined by sequencing the E5a/b, L1, URR and a segment of E2 genes. Four isolates of interest were selected for whole-genome sequencing and phylogenetically analysed to determine the presence of potentially novel isolates. Many nucleic heterogeneities and non-synonymous substitutions were identified in isolates characterised in this study. Phylogenetic analysis of the concatenated L1-URR and E5a/b-L1-URR resolved into lineages A and B; however, sub-lineage classification was unclear. Analysis of the complete genome determined the presence of a lineage B isolate and two isolates of interest. Comparative analysis of genetic variability determined that the concatenated E5a/b-L1-URR could not reliably classify isolates. A segment of the E2 gene could reliably distinguish between all lineages and sub-lineages, suggesting that this gene segment contains stable sub-lineage specific single nucleotide polymorphisms (SNPs) and may serve in sub-lineage identification. In conclusion this study provides the most comprehensive data on the genomic diversity of HPV11 in the Free State to date. Results obtained in the current study support WGS for HPV11 classification below lineage level as a standard, as it generates more information regarding genetic variants.
Open Access
Preparation and immunogenicity of a candidate replicon based yellow fever vaccine
(University of the Free State, 2014-12) Viljoen, Natalie; Burt, Felicity Jane
English: Yellow fever virus (YFV), a mosquito-borne virus that belongs to the family Flaviviridae and genus Flavivirus, is a significant cause of morbidity and mortality in yellow fever endemic areas, especially in West Africa. In humans, YFV causes yellow fever, a disease characterised by renal failure, jaundice, and/or haemorrhage. The burden of disease is highest in Africa constituting approximately 90% of reported cases worldwide. Despite the availability of highly efficacious live attenuated vaccines against YFV, the estimated prevalence for yellow fever in Africa was 130 000 severe cases and 78 000 deaths for 2013. The available live attenuated vaccines have been contraindicated for use in immunocompromised patients and individuals with hypersensitivity to eggs and/or chicken. Vaccine-associated neurotropic adverse events that result in the development of meningoencephalitis in infants and vaccine-associated viscerotropic adverse events that result in disease resembling wild-type yellow fever have been reported. Vaccine-associated viscerotropic adverse events are associated with fatality rates exceeding 40%. Therefore, there is a need for a safer alternative to complement the use of the available live attenuated vaccines. The aim of this study was to construct a DNA-launched candidate vaccine against YFV and to determine the immunogenicity of the DNA-launched pSinED-lll replicon, which would provide information regarding the applicability of DNA-launched replicons as an approach to vaccine development. The pSinGFP replicon encoding the green fluorescent protein (GFP) was kindly provided by Prof. Mark Heise. Expression of the GFP was confirmed in mammalian cell culture post-transfection with the pSinGFP replicon, thus confirming the functioning of the replicon elements and subsequent expression of the encoded protein. The gene encoding the GFP was excised and replaced with a synthesised codon-optimised gene encoding the YFV ED-lll protein using directional cloning. The nucleotide sequence was confirmed by bidirectional sequencing at the site of insertion and subsequently protein expression was confirmed in selected mammalian cell lines. Protein expression was confirmed by detection of the C-terminal histidine tag by immunofluorescence using anti-His6 mouse monoclonal antibody. Thereafter, the expressed protein was characterised by immunofluorescence using mouse immune sera previously shown to contain anti-YFV ED-lll antibody. Reactivity of the expressed protein with anti-YFV ED-lll antibody substantiated the use of the pSinED-lll replicon in a mouse immunisation study. Mice were immunised intramuscularly according to pre-approved immunisation regimes consisting of DNA only, as well as mixed modal immunisation regimes. Serum samples were collected and spleens were harvested two weeks after the administration of the final immunisations. Serum samples were screened for anti-YFV antibodies by an enzyme-linked immunosorbent assay (ELISA) and immunofluorescence to determine the induction of a humoral immune response. Five of the twenty mice in groups immunised with the DNA-launched pSinED-lll replicon developed low level antibody responses illustrating the potential of the pSinED-lll replicon to induce a humoral immune response. Splenocytes were stimulated in cell culture with concanavalin A, YFV ED-lll protein, or no stimulant to facilitate the detection of cytokine release using ELISA’s. A predominantly T-helper 1 cell-mediated immune response characterised by high level release of interferon-γ post-stimulation with the YFV ED-lll protein in vitro was elicited. The release of interleukin-10 post-stimulation may be associated with the prevention of immunologically mediated damage to the host by preventing an overzealous inflammatory immune response. Antibody-mediated anti-vector immunity should not negatively impact the immunogenicity of the DNA-launched pSinED-lll replicon as antibody directed against the encoded Sindbis virus non-structural proteins was not detected in mouse sera. In conclusion, the pSinED-lll replicon was shown to have the potential to induce both a cell-mediated and a humoral immune response post-immunisation. However, optimisation of the humoral immune response will be required.
Open Access
Preparation of multiplex immunofluorescent platform for serology
(University of the Free State, 2023) Maswanganyi, Nyiko Given; Burt, Felicity Jane; Makoah, Nigel Aminake
Arboviruses are transmitted by blood-feeding insects such as mosquitoes and ticks, and pose significant global health challenges. In South Africa there are arboviruses that are known to occur causing outbreaks annually after heavy rainfall and there are arboviruses that have been detected historically but their medical and veterinary importance is unknown. Due to the unavailability of commercial assays for many arboviruses, the development of in-house assays becomes crucial. This study aimed to develop a cost-effective multiplexing technique using immunofluorescent polyvalent antigen slides for simultaneous screening of IgG antibodies against multiple arboviruses. The nucleoprotein (NP), envelope domain III (EDIII), and capsid (C) genes for Crimean-Congo hemorrhagic fever virus (CCHFV), West Nile virus (WNV), and Sindbis virus (SINV) respectively were codon optimized and cloned into pcDNA 3.1+ plasmids. The plasmids were transfected into human embryonic kidney (HEK) 293 mammalian cells, and the expression of these genes was confirmed through immunofluorescent assay (IFA). The expressed proteins were further validated by SDS and Western blot analyses. In-house enzyme-linked immunosorbent assays (ELISAs) were developed and optimized to screen human and cattle serum samples for IgG antibodies against SINV and CCHFV. A total of 386 human serum samples and 97 cattle serum samples were screened. Polyvalent IFA slides were developed and used to screen all positive serum samples, some negative and borderline from inhouse ELISAs. The results demonstrated successful expression of CCHFV NP, confirmed through positive IFA reactions with anti-CCHF IgG. SDS and Western blot analyses further validated the size of the expressed CCHFV. WNV showed low IFA reactivity with antihis antibody and SINV showed no expression. Hence it was decided to continue using SINV-infected cells to replace SINV transfected cells, confirmed using IFA with antiSINV sera. WNV was omitted going forward due to low yield. Among the samples positive for SINV IgG ELISA, 39 exhibited concordant positive results with the polyvalent slides. Similarly, three samples positive for CCHFV IgG antibodies with CCHFV ELISA were concordant with polyvalent slides, while eight negative serum samples tested negative using the ELISAs and IFA. Three ELISA borderline samples for SINV ELISA tested positive for SINV suggesting increased sensitivity for IFA. Furthermore, specificity testing of IFA revealed an accuracy rate of 83.3%. Multiplex assays offer a low-cost and fast method for simultaneous detection of IgG antibodies against multiple arboviruses on a single platform. The IFA results were concordant with the ELISA, suggesting the reliability of the developed technique. The positive reactors among the borderline samples for SINV antibody ELISA indicate that additional screening of negative samples could improve the cut-off value. Importantly, all samples tested negative with ELISAs were also negative with the multiplex IFA. This approach enables efficient serosurveillance, particularly in resource-limited regions. The accuracy and sensitivity of the multiplex assay were evident through the confirmation of IgG presence by monovalent IFA. In conclusion, the developed cost-effective multiplexing technique using immunofluorescent polyvalent antigen slides provides a valuable tool for simultaneous screening of IgG antibodies against multiple arboviruses. The accuracy and sensitivity of the multiplex assay were validated through monovalent IFA confirmation. Subsequent research and refinement of this technique hold the potential to broaden its platform, enabling the screening of a more extensive array of viruses.
Open Access
Preparation of recombinant antigen for serological detection of African hantaviruses
(University of the Free State, 2017-07) Damane, Deborah Rethabile; Burt, Felicity Jane
Unlike other members of the Bunyaviridae family, hantaviruses are transmitted to humans through direct exposure or inhalation of virus contaminated urine or droppings from their reservoir hosts. Hantaviruses were first discovered in 1976 with the identification of Hantaan virus (HNTV) from the reservoir Apodemus agarius in Asia and later in North America. In 2006, Sangassou virus (SANGV) was the first to be isolated in Africa in the African house mouse, Hylomyscus sinus and subsequently followed by the identification of ten more African hantaviruses in both rodent and insectivore hosts through reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). Hantaviruses are a public health concern with annual cases of disease reported to be approximately 200,000 per year, with most cases reported in Asia. In Africa, disease associated with hantaviruses is not well defined. Culturing the virus and preparing reagents using native virus requires the use of biosafety level (BSL) 3 or 4 laboratories limiting the number of facilities with capability to prepare serological assays. Hence, the use of recombinant antigens that are safe to use in a BSL 1 laboratory that have application as serological tools for surveillance are required. The aim of the study was to develop serological assays to test for antibodies against hantaviruses in human serum samples collected in the Free State, South Africa using a recombinant nucleocapsid protein (NP) of SANGV as a representative of African hantaviruses. Transiently transfected cells were used to prepare antigen slides for IFA and expressed protein was used in an in-house enzyme linked immunosorbent assay (ELISA). In-house assays and commercially available ELISA kits were used to screen human serum samples. There are limited seroprevalence studies performed in Africa to detect IgG antibodies against hantaviruses in humans and no commercial serological assays are available using an African antigen. Hence, it was considered that the preparation of a recombinant African hantavirus antigen based on SANGV could have application in serological surveillance studies. The S gene segment of the SA14 strain of SANGV was modified and codon optimized for enhanced expression and detection. The construct was sequenced and aligned to the native S gene. It was used to transfect baby hamster kidney cells (BHK-21). Expression of a 50kDA protein was confirmed by SDS-PAGE and Western blot assay. Antigen slides were prepared from transfected cells fixed on 12 well chamber slides. Positive controls from the commercially available ELISA kits were used in the IFA. Four of the 176 serum samples tested gave a positive test. For the in-house ELISA, protein was harvested from T75 culture flasks. The antigen was tested using positive and negative controls from the commercial ELISA kits. A suitable differentiation between positive and negative samples was not detectable despite attempts to optimize the in house ELISA. It is likely that the protein yield was insufficient for the ELISA and further attempts, beyond the scope of this project, to increase the protein yield will be investigated using a stable cell line. Commercially available ELISA kits comprising of HNTV, Dobrava (DOBV) and Puumala (PUUV) recombinant NP antigens for Europe and Asia group and Andes virus (ANDV) and SNV for the American group were used to screen acute human sera in the laboratory. Positive reactors were identified using both kits. The significance of the results is difficult to interpret as there was lack of concordance. However, it does suggest that hantaviruses are to be found in this area and that the use of a homologous antigen for serological surveillance is essential. Results confirmed some serological cross-reactivity between heterologous Asian, American and African hantaviruses and a potential application for an African hantavirus as a tool for surveillance.
Open Access
Zoonotic diseases in high-risk populations in the Free State province, South Africa
(University of the Free State, 2021-05) Van der Westhuizen, Cornelius Gerhardus; Musoke, Jolly; Burt, Felicity Jane
Zoonotic diseases are infectious diseases transmitted from vertebrate animals to humans and are accountable for more than 60% of all recognized human diseases and 75% of all new or emerging infectious diseases (EID). In South Africa (SA), endemic zoonoses include Mycobacterium bovis (M. bovis), Brucella sp., and Leptospira sp. The prevalence and burden of other pathogens, such as hantaviruses, are unknown. Therefore, identifying high-risk occupations and other risk factors are important for control and preventative measures to decrease the disease burden of these zoonoses. Thus, this study aimed to investigate the incidence rate of M. bovis and Brucella sp. in cattle and farm workers in two different farming communities (communal and commercial), as well as their associated risk factors. This study aimed to document occupational exposure to Brucella sp., Leptospira sp. and hantaviruses across the Free State province, South Africa. Four commercial farms and a rural cattle farming community within the Moqhaka and Ngwathe municipal regions were selected for the purpose of this study. From these farms, sputum and blood specimens were collected from 13 commercial farm workers and 13 communal farm workers. Sputum samples were screened for M. bovis through Mycobacteria Growth Indicator Tube (MGIT) culture. Blood specimens from these 26 farm workers, in addition to 301 archived sera, were screened for Brucella sp., hantaviruses, and Leptospira sp. antibodies using commercially available enzyme-linked immunosorbent assays (ELISA). From the 26 farm workers, no M. bovis was isolated. Out of the 327 sera screened, 35/327 (10.7%) were Brucella sp. IgG positive, 17/327 (5.2%) Leptospira sp. IgM positive, and 38/327 (11.6%) hantavirus IgG positive. A combined total of 321 cattle were screened for M. bovis through tuberculin skin testing (TST); 71 cattle were from communal farms and 250 from commercial farms. Additionally, blood samples collected from 69 and 1793 cattle within the communal and commercial farms, respectively, were screened for Brucella sp. using a Rose Bengal test (RBT) and complement fixation test (CFT). A total of 8/321 (2.5%) cattle reacted positive to the TST, and two were positive using the interferon-gamma release assay. Initial RBT screening resulted in 52/1859 (2.8%) positive results, further testing using a CFT identified 19/1859 (1%) brucellosis-positive cattle. A higher percentage of brucellosis-positive animals were from communal cattle (6/69; 8.7%) compared to commercial cattle (13/1859; 1.1%). Statistical analysis and probability values were calculated using a chi-squared or Fisher's exact test in the case of sparse data. Analysis identified higher Brucella sp. occupational exposure in veterinarians (p-value = 0.0006) and laboratory workers (p-value = 0.031). Further analysis showed a statistically significant correlation between people who reported illness post-exposure to animal blood/tissue (p-value = 0.029); and older age (p-value = 0.0008) with Brucella sp. seropositivity. Working at the abattoir (p-value = 0.024) was identified as a high-risk occupation for contracting Leptospira sp. In conclusion, the low incidence rate of M. bovis in cattle suggests limited contact with known reservoirs (i.e. buffalo or other wildlife). However, the higher incidence rate of brucellosis in communal cattle highlights the importance of implementing mass herd vaccination campaigns, particularly in communal settings. This report documents the seroprevalence of Leptospira sp., Brucella sp. and hantaviruses in various high-risk occupations in the Free State province and can be used as a basis for the development and establishment of adequate preventive or control measures.

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