Browsing by Author "Mnyamana, Yanga Eddie"

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    Antibody production with synthetic peptides against human Coronavirus-NL63 ORF3 protein
    (University of the Western Cape, 2025) Mnyamana, Yanga Eddie
    Human Coronavirus (HCoV) NL63 was detected in an infant in the Netherlands in 2004 and will hereafter be referred to as CoV-NL63. Four years later, it was classified into Group 1 coronaviruses by the International Committee for Taxonomy of Viruses (ICTV), which was modified into alphacoronavirus in 2009. It appears that CoV‐NL63 originates from bats and its hosts are bats and palm civets. A 27,553‐bp CoV‐NL63 genome structure and the spike glycoprotein (S1 domain) use angiotensin‐converting enzyme 2 (ACE2) for infectious entry. Furthermore, CoV‐NL63 simplifies binding to ACE2 by attaching to heparan sulphate proteoglycans on the cell surface. ACE2 has a significant expression in airway differentiated epithelial cells and CoV‐NL63 enter these cells from their apical surface. It is imperative to note that CoV‐NL63, CoV‐229E, CoVHKU1 and CoV‐OC43 infect polarized epithelial cells, and the entry and release of these viruses are related to apical cells in addition to respiratory signs, there are two non-respiratory symptoms including gastroenteritis and Kawasaki disease, both of which are associated with CoV‐NL63. Moreover, neurological manifestations of CoV-NL63 have been reported, especially in children. The main aim of this research study was to generate monoclonal antibodies (mAbs) against the human coronavirus NL63 Open Reading Frame 3 (ORF3) protein. Firstly, bioinformatic tools were used to assist in identifying antigenic determinants and antigenic epitopes were used to generate peptides of ORF3 protein. Thereafter, the ORF3 protein was characterized using various molecular techniques across the bacterial and mammalian expression systems. A CoV-NL63 full-length ORF3 protein (Protein Identity Number: ABE97138.1) was utilized for the synthesis of the peptide from the N-terminus and peptides conjugated with a MAP; KLH and MAP+KLH linkage on the carboxyl end. The mAbs were generated in vivo by immunizing BALB/c mice. Antigen-specific ELISA assessed the antibody specificity and sensitivity. The mAbs that exhibited the highest specificity and sensitivity for antigenic peptide (s) were used to characterize the ORF3 protein by Western blot and immunofluorescence analysis (IFA).
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    Expression of human coronavirus NL63 and SARS-CoV nucleocapsid proteins for antibody production
    (University of the Western Cape, 2012) Mnyamana, Yanga Eddie; Fielding, Burtram C.; McBride, Ruth; Dept. of Medical BioSciences
    Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV) showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi ® vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the Western Cape.