No description available.

<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

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.

No description available.

Thesis (M. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

As aves silvestres são consideradas importantes reservatórios de diversos vírus aviários que podem afetar aves comerciais. Dessa forma, o monitoramento das aves silvestres é fundamental para garantir a sanidade dos plantéis avícolas brasileiros. Nos últimos anos, o número de espécies de aves nas quais os coronavírus aviários foram encontrados aumentou vertiginosamente em diversos países. Contudo, poucos estudos envolvendo a detecção de coronavírus aviários em aves de cativeiro e aves silvestres ou sinantrópicas foram realizados no Brasil. Assim, o presente estudo teve como objetivo identificar a presença dos coronavírus aviários em aves silvestres no Brasil e caracterizá-los molecularmente. As amostras foram testadas através do teste de RRT-PCR para detecção do gene UTR do IBV, bem como uma nested-PCR para detecção do gene S1 dos coronavírus aviários. O sequenciamento de alto desempenho foi utilizado para caracterizar os vírus detectados. No total, foram testadas 300 amostras de aves silvestres (147 suabes orofaringeanos e 153 suabes cloacais). No total, 27 amostras foram positivas pelo teste RRT-PCR. Duas amostras de Anseriformes das amostras positivas no teste de RRT-PCR foram selecionadas para sequenciamento de alto desempenho. Em ambas as amostras sequenciadas foi constatada a co-infecção pelos vírus da bronquite infecciosa e vírus da doença de Newcastle. A análise das amostras demonstrou alta identidade com vírus vacinais, o que demonstra que estirpes vacinais utilizadas na imunização de aves de produção circulam em aves silvestres e de produção de subsistência. / Wild birds are an important reservoir of different viruses that can affect poultry. Viral surveillance in wild birds is, thus, extremely important to ensure the poultry heal in Brazil. In recent years, the number of species of birds in which avian coronaviruses have been found skyrocketed in several countries. However, few studies involving the detection of avian coronaviruses in captive wild birds or wild life birds were conducted in Brazil. Thus, the present study aimed to identify the presence of avian coronaviruses in Brazil and characterize them molecularly. Samples were tested by RRT-PCR test for detection of the UTR gene of IBV, as well as a nested-PCR for detection of S1 gene. In total, 300 samples of wild birds (147 oropharyngeal swabs and 153 cloacal swabs) were tested. In total, 27 samples were positive in RT-PCR assay. Two positive samples in RRT-PCR assay were selected for Next-generation sequencing. In both sequenced samples, co-infection with infectious bronchitis virus and Newcastle disease virus was found. The analysis of samples showed identity with vaccinal strains used in immunization of commercial flocks circulate in wild birds and subsistence flocks.