<p>Human Coronavirus NL-63 is a respiratory virus with a high incidence rate, causing mild respiratory infections in children under the age of 18. The outbreak of Sever Acute Respiratory&nbsp / Syndrome (SARS) in 2003 sparked increased interest into the field of coronavirology and respiratory diseases subsequently led to the discovery of this novel Human Coronavirus (HCoV) by a group of scientists in Holland. The membrane protein (M) of NL-63 has been shown to interact with the nucleocapsid, spike and envelope proteins of the virus when expressed ex vivo. In contrast, the envelope protein (E) is shown to exhibit ion channel activity, interacts with the membrane protein during the formation of viral-like particles. The functions of the open reading frame 3 (ORF3) proteins remains a mystery. Research does, however, indicate that this protein is needed for in vivo infectivity and pathogenesis. Bioinformatic analysis indicates that both the ORF3 and M proteins posses at least 3&rsquo / C-terminal transmembrane regions. To further characterize the biological activity of these three proteins in clinical and laboratory samples, sensitive and specific antibodies are required. Thus, the antigenic regions of ORF3, M and the entire E gene were amplified by PCR and ligated into a bacterial expression vector for expression and subsequent generation of antibodies in a mouse system. The identities of the cloned genes were confirmed by sequencing before being expressed in an in vitro bacterial system. Western Blots were used to identify the expression of the 41kDa, 42kDa and 34kDa GST-tagged viral proteins which were consistent with the bioinformatically predicted protein species. Verified fusion proteins were expressed in large quantities, quantified and concentrated for in vivo antibody production. Inoculation of 9 healthy, female Balb/C mice with the purified fusion proteins yielded high titers of polyclonal antibodies. Western Blotting was once again used to validate the production of the antibodies before their specificity was quantitatively measured using a modified competition ELISA.</p>

<p>Human Coronavirus NL-63 is a respiratory virus with a high incidence rate, causing mild respiratory infections in children under the age of 18. The outbreak of Sever Acute Respiratory&nbsp / Syndrome (SARS) in 2003 sparked increased interest into the field of coronavirology and respiratory diseases subsequently led to the discovery of this novel Human Coronavirus (HCoV) by a group of scientists in Holland. The membrane protein (M) of NL-63 has been shown to interact with the nucleocapsid, spike and envelope proteins of the virus when expressed ex vivo. In contrast, the envelope protein (E) is shown to exhibit ion channel activity, interacts with the membrane protein during the formation of viral-like particles. The functions of the open reading frame 3 (ORF3) proteins remains a mystery. Research does, however, indicate that this protein is needed for in vivo infectivity and pathogenesis. Bioinformatic analysis indicates that both the ORF3 and M proteins posses at least 3&rsquo / C-terminal transmembrane regions. To further characterize the biological activity of these three proteins in clinical and laboratory samples, sensitive and specific antibodies are required. Thus, the antigenic regions of ORF3, M and the entire E gene were amplified by PCR and ligated into a bacterial expression vector for expression and subsequent generation of antibodies in a mouse system. The identities of the cloned genes were confirmed by sequencing before being expressed in an in vitro bacterial system. Western Blots were used to identify the expression of the 41kDa, 42kDa and 34kDa GST-tagged viral proteins which were consistent with the bioinformatically predicted protein species. Verified fusion proteins were expressed in large quantities, quantified and concentrated for in vivo antibody production. Inoculation of 9 healthy, female Balb/C mice with the purified fusion proteins yielded high titers of polyclonal antibodies. Western Blotting was once again used to validate the production of the antibodies before their specificity was quantitatively measured using a modified competition ELISA.</p>

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Human Coronavirus NL-63 is a respiratory virus with a high incidence rate, causing mild respiratory infections in children under the age of 18. The outbreak of Sever Acute Respiratory Syndrome (SARS) in 2003 sparked increased interest into the field of coronavirology and respiratory diseases subsequently led to the discovery of this novel Human Coronavirus (HCoV) by a group of scientists in Holland. The membrane protein (M) of NL-63 has been shown to interact with the nucleocapsid, spike and envelope proteins of the virus when expressed ex vivo. In contrast, the envelope protein (E) is shown to exhibit ion channel activity, interacts with the membrane protein during the formation of viral-like particles. The functions of the open reading frame 3 (ORF3) proteins remains a mystery. Research does, however, indicate that this protein is needed for in vivo infectivity and pathogenesis. Bioinformatic analysis indicates that both the ORF3 and M proteins posses at least 3 C-terminal transmembrane regions. To further characterize the biological activity of these three proteins in clinical and laboratory samples, sensitive and specific antibodies are required. Thus, the antigenic regions of ORF3, M and the entire E gene were amplified by PCR and ligated into a bacterial expression vector for expression and subsequent generation of antibodies in a mouse system. The identities of the cloned genes were confirmed by sequencing before being expressed in an in vitro bacterial system. Western Blots were used to identify the expression of the 41kDa, 42kDa and 34kDa GST-tagged viral proteins which were consistent with the bioinformatically predicted protein species. Verified fusion proteins were expressed in large quantities, quantified and concentrated for in vivo antibody production. Inoculation of 9 healthy, female Balb/C mice with the purified fusion proteins yielded high titers of polyclonal antibodies. Western Blotting was once again used to validate the production of the antibodies before their specificity was quantitatively measured using a modified competition ELISA. / South Africa

<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

Philosophiae Doctor - PhD / Human Coronavirus NL63 (HCoV-NL63) is one of seven coronaviruses (CoVs) that cause respiratory disease in the global population. The Membrane (M) and Nucleocapsid (N) proteins are part of the core CoV-structural proteins, crucial in viral replication and virion assembly. Here the expression of HCoV-NL63 M and N was characterized across multiple in vitro systems including bacterial, insect and mammalian. To detect untagged proteins in viral structural studies, anti-peptide antibodies were generated in a mouse model. Polyclonal antisera and hybridoma-secreted antibodies exhibited specific binding to their respective full length protein antigens. Anti-peptide monoclonal antibodies were successfully generated against the HCoV-NL63 M and N proteins. During CoV infection, the interaction of CoV M and N is necessary for the production of infectious virions. For the first time, co-expressed, full length HCoV-NL63 M and N were assayed for protein-protein interaction in a mammalian cell system, allowing for native protein folding and modification. M protein formed higher order homomultimers in the presence and absence of co-expressed N.

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.

<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>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>