<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>

The Severe Acute Respiratory Syndrome (SARS) virus encodes several unusual RNA processing enzymes, including Nsp15, an endoribonuclease that preferentially cleaves 3? of uridylates through a Ribonuclease A-like mechanism. Crystal structures of Nsp15 confirmed that the Nsp15 active site is structurally similar to that of Ribonuclease A. These similarities and our molecular docking analysis lead us to hypothesize that previously characterized Ribonuclease A inhibitors will also inhibit the SARS-CoV Nsp15. Benzopurpurin B, C-467929, C-473872, N-36711, N-65828, N-103018 and Congo red were tested for effects on Nsp15 endoribonuclease activity. A real-time fluorescence assay revealed that the IC50 values for inhibiting Nsp15 were between 0.2 ?M and 40 ?M. Benzopurpurin B, C-473872, and Congo red are competitive inhibitors, according to kinetic studies and were demonstrated to bind SARS-CoV Nsp15 by a differential scanning fluorimetry assay. Benzopurpurin B also inhibited the Nsp15 orthologs from two other coronaviruses: mouse hepatitis virus (MHV) and infectious bronchitis virus. The three compounds reduced infectivity of MHV in L2 cells by 8 to 26 fold. The more effective drugs also caused a decrease in MHV RNA accumulation.

An infectious cDNA clone of Vero cell adapted Beaudette strain of IBV was constructed using in vitro assembly of cDNA fragments. The entire genome of IBV was RT-PCR amplified into seven fragments, with each piece overlapping about 10 nucleotides. The fragments were ligated and transcribed to synthesize RNA, which was transfected into BHK-21 cells. These cells were then overlaid onto IBV susceptible Vero cells. After five days transfection, the virus was successfully rescued from the transfected cells. The cDNA clone from our laboratory strain has a five nucleotide insertion not present in the originally sequenced virus, resulting in total genome size of 27,613 nucleotides. The infectious cDNA clone was further manipulated to demonstrate its potential as a gene transfer vector, by replacing the ORF5a open reading frame with enhanced green fluorescent protein. The recombinant infectious cDNA clone was also successfully rescued after three days transfection of BHK-21 cells followed by co-culturing with Vero cells. This study showed that the 5a protein, whose function is not known, is not necessary for in vitro IBV replication. This study also showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

abstract / Microbiology / Master / Master of Philosophy

<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>

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 198-221). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-18Bitstream added on 2014-06-13T18:31:18Z : No. of bitstreams: 1 rosa_acg_me_araca.pdf: 260018 bytes, checksum: 83d376eb61dc20b26646b886718270e8 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo descreve o desenvolvimento e a aplicação da reação de transcrição reversa in situ em cadeia da polimerase (RT-PCR in situ), para detectar a co-infecção experimental de perus de 1-dia de idade com Coronavirus (TCoV) e Astrovirus (TAstV-2) isolados de casos clínicos no Brasil. A primeira etapa da reação consistiu na preparação específica de sondas de DNA biotiniladas homólogas ao gene da polimerase viral do TAstV-2 e da região 3'UTR do TCoV. Foram utilizados cortes histológicos de intestino correspondendo ás regiões do íleo, junção íleo-ceco e ceco para avaliar a reação de RT-PCR in situ. Para permeabilização tecidual uma digestão foi aplicada com 10 μg/μl proteinase K por 30 min. Na etapa de hibridização, as sondas de DNA homólogo às regiões genômicas virais ligadas à biotina foram diluídas na concentração de 2μg/μl na solução de hibridização e incubadas overnight à 42ºC. Em seguida, uma diluição ótima do anticorpo monoclonal anti-biotina acoplado a fosfatase alcalina e a peroxidase foram aplicados, para TAstV-2 e TCoV, respectivamente. O substratos diaminobenzidina 3,3 (DAB) e FastRed Ò foram utilizados para identificar a hibridização das regiões homólogas correspondentes ao TCoV e TAstV-2, respectivamente. A reação positiva foi visualizada por deposição de pigmentos vermelhos (TAstV-2) e marrom acastanhado (TCoV). Em relação à localização dos genes virais amplificados, foram confirmados nas células da base (células caliciformes) e ao longo das vilosidades intestinais principalmente no citoplasma dos enterócitos de forma difusa para ambos os vírus. Marcações positivas também foram evidenciadas na submucosa próximas às regiões com intensa congestão vascular... / This study describes the development and application of the reaction in situ reverse transcription polymerase chain reaction (RT-PCR in situ) to detect co-infection of turkeys to experimental 1-day-old with Coronavirus (TCoV) and Astrovirus ( TAstV-2) isolated from clinical cases in Brazil. The first step of the reaction has been to prepare specific biotinylated DNA probes homologous to the viral polymerase gene of TAstV-2 and the 3'UTR region of TCoV. We used histological sections of intestine corresponding to the regions of the ileum, ileum-cecum junction and cecum to evaluate the reaction of RT-PCR in situ. For permeabilization tissue digestion was applied with 10 g / uL proteinase K for 30 min. In step hybridization, DNA probes homologous to the viral genomic regions linked to biotin were diluted to the concentration of 2μg/μl in hybridization solution and incubated overnight to 42 ° C. Then, an optimal dilution of monoclonal anti-biotin coupled to alkaline phosphatase and peroxidase were applied to TAstV-2 and TCoV, respectively. 3.3 The substrate were used to identify the hybridization ofÒdiaminobenzidine (DAB) and FastRed homologous regions corresponding to TCoV and TAstV-2, respectively. The positive reaction was visualized by deposition of red pigment (TAstV-2) and brown brown (TCoV). Concerning the location of the amplified viral genes was confirmed by the base cells (goblet cells) and along the intestinal villi in the cytoplasm of enterocytes diffusely to both viruses. Tags positive were also demonstrated in the submucosa close to the areas with intense vascular congestion. In conclusion, the RT-PCR in situ standard in this study showed good ability to detect viral RNA promoting a desirable... (Complete abstract click electronic access below)