Magister Scientiae (Medical Bioscience) - MSc(MBS) / The human coronavirus NL63 was discovered in 2004 by a team of researchers in Amsterdam. Since its discovery it has been shown to have worldwide spread and affects mainly children, aged 0-5 years old, the immunocompromised and the elderly. Infection with HCoV-NL63 commonly results in mild upper respiratory tract infections and presents as the common cold, with symptoms including fever, cough, sore throat and rhinorrhoea. Lower respiratory tract findings are less common but may develop into more serious complications including bronchiolitis, pneumonia and croup. The primary function of the HCoV-NL63 nucleocapsid (N) protein is the formation of theprotective ribonucleocapsid core. For this particle to assemble, the N-protein undergoes N-N dimerization and then interacts with viral RNA. Besides the primary structural role of the Nprotein, it is also understood to be involved in viral RNA transcription, translation and replication, including several other physiological functions. The N-protein is also highly antigenic and elicits a strong immune response in infected patients. For this reason the N-protein may serve as a target for the development of diagnostic assays. We have used bioinformatic analysis to analyze the HCoV-NL63 N-protein and compared it to coronavirus N-homologues. This bioinformatic analysis provided the data to generate recombinant clones for expression in a bacterial system. We constructed recombinant clones of the N-protein of SARS-CoV and HCoV-NL63 and synthesized truncated clones corresponding to the N- and C-terminal of the HCoV-NL63 N-protein. These heterologously expressed proteins will serve the basis for several post-expression studies including characterizing the immunogenic epitope of the N-protein as well identifying any antibody crossreactivity between coronavirus species. / South Africa

>Magister Scientiae - MSc / 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. / South Africa