Masters Degrees (Medical Microbiology)

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Browsing Masters Degrees (Medical Microbiology) by Author "Burt, F. J."

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    Development of detection assays for sindbus virus and investigating in vitro infection of mammalian cells
    (University of the Free State, 2013-08) Hanekom, Hermanus Albertus; Burt, F. J.
    Sindbis virus (SINV) is a member of the Alphavirus genus and belongs to the family Togaviridae. The virus has a positive sense RNA genome of 11700 bases which encodes for both structural and non structural proteins. Infections are frequently diagnosed based on clinical, epidemiological and laboratory criteria. Laboratory confirmation is essential as SINV infections must be distinguished from various conditions that share similar clinical manifestations. The most frequently used methods for identification are haemagglutination inhibition, enzyme-linked immunosorbent assay, plaque reduction neutralization tests as well as conventional in-vitro neutralization assays. Serological assays for the detection of SINV are not readily available commercially and due to the non-specific symptoms caused by SINV infection the number of infections per annum may be under diagnosed. The purpose of this study was to develop serological assays such as ELISA and a novel neutralization assay that could be used in serological surveys for the detection of IgG antibodies against SINV. Furthermore to develop assays that could be used to determine the level of viral replication in mammalian cells for characterizing infection in mammalian cells as well as investigate the influence of interferon on viral replication and look for evidence of apoptosis caused by SINV infection. An in house ELISA was developed and used to screen 146 sera for IgG antibodies against SINV. The in-vitro neutralization assay is the gold standard for serology and 43 samples in total were tested in both the ELISA and the in-vitro neutralization assay. Analysis and comparison of the results obtained using the in-house ELISA and the neutralization assay indicated that the sensitivity of the ELISA was 68.9% and the specificity of the in house ELISA was 78.57 - 85.71% depending on the use of the percentage positive or optical density values to differentiate positive and negative samples. A forward and reverse primer for the amplification of a conserved 181bp region of the nsp2 gene encoding the nsp2 protein of SINV were designed along with a TaqMan hydrolysis probe to be used in a real time quantitative TaqMan PCR. The infection of mammalian cells, human macrophages and HeLa cells, was determined by measuring viral loads with a real time quantitative TaqMan RT-PCR. Two strains of SINV were used in attempts to infect macrophages, a strain from Egypt and a strain from South Africa. Small increases in viral load suggested possible low levels of viral replication but were considered insufficient to warrant further investigation and insufficient to investigate occurrence of antibody dependent enhancement of disease in macrophages. The mechanism possibly interfering with replication of virus in the human macrophages was investigated. Supernatant fluid samples from macrophage infections were tested for the release of interferon gamma which could inhibit viral replication. There were nine to fifteen fold differences in the concentration of 2 interferon gamma detected in the supernatant fluid at baseline and 24h after infection. HeLa cells were treated with similar concentrations of human interferon gamma at different time intervals. Pretreatment and concurrent treatment with infection showed reduced levels of viral load compared with no treatment or delay in treatment. Hence the suggestion that interferon could have played a role in inhibiting viral replication in the human macrophages. DNA was extracted from HeLa cells infected with SINV and the DNA fragments separated through agarose gel electrophoreses. There were multiple bands visible in the infected samples whereas the negative control did not show multiple bands, only one large band of genomic DNA. The presence of multiple DNA fragments in infected cells and absence of those fragments from uninfected cells were suggestive of virus induced apoptosis.
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    Development of molecular and serological assays for diagnosis and surveillance of Crimean-Congo haemorrhagic fever virus
    (University of the Free State, 2015-05) Pieters, Danelle; Burt, F. J.; Jansen van Vuren, P.
    Crimean-Congo haemorrhagic fever virus (CCHFV) an arthropod-borne virus associated with haemorrhagic disease in humans. The global distribution of CCHFV correlates with that of ticks from the Hyalomma genus. CCHFV infection is diagnosed by detection of viral nucleic acid using reverse-transcription polymerase-chain-reaction (RT-PCR) or other molecular assays, by virus isolation from infected cell culture or suckling mouse brain or by detection of anti-CCHFV antibodies using enzyme-linked immunosorbent assay (ELISA) or immunofluorescence assay (IFA). High biocontainment facilities are required for virus isolation and preparation of whole virus native antigen for use in serological assays. Currently, treatment is limited to supportive therapy. CCHFV is currently emerging and re-emerging in many regions, which emphasize the requirement for safe, reliable and inexpensive assays to increase diagnostic capacity and monitor emergence of the virus. A nucleic acid sequence-based amplification (NASBA) molecular assay for detection of CCHFV ribonucleic acid (RNA) was developed. The assay can be performed without the requirement for sophisticated laboratory equipment. A commercially available enzyme mixture and buffer were compared with a more cost effective and easier to obtain in-house enzyme mixture and amplification buffer. Specificity of the NASBA assays were determined by testing viral RNA extracted from Vero cell culture infected with genetically diverse southern African CCHFV strains. A total of 41/48 samples tested were positive. Sensitivity of the NASBA assays was determined using dilutions of viral RNA and transcribed RNA to detect minimal copy number that could be amplified. The NASBA assay was able to detect at least 3.7 RNA copies. Diagnostic application of the NASBA assays was investigated by amplifying RNA extracted from clinical samples and the results compared with two commercial real-time RT-PCR assays. A total of 20/22 samples tested positive using the NASBA whereas the commercially available assays were able to amplify 22/22 samples. Subsequently, the inhibitory effect of sera on the amplification of CCHFV RNA using the NASBA assay was investigated using sera spiked with transcribed RNA. Two expression systems were investigated for the expression of recombinant CCHFV nucleocapsid protein (NP) for use in serological assays. The baculovirus expression system was initially investigated. The open reading frame of the S segment of a CCHFV strain was codon optimized for expression in insect cells. A pFastBac HT B transfer vector containing the optimized CCHFV NP gene was prepared and used to transform DH10Bac™ Escherichia coli cells to transpose the optimized CCHFV NP gene to a bacmid. The recombinant bacmid was utilized to transfect Spodoptera frugiperda 9 cells. The cell lysates were analysed, however, no expression of the CCHFV NP could be confirmed. A mammalian expression system was subsequently investigated. A pcDNATM 3.1D/V5-His-TOPO.CCHFV.NP construct was used to transfect baby hamster kidney-21 cells. Expression of CCHFV NP was detected in transiently transfected cells using IFA and serum collected from a convalescent CCHFV patient. To profile the immune response against CCHF viral proteins, 15 sera collected from convalescent patients at various times after onset of illness were tested for antibody against CCHFV NP and glycoproteins (GP) using commercially available slides. The antigen slides were prepared from transfected cells expressing recombinant CCHFV NP and GP. Antibody against CCHFV GP and NP were detected in all samples. End point titers of anti-CCHFV NP and GP were determined for two serum samples. Commercially available slides are expensive and therefore have limited application for testing large numbers. Application of in-house antigen slides prepared from transfected cells expressing CCHFV NP were tested using IFA and 14 sera collected from convalescent CCHFV patients. All sera tested positive, suggesting that preparation of a stable cell line expressing CCHFV NP is warranted for application in detection of antibody against CCHFV.
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    Identification of antigen-specific serological cross-reactivity among survivors of Crimean-Congo Haemorrhagic fever
    (University of the Free State, 2013) Rangunwala, Azeeza; Burt, F. J.
    Abstract not available
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    Identification of antigenic regions and linear B cell epitopes on yellow fever virus
    (University of the Free State, 2013-02) Smouse, Shannon Lucrecia; Burt, F. J.
    English: Yellow fever virus (YFV) virus is an arthropod-borne virus that causes viral hemorrhagic fever in humans in the tropical parts of both Africa and South America. The virus belongs to the family Flaviviridae, of the genus Flavivirus comprising of approximately 70 viruses. It is transmitted to vertebrates by the bite of an infected female mosquito, primarily the Aedes species. It is a re-emerging pathogen with case-fatality rates that can exceed 50% in humans. YFV can cause an acute febrile illness in humans which can progress to severe disease with hepatic and renal failure. The diagnosis of infection and testing of the immune status of vaccinees require reagents that are prepared in biosafety level (BSL) three and four facilities. Therefore the development of a recombinant antigen that does not require BSL three facilities for preparation and is safe to use, would have an important role in a diagnostic laboratory for detecting antibodies in infected individuals and vaccinees. Despite the availability of a live-attenuated efficacious vaccine, it is not recommended for immunocompromised individuals, thus development of new generation vaccines would have important public health implications. Identification and mapping of antigenic regions and viral epitopes is important for development of subunit vaccines and improved diagnostics. Subunit vaccines focusing on antigens that induce a protective immune response provide a safe approach to the development of vaccines against diseases causing severe and frequently fatal haemorrhagic fevers. The aim of this study was to identify immunodominant viral proteins that induce detectable antibody responses that could be used for developing diagnostic assays and to identify linear B cell epitopes on selected viral proteins. The complete open reading frame of the genes encoding the domain III (EDIII) region of the envelope protein, capsid (C) and NS4a proteins of YFV were amplified, from the 17D strain of YFV, by RT-PCR using primers specifically designed from sequence data retrieved from GenBank. Oligonucleotide primers were modified with BamHI and HindIII restriction enzyme sites that facilitated downstream cloning. Each amplicon was cloned into the pGEM®-T Easy cloning vector using T/A cloning. Each gene was rescued from the recombinant plasmid using BamHI and HindIII restriction enzyme sites and ligated into bacterial expression system, pQE-80L vector. In a previous study, the YFV EDIII gene was cloned into pQE-80L and expressed in JM109 Escherichia coli cells however extremely low yields were obtained. In this study the expression levels were improved using different cell lines and optimizing incubation conditions. An insoluble 13 kDa protein was expressed from the construct and confirmed by Western blot analysis. The protein was expressed with a 6 x Histidine tag that was used to facilitate purification using a Ni2+ column under denaturing conditions. Attempts to express the YFV C and NS4a proteins were not successful and expression was abandoned. In an attempt to improve solubility the YFV EDIII gene was excised from the pGEM®-T Easy vector and subsequently cloned into pCold TF bacterial expression vector. A ~65 kDa soluble protein was expressed from the construct and purified under native conditions. The functional activity of the recombinant antigens in ELISA was compared with whole cell lysate antigen prepared from cell cultures infected with YFV. The biological activity of the recombinant YFV pQE-80L-EDIII antigen was confirmed in immunoassays using serum samples from humans vaccinated with YFV vaccine. Positive sera failed to react in ELISA using pCold TF expressed antigen and this antigen was excluded from further assays. A total of 20/24 serum samples from human vaccinees collected at varying stages after vaccination reacted in an ELISA with the recombinant YFV pQE-80L-EDIII protein and 24/24 reacted in ELISA with whole cell lysate antigen. The EDIII region of the envelope protein was shown to be able to differentiate between West Nile Virus infection and YFV infection in a limited number of convalescent horse sera. The recombinant EDIII protein was used to immunize mice. Serum samples collected from the mice reacted against whole cell lysate antigen in ELISA and was shown to have neutralising antibodies using an in vitro neutralisation assay. Hence the EDIII region of the envelope protein likely induces an important protective immune response. Finally, bioinformatics was used to predict possible epitope regions and using peptide libraries spanning predicted sites, one potential epitopic region was identified in the EDIII protein. Putative epitopic and antigenic regions along the length of the C, NS4a and EDIII proteins of each strain were predicted using the BCPREDS and ABCpred software. In conclusion, the EDIII protein, an immunodominant antigen of YFV, prepared in this study has some potential for differentiation of flavivirus antibodies although it lacks sensitivity for routine diagnosis. A potential epitope, TGHGTVVMQ, from amino acid 21 to 29 on the EDIII protein was identified using bioinformatics and was shown to have reactivity against immune sera. The significance of this epitope needs further investigation. Finally the EDIII region of the YFV protein shows potential as a target region for vaccine development as shown for other flaviviruses but which has not previously been published for YFV.
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    Preparation of recombinant antigens for demonstrating antibody responses in patients with Crimean-Congo haemorrhagic fever virus infections
    (University of the Free State, 2011-06) Samudzi, Rudo Ruth; Burt, F. J.
    Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely distributed in Africa, Asia, Russia and the Balkans. The causative agent, CCHF virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Cases of CCHF are diagnosed annually in southern Africa. Increasing numbers of cases are seen in regions of Asia and in the past ten years CCHFV has emerged in several countries in the Balkans and re-emergence in south-western regions of the Russian Federation. Diagnosis of CCHFV infections during the acute phase is based on isolation of the virus or amplification of viral RNA. Patients that survive the infection have a demonstrable IgG and IgM antibody response, usually from day 5 to 7 after onset of illness. Current serological diagnostic assays based on ELISA or IF use inactivated virus which requires biosafety level 4 facilities for culturing the virus and therefore limits the number of laboratories that can prepare suitable reagents. Preparation of recombinant antigens would enable laboratories to perform serological diagnosis of CCHFV infections and surveillance studies. The purpose of this study was to prepare a recombinant CCHFV nucleoprotein using a bacterial expression system, to determine if the protein was immunogenic and to determine if the protein was able to detect IgG antibodies in survivors of CCHFV infection. The complete open reading frame of the gene encoding the NP of CCHFV was amplified by RT-PCR using primers specifically designed with restriction sites engineered to the primers to facilitate cloning. The amplicon was cloned into pGEM® T Easy vector using T/A cloning and the gene sequenced to confirm that the correct gene had been amplified and cloned into the vector for downstream cloning and expression applications. Initially we aimed to express the native gene using a bacterial expression system and the NP gene was rescued from the recombinant plasmid and cloned into pQE-80L vector using the BamH1 and Pst1 restriction sites present in the multiple cloning site on the vector. Various attempts were made to express the CCHFV NP protein however no protein was detectable using SDS PAGE methods or Western blot. The nucleotide sequence that we had determined for the open reading frame of our gene encoding the NP was analysed using the Rare Codon Analysis Tool software and we elected to codon optimize the gene for expression in E. coli. The optimized gene was synthesized by GenScript and supplied cloned in the multiple cloning site of pUC57. The optimized gene was excised from pUC57 and cloned into pColdTF bacterial expression vector. A 106 kDa protein was expressed from the construct likely representing the HIS tagged TF chaperone protein fused to the CCHFV NP protein and confirmed by Western blot analysis. A higher yield of the protein was present in the insoluble phase and as optimization of the growth and induction conditions did not significantly alter the insoluble to soluble ratio of the expressed protein, the protein was harvested from the insoluble phase by denaturing, purification and refolding of the protein. The biological activity of the recombinant protein was confirmed using immunoassays and by immunizing mice to determine if the antibodies induced by the recombinant protein could be detected using an antigen prepared from the whole virus. Four of five mice immunized with the recombinant NP had a detectable antibody response using an immunofluorescent assay. Serum samples from acute and convalescent patients collected at varying stages after onset of illness were reacted in a Western blot with the recombinant CCHFV NP protein. The recombinant antigen was able to detect IgG antibody in all the convalescent patient sera except two sera collected on days 14 and 15 during the acute phase. In contrast all the samples were detected using the recombinant antigen in an ELISA. Due to the potential biohazardous nature of samples only samples collected two weeks after onset of illness were tested. The results showed 100% concordance with the results obtained in an ELISA using mouse brain derived antigen. The assay was shown to be reproducible and stability studies showed that four months after preparation the protein was still active. A full validation of the protein using a large panel of serum samples from confirmed CCHF patients is now required. The results suggest that bacterially expressed proteins lacking post translational modifications and folding that occur with mammalian and baculovirus expression can be used in ELISA to detect IgG antibody against CCHFV in human sera which finds application in diagnostics, epidemiologic and surveillance studies.