Masters Degrees (Medical Microbiology)

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Browsing Masters Degrees (Medical Microbiology) by Subject "Arbovirus infections"

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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.