<p>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.</p>

<p>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.</p>

Magister Scientiae - MSc / Respiratory 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. / South Africa

Masters of Science / The Coronaviridae family consists of RNA viruses within the order Nidovirales. The family is classified into two genera, namely the corona- and toroviruses. Coronaviruses are enveloped, single stranded, positive sense RNA viruses with genomes ranging between 27-32kb in size. The 5’ two-thirds of the genome encodes for the 1a/b polyprotein, while the 3’ one-third of the genome encodes for the structural proteins that mediate viral entry into the host cell. These structural proteins include the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The nucleocapsid protein is expressed at high levels within an infected cell. Studies have shown that this protein plays a key regulatory role in different cellular pathways, including the inhibition of interferon production and the up-regulation of the AP1 signal transduction pathway, amongst others. Also, the N protein is vital in the formation of the ribonucleocapsid core by binding to the viral RNA during virion assembly. The focus of this study is the immune response in whole blood cultures to the presence of human coronavirus (HCoV) NL63 N protein. To characterise the stimulation of the immune activity against HCoV-NL63 N in blood cultures, the HCoV-NL63 N gene was expressed in a bacterial system. In this pilot study, GSTtagged N constructs were then purified and used to treat whole blood cultures from three volunteers. ELISAs were used to measure the cytokine response in these treated whole blood cultures. Results showed that the nucleocapsid protein has an inflammatory response on whole blood cultures. These results have generated vital information in the potential function of the HCoV-NL63 N protein on the immune system. It is suffice to say that the HCoV-NL63 N protein is able to elicit an effective inflammatory response within the host cell. Future studies into the cellular pathways affected by the HCoV-NL63 N protein will clarify its exact role in stimulating the host immune system.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Coronaviridae 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.

<p>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.</p>

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / 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)&nbsp / 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&nbsp / 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&nbsp / 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&nbsp / 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 &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / 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&nbsp / 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&nbsp / 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&nbsp / 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&nbsp / Western Cape.</p>

<p>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.</p>

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / 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)&nbsp / 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&nbsp / 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&nbsp / 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&nbsp / 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 &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / 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&nbsp / 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&nbsp / 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&nbsp / 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&nbsp / Western Cape.</p>

>Magister Scientiae - MSc / Acute respiratory infections (ARI) continue to be the leading cause of acute illnesses worldwide and remain the most important cause of infant and young children mortality. Many viruses such as rhinoviruses, influenza viruses, parainfluenza viruses, respiratory syncytial viruses, adenoviruses and coronaviruses are deemed to be the etiological agents responsible for ARI’s in children. The recently discovered coronaviruses HCoV-HKU1 and HCoV-NL63 contribute significantly to the hospitalization of children with ARI’s. HCoV-NL63 was first identified in 2004, as the pathogen responsible for the hospitalization of a 7 month old child presenting with coryza, conjunctivitis and fever. Since then a significant amount of knowledge has been gained in the clinical spectrum on this virus, however HCoV-NL63 is still not well characterized on the molecular and proteomic level. This dissertation focuses on bringing about this characterization by cloning the HCoV-NL63 Nucleocapsid gene to be expressed in a bacterial system and transfecting the Nucleocapsid, Membrane and Envelope genes into a Mammalian cell culture system in order for its respective proteins to be expressed. With the use of Bioinformatic analytic tools certain characteristics of HCoV-NL63 Nucleocapsid, Membrane and Envelope proteins are able to be identified, as well as certain motifs and/or regions that are important in the functioning of these proteins. By comparing the results obtained for HCoV-NL63 N,M and E to other well studied coronavirus homologous will enlighten us on the potential role(s) of these proteins in determining HCoV-NL63 pathogenicity and infectivity. vi Although certain functions of these proteins can be deduced by the means of bioinformatics analysis, it is still imperative for it to be extensively characterized In Vitro. This will therefore form a fundamental step in the development of many other projects, which unfortunately fall outside the scope of this M.Sc thesis.