Browsing by Author "Fielding, Burtram C."
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Item Acute toxicity studies of the South African medicinal plant Galenia africana(Elsevier, 2018) Ng’uni, Tiza; Klaasen, Jeremy A.; Fielding, Burtram C.Background: Medicinal plants are used by a large proportion of the global population as complementary and alternative medicines. However, little is known about their toxicity. G. africana has been used to treat wounds, coughs and skin diseases and is used in cosmetic formulations such as lotions and shampoos. Methods: The acute oral and dermal toxicity potential of G. africana was analyzed after a single administration of 300 and 2000 mg/kgbw for acute oral toxicity and 2000 mg/kgbw for acute dermal toxicity. Female Sprague- Dawley rats were used for the acute oral toxicity study whereas both male and female Sprague-Dawley rats were used for the acute dermal toxicity study. In the Episkin skin irritation test, the irritation potential of G. africana (concentrate) and G. africana (in-use dilution) extracts were assessed using the Episkin reconstituted human epidermis. In the dermal sensitization study, female CBA/Ca mice were treated with G. africana concentrations of 50, 100 and 200 mg/ml respectively. The vehicle of choice was dimethylformamide which acted as a control. Results: The results of the acute oral and dermal toxicity studies revealed that the median lethal dosage (LD50) for G. africana extract in Sprague-Dawley rats was considered to exceed 2000 mg/kgbw. In the irritation test, the G. africana (concentrate) and G. africana (in-use dilution) extracts were non-irritant on the Episkin reconstituted human epidermis. In the dermal sensitization study, the stimulation index (SI) values for the mice treated with the G. africana extract at concentrations of 50, 100 and 200 mg/ml/kgbw, when compared to the control group, were 1.3, 0.9 and 1.3 respectively. The open application of the extract at the various concentrations did not result in a SI of ≥ 3 in any group. Hence, it did not elicit a hypersensitivity response. Conclusion: These findings demonstrate that the acute toxicity profile for G. africana is acceptable and can subsequently be used for single use in the pharmaceutical and cosmetic industries.Item Additive antibacterial activity of naringenin and antibiotic combinations against multidrug resistant Staphylococcus aureus(Academic Journals, 2015) Ng'uni, Tiza; Mothlalamme, Thato; Daniels, Raymond; Klaasen, Jeremy; Fielding, Burtram C.Methicillin-resistant Staphylococcus aureus has been causing numerous problems in the health care sector. This is mainly due to its ability to develop resistance to a number of antibiotics used to treat staphylococcal infections. Medicinal plants have been used to treat various ailments over the years and are generating a lot of interest as alternative treatment options. Naringenin is a plant derived flavonoid that possesses antibacterial properties, among others. This study assessed the effect of combinations of naringenin and four antibiotics against two Staphylococcus aureus strains. The minimum inhibitory concentrations were determined using the disk diffusion and broth microdilution assays. In the disk diffusion assay, naringenin did not inhibit bacterial growth, nor did it enhance the antibacterial activity of the antibiotics in the combination study. This was attributed to its slow rate of diffusion out of the disks. On the contrary, in the broth microdilution assay, naringenin exhibited additive effects when combined with the antibiotics (at sub-inhibitory concentrations). These results show the potential of naringenin as an antibacterial agent. Furthermore, the additive effects observed at low naringenin concentrations showed that it can potentially be used in combination with antibiotics against multidrug resistant bacteria.Item Airborne transmission: How much do we actually know about Covid-19?(University of the Western Cape, 2020) Fielding, Burtram C.Item Amino acids 1055 to 1192 in the S2 Region of severe acute respiratory syndrome Coronavirus S Protein induce neutralizing antibodies: Implications for the development of vaccines and antiviral agents(American Society for Microbiology, 2005) Keng, Choong-Tat; Zhang, Aihua; Shen, Shuo; Lip, Kuo-Ming; Fielding, Burtram C.; Tan, Timothy H.P.; Chou, Chih-Fong; Loh, Chay Boon; Wang, Sifang; Fu, Jianlin; Yang, Xiaoming; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-JooThe spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-S 10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.Item Antimicrobial-resistant Klebsiella species isolated from free-range chicken samples in an informal settlement(Termedia Publishing House, 2012) Fielding, Burtram C.; Mnabisa, Amanda; Gouws, Pieter A.; Morris, ThureyahSub-therapeutic doses of antimicrobial agents are administered routinely to poultry to aid growth and to prevent disease, with prolonged exposure often resulting in bacterial resistance. Crossover of antibiotic resistant bacteria from poultry to humans poses a risk to human health. In this study, 17 chicken samples collected from a vendor operating in an informal settlement in the Cape Town Metropolitan area, South Africa were screened for antimicrobial-resistant Gram-negative bacilli using the Kirby Bauer disk diffusion assay. In total, six antibiotics were screened: ampicillin, ciprofloxacin, gentamicin, nalidixic acid, tetracycline and trimethoprim. Surprisingly, Klebsiella ozaenae was identified in 96 and K. rhinoscleromatis in 6 (n = 102) of the samples tested. Interestingly, ~40% of the isolated Klebsiella spp. showed multiple resistance to at least three of the six antibiotics tested. Klebsiella ozaenae and K. rhinoscleromatis cause clinical chronic rhinitis and are almost exclusively associated with people living in areas of poor hygiene.Item Barely explained - National coronavirus lockdown: We speak with an expert(University of the Western Cape, 2020) Fielding, Burtram C.Item Cellular Characterization of SARS Coronavirus Nucleocapsid(Leibniz Research Centre for Working Environment and Human Factors, 2004) Goh, Phuay-Yee; Choi, Yook-Wah; Shen, Shuo; Tan, Yee-Joo; Fielding, Burtram C.; Tan, Timothy H.P.; O, Eng-Eong; Lim, Seng Gee; Hong, WanjinThe Severe and Acute Respiratory Syndrome coronavirus (SARS CoV) is a newly-emerged virus that caused an outbreak of atypical pneumonia in the winter of 2002-2003. Polyclonal antibodies raised against the nucleocapsid (N) of the SARS CoV showed the localization of N to the cytoplasm and the nucleolus in virus-infected and N-expressing Vero E6 cells. Like other coronavirus N proteins, the SARS N is probably a phosphoprotein. N protein expressed in mammalian cells is apparently able to “spread” to neighboring cells. For N to spread to neighboring cells, it must be exported out of the expressing cells. This is shown by the immunoprecipitation of N from the culture medium of a stable cell line expressing myc-N. Deletion studies showed that the 27 kD C-terminal domain of N (C1/2) is the minimal region of N that can spread to other cells. The nucleolar localization and spreading of N are artefacts of fixation, reminiscent of other protein-transduction domain (PTD)-containing proteinsItem Characterisation of human coronavirus-NL63 nucleocapsid protein(Academic Journals, 2012) Berry, Michael; Manasse, Taryn-Lee; Tan, Yee-Joo; Fielding, Burtram C.Coronavirus N is a multifunctional protein that plays an essential role in enhancing the efficiency of virus transcription and assembly. This manuscript reports the analysis of HCoV-NL63 N protein by comparing the amino acid sequences of coronavirus N-homologues. A ~50 kDa protein was expressed in both a mammalian cell and bacterial cell system that is similar in size to the predicted ~42.6 kDa HCoV-NL63 N protein. PSORTII identified two putative nuclear localisations signals and PONDR identified one disordered region in HCoV-NL63 N. The reported protein analysis serves as a prelude to laboratory analysis to understand the processing of HCoV-NL63 N.Item The characterization and phylogenetic relationship of the trichoplusia ni single capsid nuclear polyhedrosis virus polyhedrin gene(Kluwer Academic Publishers, 1999) Fielding, Burtram C.; Davison, SeanThe polyhedrin gene (polh) was identified from the Trichoplusia ni (Tni) single capsid nuclear polyhedrosis virus (SNPV). An EcoRI fragment containing the truncated polyhedrin gene was detected by hybridization with an AcMNPV expression vector probe; the remaining portion of the gene was amplified by reverse PCR. An open reading frame (ORF) of 741 nucleotides (nt), encoding a putative protein of 246 amino acids (a.a) with Mr 28,780 Da was identified. The 50-noncoding region contained the putative late (TAAG) transcription initiation motif. The 30 end, downstream of the translation stop codon, lacked an obvious putative poly (A) signal. Nucleotide and amino acid homology are greater than 80% to that of Mamestra brassicae polyhedrin sequences.Results suggest that T. niSNPV is a member of the group II nuclear polyhedrosis viruses.Item Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus(American Society for Microbiology, 2004) Fielding, Burtram C.; Tan, Yee-Joo; Shen, Shuo; Tan, Timothy H.P.; Ooi, Eng-Eong; Lim, Seng Gee; Hong, Wanjin; Goh, Phuay-YeeA novel coronavirus (CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV genome encodes the characteristic essential CoV replication and structural proteins. Additionally, the genome contains six group-specific open reading frames (ORFs) larger than 50 amino acids, with no known homologues. As with the group-specific genes of the other CoVs, little is known about the SARS-CoV group-specific genes. SARS-CoV ORF7a encodes a putative unique 122-amino-acid protein, designated U122 in this study. The deduced sequence contains a probable cleaved signal sequence and a C-terminal transmembrane helix, indicating that U122 is likely to be a type I membrane protein. The C-terminal tail also contains a typical endoplasmic reticulum (ER) retrieval motif, KRKTE. U122 was expressed in SARS-CoVinfected Vero E6 cells, as it could be detected by Western blot and immunofluorescence analyses. U122 is localized to the perinuclear region of both SARS-CoV-infected and transfected cells and colocalized with ER and intermediate compartment markers. Mutational analyses showed that both the signal peptide sequence and ER retrieval motif were functional.Item Circuit breakers the mini lockdown needed for hotspots(SABC, 2020) Fielding, Burtram C.Circuit breakers form part of five-point plan from the department of health that Premier Alan Winde be presented to the provincial cabinet on Tuesday for adoption. Professor Burtram Fielding, molecular biologist and Director of Research Development at UWC will explain what are circuit breakers and how they could help ease off the rise of infections in hotspots.Item Circuit breakers the mini lockdown needed for hotspots(University of the Western Cape, 2020) Fielding, Burtram C.Item Cloning and expression of human cyclophilin A and its interaction with human coronavirus NL63 nucleocapsid protein(University of the Western Cape, 2011) Gela, Anele; Fielding, Burtram C.; Willemse, CCoronaviridae family is composed of a number of ribonucleic acid (RNA)-containing viruses currently classified into two genera, the coronavirus and torovirus. The family is classified together with the Arteviridae in the order Nidovirales. Coronaviruses are enveloped single stranded positive sense RNA viruses about 80-160 nm in diameter. The coronavirus is, as in the case of all positive sense RNA virus, a messenger, and the naked RNA is infectious. The 5′-two thirds of the genome encodes for a polyprotein that contains all the enzymes necessary for replication, whereas the 3′-one third encodes for all the structural proteins that mediate viral entry into the host cell. The structural proteins include spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins.Nucleocapsid protein is one of the most crucial structural components of coronaviruses;hence major attention has been focused on characterization of this protein. Some laboratories have demonstrated that this protein interferes with different cellular pathways, thus implying it to be a key regulatory component of the virus (Zakhartchouk, Viswanathan et al. 2005). Furthermore, it has been shown that severe acute respiratory syndrome (SARS)-N protein interacts with cellular proteins, including cyclophilin A (CypA), heterogenous nuclear ribonucleoprotein (hnRNP) A1, human ubiquitin-conjugating enzyme, cyclin dependent kinase (CDK)-cyclin complex protein, Ikappaßalpha (IkBα), cytochrome (Cyt) P450 etc. For the purpose of this study, the focus is based on CypA interaction with human coronavirus (HCoV) NL63-N protein. These interactions might play a role in the pathology of HCoV-NL63. Using glutathione-S-transferase (GST), the interaction of CypA with the nucleocapsid protein can be clearly demonstrated to be direct and specific. Since the N protein is involved in viral RNA packaging to form a helical core, it is suffice to say that both NL63-N and CypA are possibly within the HCoV-NL63 replication/transcription complex and NL63-N/human CypA interaction might function in the regulation of HCoV-NL63 RNA synthesis. In addition, the results will demonstrate that HCoV-NL63-N has only a specific domain for interacting with CypA.Item Cloning and expression of the M-gene from the human coronavirus NL-63 in different expression systems(University of the Western Cape, 2008) Lubbe, Lizel; Fielding, Burtram C.; Dept. of Medical BioSciences; Faculty of ScienceRespiratory tract infections are one of the leading causes of morbidity and mortality across the world. This is especially true for infants, young children, the elderly and the immunocompromised. The strain placed on economies and health care systems of all countries by these diseases are phenomenal. Although we are familiar with various agents leading to these kinds of infections (e.g. rhino-, influenza-, parainfluenza, human metapneumo-, respiratory syncytial-, adeno- and coronaviruses), the cause of a substantial portion, (48-70%) of cases remain unidentified (Van der Hoek et al, 2004; Fouchier et al, 2004; File, 2003; Fine et al, 1999; Shay et al, 1999, Henrickson et al 2004; Murray et al 2001). In the past, human coronaviruses have not been known to cause severe disease in humans. For this reason, little research was performed on these viruses, with research focusing on the animal coronaviruses that are of veterinary importance. However, with the outbreak of SARS in 2003, the field of human coronavirus research has received significantly more attention. Also, the subsequent identification of two additional novel human coronaviruses (NL63 and HKU1) has led to an increased awareness of the potential threat of these viruses. With the discovery of these new human coronaviruses, it has become clear that the potential for another outbreak by a yet unknown human coronavirus is a very real possibility. This has made research into the pathogenesis and the role of the various coronavirus genes in the pathogenesis of these viruses of utmost importance. HCoV-NL63 was first discovered in January 2003 in the Netherlands. It causes upper and lower respiratory tract disease in young children, the elderly and immunocompromised individuals. The disease is also associated with croup and has even been implicated as a possible cause of the childhood vascular ailment Kawasaki Disease. HCoV-NL63 is frequently found in combination with other respiratory viruses leading to superinfections. It is still unclear whether HCoV-NL63 is an opportunistic virus or whether it leads the way for co-infection with other respiratory viruses. This particular virus is also the only coronavirus sharing the same cellular receptor as SARS-CoV. The virus is found all over the world and has been identified in countries like Australia (Arden et al, 2005), Japan (Ebihara et al., 2005; Suzuki et al., 2005), Belgium (Moës et al., 2005), Hong Kong (Chiu et al., 2005), Taiwan (Wu et al.,2007) Korea (Choi et al., 2006), Canada (Bastien et al., 2005), France (Vabret etal., 2005), Switzerland (Kaiser et al., 2005; Garbino et al., 2006), Germany (Vander Hoek et al., 2005), Sweden (Koetz et al., 2006) and South Africa (Smuts andHardie, 2006). In this study, the HCoV-NL63 genome was transcribed from RNA to DNA from which the M gene was amplified with various primers designed for use in specific expression systems. The various genes were cloned into the pGEM vector and confirmed by sequencing. The genes were now expressed in cloning vectors suited for each expression system (pFastBac for baculovirus expression, pFlexi for bacterial expression and pCMV for mammalian expression). Clones were sequenced for a second time. The recombinant clone in pFlexi was expressed in KRX cells and a 36hr time course was performed. The recombinant pFastBac clone was used to infect Sf9 insect cells and P1 and P2 viral stocks were obtained. The recombinant pCMV clone was used to transfect Cos1 mammalian cells. The genome was successfully transcribed and the M gene amplified and cloned into pGEM and confirmed by sequencing. Subsequent cloning of the various M genes into pFastBac for baculovirus expression, pFlexi for bacterial expression and pCMV for mammalian expression was achieved and sequencing confirmed the presence of the inserts in frame. pFlexi clones were successfully expressed in bacterial KRX cells with expression of the M protein in the pellet of the lysed bacterial cells. No M protein was seen in the supernatant of the lysed cells. Sf9 insect cells were infected with the recombinant pFastBac clones and P1 and P2 viral stocks were obtained. Protein expression occurs in KRX bacterial cells with optimal expression at approximately 24 hours. The M protein expresses on the cell membrane as can be concluded from the product obtained in the pellet of the lysed bacterial cells. Very little of the expressed protein is present in the plasma of the cell as evidenced by the absence of protein in the supernatant of the lysate.Item Comparative analysis of human coronavirus-NL63 ORF3 protein homologues(Academic Journals, 2009) Fielding, Burtram C.; Suliman, TasnimIt has been reported in some studies that the newly discovered human coronavirus NL-63 (HCoVNL63) is one of the most common coronaviruses associated with acute respiratory infections. HCoVNL63 was first isolated in 2004 from a 7 month old infant in Holland. The HCoV-NL63 genome encodes for one accessory protein, ORF3. This reports the computational analysis of human coronavirus NL63 ORF3 by comparing the amino acid sequences of coronavirus ORF3-homologues. The HCoV-NL63 ORF3 gene was found to encode a putative protein ~25.6 kDa in size. ORF3 was predicted to contain three potential transmembrane regions. The amino acid sequence of HCoVNL63 ORF3 was shown to be most similar to HCoV 229E ORF4 (43% identity; 62% similarity).Item The Coronavirus Nucleocapsid is a Multifunctional Protein(MDPI, 2014) McBride, Ruth; van Zyl, Marjorie; Fielding, Burtram C.The coronavirus nucleocapsid (N) is a structural protein that forms complexes with genomic RNA, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly. Recent studies have confirmed that N is a multifunctional protein. The aim of this review is to highlight the properties and functions of the N protein, with specific reference to (i) the topology; (ii) the intracellular localization and (iii) the functions of the protein.Item Covid-19 AMA (Ask Me Anything)(University of the Western Cape, 2020) Fielding, Burtram C.; Kiewit, Lester; Goldstein, SueItem COVID-19 schools of thought(SABC, 2020) Fielding, Burtram C.Interview with Prof Bertram C. Fielding.Item COVID-19 schools of thought(University of the Western Cape, 2020) Fielding, Burtram C.Item Decoding COVID-19(University of the Western Cape, 2020) Fielding, Burtram C.