Doctor of Philosophy / Department of Chemical Engineering / Peter Czermak / Peter H. Pfromm / Large-scale insect cell culture utilizing the baculovirus expression vector system (BEVS) can be used to produce biopharmaceuticals such as vaccines and therapeutic proteins. Biopharmaceutical production processes are generally complex and sensitive to many process parameters and changes, but on-line monitoring in this area is relatively limited and the fundamental understanding of the intricate relationships between significant process parameters and the process outcome, especially on the multi-liter or multi-m3 scale, is rarely conclusive. Dielectric spectroscopy (DS), which is based on the frequency dependent measurement of the passive dielectric properties of materials, was applied to large-scale insect cell cultures infected with a baculovirus under low multiplicity of infection conditions to produce a recombinant protein of the virus-like particle class. DS not only allowed the qualitative monitoring of the infection and recombinant protein production process within the culture in real-time but also the detection of important culture events, e.g. the peak in baculovirus production/concentration. Additionally, DS seemed to be able to serve as a predictive tool for the overall recombinant protein yield early in the process. Partial Least Square models were successfully developed allowing monitoring of the cultures progress in terms of cell density, size, and even nutrient concentration replacing the need for discrete sampling and therefore reducing contamination risks. In summary, DS has been demonstrated to have the potential to increase bioprocess understanding and the repeatability of recombinant protein production in the BEVS but ultimately also to satisfy the increased requirements for process monitoring as delineated recently in the Process Analytical Technology initiative by the Food and Drug Administration.

The application of variety of virological techniques to the study of Wesselsbron virus hos resulted in the compiling of much worthwhile information on the biophysical, biochemical and serological properties of this virus. Wesselsbron virus , like most arboviruses, showed marked sensitivity to the action of bother ether and sodium deoxycholate.

Thèse d'exercice : Médecine. Médecine interne : Université de Nantes : 2003. / Bibliogr. f. 51-55 [43 réf.].

Thèse doctorat : Virologie : Paris 12 : 2003. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 209 réf.

Thèse (M.Sc.)--Université Laval, 2009. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Influenza A virus is one of the most common human pathogens which caused considerable disease burdens through annual epidemics and occasional pandemics. The consequences vary from mild to severe or even fatal. What are the host and viral elements which determine the consequence of infection? In the past 15 years, several avian influenza A viruses including H5N1, H9N2, H7N7 and H7N9 subtypes were found to cross host barrier and infect humans. Question about how avian influenza A viruses gained the ability to replicate in human cells remains unanswered. Studies on host factors associated with virus replication would provide important information for understanding host restriction, virus pathogenesis and for antiviral drug development. Nuclear factor 90 (NF90) is a host protein identified in our previous study to inhibit influenza A virus replication. Antiviral activity of NF90 was also found for other viruses. However, detailed mechanisms for the antiviral function of NF90 remains largely unknown. This study is focused on NF90’s antiviral functions through exploring its relationship with PKR activation and stress granules formation using influenza A virus as a model. I characterized the interaction between NF90 and PKR, and showed the C-terminal of NF90 interacts with PKR in an RNA-binding dependent manner. Using transient and stable NF90 knockdown cells, I found that NF90 is required for PKR activation upon stimulation by dsRNA or infection with a NS1 mutated virus. PKR activation leads to the formation of stress granules and stall of protein translation. I found that NF90 is a core component of stress granules, which may underlie the mechanism for the antiviral activity of NF90. However, NF90 may also complete with PKR for RNA binding and regulate PKR activation. To further delineate the interaction between NF90 and PKR by using influenza A virus, my study constructed a panel of NS1 mutant viruses which were attenuated in antagonizing specific host antiviral pathways. I characterized the NS1 123-127 mutant virus which is unable to inhibit PKR phosphorylation but retained other functions unaffected. It was demonstrated that NF90 mediates PKR-dependent antiviral pathway since NS1 123-127 mutant virus replicated to a comparable level as wild type virus in the NF90 knockdown but not scramble knockdown 293T cells or in the interferon deficient Vero cells. This study for the first time found NF90 serves as a regulator of PKR antiviral pathway. To understand the mechanism for NF90 inhibition of influenza A virus replication, I found that NP, but not the other polymerase subunits, of influenza A virus was targeted to the stress granules. Since NF90 interacts with NP, it is reasonable to postulate that NF90 mediates the localization of NP, and possibly viral mRNA, to the stress granules in order to inhibit influenza A virus replication through regulation of proteins synthesis. In summary, my study provided comprehensive evidence to support a novel NF90-PKR antiviral pathway and suggests that NF90 may play critical roles to balance PKR phosphorylation in response to virus infection in cells. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The aims of this study were to investigate the role of the diversity of dengue virus populations in changing patterns of virus transmission and disease. Prior to the commencement of this study, dengue 2 virus (DENV-2) had been associated most frequently with severe disease, so the study commenced with this serotype. Because it was not possible to quantitate diversity in the entire 11 kb of the viral genome, the study focussed on the envelope (E) gene, because the E protein is the major protein on the surface of the virion and thus might be under strong selective pressure from the host immune system and from the requirement to engage specific receptors on host cells. This study was the first direct quantification of the diversity of dengue virus populations in individual hosts. The nucleotide sequences of more than 70 per cent of the E genes in each virus population differed from the consensus nucleotide sequence for the population. In the course of quantitating genetic diversity in DENV-2 virus populations in patients and in mosquitoes, recombinant DENV-2 and both parental virus populations were detected in a single mosquito. This was the first such report. In 2001, just after the commencement of this study, Myanmar had the largest outbreak of dengue on record. Unlike previous outbreaks, 95 per cent of dengue viruses isolated from patients were of a single serotype, DENV-1. Despite the large number of cases of dengue, the proportion of patients with severe dengue was low. In the light of these observations, the direction of this study changed to focus on DENV-1. Phylogenetic analysis of the E genes of DENV-1 collected before and after the 2001 dengue outbreak suggested that some time before 1998, an early lineage of DENV-1 had become extinct and had been replaced by two new lineages. There was no evidence that these changes were due to selection or to recombination within the E protein genes of the old clade of viruses and the newly introduced viruses. A more detailed analysis was undertaken, of the entire genome of 11 human DENV-1 isolates and of 4 from mosquitoes recovered in Yangon between 1971 and 2002, to determine whether the extinction of the pre-1998 lineage of DENV-1 (clade A) and the appearance of the two new lineages (clades B and C) could have been due to selective pressures acting on genes other than E. Evidence of only weak selection was found in the NS5 gene (at amino acids 127,135 and 669) but the resultant amino acid changes did not distinguish all recent viruses from viruses belonging to the extinct clade. The phylogenetic relationships between individual genes from these viruses and between the open reading frames were similar. No evidence was found of recombination that might have given rise to two new clades of virus with enhanced fitness. Collectively, these data suggested that the extinction of clade A viruses and their replacement by the two new clades, between 1998 and 2000 was a stochastic event in an inter-epidemic period when rates of virus transmission were low. This was the first report of such an extinction of a lineage of DENV-1 and its replacement by new lineages. At about the same time as the 2001 outbreak of DENV-1 infection in Myanmar, an outbreak of DENV-1 began in the Pacific. A comparison of the nucleotide sequences of the E genes of viruses from the Pacific with those of viruses from throughout south-east Asia suggested that the outbreak in the Pacific was due to the introduction of multiple genotypes of DENV-1 from Asia and that some of these DENV-1 could have originated in Myanmar. The principal observations from this study are: - (a) Dengue virus populations in individual hosts are extremely heterogenous and may contain a significant proportion of non-infectious genomes. (b) Intra-serotypic recombination between dengue viruses may be far more common than the literature suggests but it may not be detected because of the almost universal use of consensus nucleotide sequences. (c) Significant changes in dengue virus genotypes that occur at single localities may be due to genetic bottlenecks rather than to selection or to recombination. (d) Dengue viruses can be transported more than 10,000 km to cause outbreaks in non-endemic areas. Key words: Dengue viruses, diversity, recombination, selection, genetic bottleneck

Thesis (M.S.)--University of Wyoming, 2007. / Title from PDF title page (viewed on June 10, 2009). Includes bibliographical references (p. 97-102).

Made available in DSpace on 2014-06-11T19:28:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2003-01Bitstream added on 2014-06-13T19:58:15Z : No. of bitstreams: 1 cezar_ma_me_botfca.pdf: 763015 bytes, checksum: ee0660882c5afc5f1bb0880fde94205f (MD5) / O objetivo deste trabalho foi caracterizar isolados de tobamovírus coletados em campos de produção comercial de pimentão e pimenta nas regiões de Lins, Sorocaba e Salto, Estado de São Paulo. A coleção de isolados foi submetida a testes sorológicos utilizando-se antissoros policlonais para o ToMV e PMMoV e em seguida à purificação biológica por meio de lesões monolesionais em Nicotiana glutinosa, onde algumas das lesões foram transferidas para Nicotiana cleevelandi, hospedeira multiplicadora de tobamovírus. Os isolados foram mantidos em plantas de Nicotiana cleevelandi por meio de sucessivas passagens mecânicas. Os isolados foram inoculados em uma série diferencial de Capsicum spp: C. annuum cv. ECW (L+L+); C. annuum cv. Magda (L+L+); C. annuum cv. Tisana (L1L1); C. frutescens cv. Tabasco (L2L2); C. chinense PI 159236 (L3 L3); C. chacoense PI 260429 (L4L4). Foram inoculados três plantas de cada genótipo diferencial no estádio de primeira folha verdadeira e estas avaliadas durante 21 dias. Realizou-se a identificação molecular destes isolados utilizando-se oligonucleotídeos universais e específicos para as espécies de TMV, ToMV e PMMoV em reação de RT-PCR de uma só etapa, utilizando-se a transcriptase reversa AMV e a Taq DNA polimerase. A especificidade dos oligonucleotídeos foi inicialmente avaliada por meio de comparação da seqüência nucleotídica dos oligonucleotídeos com a do genoma de espécies de tobamovírus disponíveis no Genbank.O teste biológico confirmou a presença de tobamovírus nas amostras coletadas a partir de pimentão e pimenta, entretanto não permitiu diferenciar as espécies de vírus presentes. De acordo com os resultados obtidos através da inoculação dos isolados em genótipos diferenciais, observou-se que os isolados Pe-01, Pe-03, Pe-05 e Pe-12... . / The objective of this work was to characterize virus isolates belonging to the Tobamovirus genus collected from commercial sweet and hot peppers fields surrounding the cities of Lins, Sorocaba and Salto, São Paulo State. The collection of isolates was subjected to serological detection using ToMV and PMMoV antisserums, followed by single local lesion passages in Nicotiana glutinosa. Some of the local lesion isolates were selected and inoculated in Nicotiana cleevelandi plants, a propagative host of tobamoviruses. The isolates were maintained in Nicotiana cleevelandi plants through successive sap-inoculations. These isolates were further inoculated on the differential genotypes of Capsicum spp: C. annuum cv. ECW (L+L+); C. annuum cv. Magda (L+L+); C. annuum cv. Tisana (L1L1); C. frutescens cv. Tabasco (L2L2); C. chinense PI 159236 (L3 L3); C. chacoense PI 260429 (L4L4). Three plants of each differential genotypes were inoculated at the first leaf stage, and evaluated during 21 days. The same isolates were submitted to the molecular identification using degenerated and specific primers for the TMV, ToMV and PMMoV species, in a one step RTPCR protocol, using the AMV reverse transcriptase and the Taq DNA polymerase. The specificity of the primers where first evaluated by a nucleotide sequence alignment of the primers with the sequence of the genome of tobamovirus disposable in the Genbank. The biological test has confirmed the presence of tobamovirus in the sweet pepper and hot pepper samples, but could not differentiate the species of virus. Using the differential genotypes of Capsicum spp, the isolates Pe-01, Pe-03, Pe-05 andPe-12 could be classified as belonging to the P1-2 pathotype. The isolate Pe-08 belongs to the P1 and Pe-02, Pe-04, Pe-06, Pe-07, Pe-09, Pe-10, Pe-11 and Pe-13 as P0 pathotype. By the RT-PCR the isolates Pe-06, Pe-07, Pe-09, Pe-10... (Complete abstract, click electronic address below).

Orientador: Renate Krause Sakate / Resumo: O objetivo deste trabalho foi caracterizar isolados de tobamovírus coletados em campos de produção comercial de pimentão e pimenta nas regiões de Lins, Sorocaba e Salto, Estado de São Paulo. A coleção de isolados foi submetida a testes sorológicos utilizando-se antissoros policlonais para o ToMV e PMMoV e em seguida à purificação biológica por meio de lesões monolesionais em Nicotiana glutinosa, onde algumas das lesões foram transferidas para Nicotiana cleevelandi, hospedeira multiplicadora de tobamovírus. Os isolados foram mantidos em plantas de Nicotiana cleevelandi por meio de sucessivas passagens mecânicas. Os isolados foram inoculados em uma série diferencial de Capsicum spp: C. annuum cv. ECW (L+L+); C. annuum cv. Magda (L+L+); C. annuum cv. Tisana (L1L1); C. frutescens cv. Tabasco (L2L2); C. chinense PI 159236 (L3 L3); C. chacoense PI 260429 (L4L4). Foram inoculados três plantas de cada genótipo diferencial no estádio de primeira folha verdadeira e estas avaliadas durante 21 dias. Realizou-se a identificação molecular destes isolados utilizando-se oligonucleotídeos universais e específicos para as espécies de TMV, ToMV e PMMoV em reação de RT-PCR de uma só etapa, utilizando-se a transcriptase reversa AMV e a Taq DNA polimerase. A especificidade dos oligonucleotídeos foi inicialmente avaliada por meio de comparação da seqüência nucleotídica dos oligonucleotídeos com a do genoma de espécies de tobamovírus disponíveis no Genbank.O teste biológico confirmou a presença de tobamovírus nas amostras coletadas a partir de pimentão e pimenta, entretanto não permitiu diferenciar as espécies de vírus presentes. De acordo com os resultados obtidos através da inoculação dos isolados em genótipos diferenciais, observou-se que os isolados Pe-01, Pe-03, Pe-05 e Pe-12... (Resumo completo, clicar acesso eletrônico abaixo). / Abstract: The objective of this work was to characterize virus isolates belonging to the Tobamovirus genus collected from commercial sweet and hot peppers fields surrounding the cities of Lins, Sorocaba and Salto, São Paulo State. The collection of isolates was subjected to serological detection using ToMV and PMMoV antisserums, followed by single local lesion passages in Nicotiana glutinosa. Some of the local lesion isolates were selected and inoculated in Nicotiana cleevelandi plants, a propagative host of tobamoviruses. The isolates were maintained in Nicotiana cleevelandi plants through successive sap-inoculations. These isolates were further inoculated on the differential genotypes of Capsicum spp: C. annuum cv. ECW (L+L+); C. annuum cv. Magda (L+L+); C. annuum cv. Tisana (L1L1); C. frutescens cv. Tabasco (L2L2); C. chinense PI 159236 (L3 L3); C. chacoense PI 260429 (L4L4). Three plants of each differential genotypes were inoculated at the first leaf stage, and evaluated during 21 days. The same isolates were submitted to the molecular identification using degenerated and specific primers for the TMV, ToMV and PMMoV species, in a one step RTPCR protocol, using the AMV reverse transcriptase and the Taq DNA polymerase. The specificity of the primers where first evaluated by a nucleotide sequence alignment of the primers with the sequence of the genome of tobamovirus disposable in the Genbank. The biological test has confirmed the presence of tobamovirus in the sweet pepper and hot pepper samples, but could not differentiate the species of virus. Using the differential genotypes of Capsicum spp, the isolates Pe-01, Pe-03, Pe-05 andPe-12 could be classified as belonging to the P1-2 pathotype. The isolate Pe-08 belongs to the P1 and Pe-02, Pe-04, Pe-06, Pe-07, Pe-09, Pe-10, Pe-11 and Pe-13 as P0 pathotype. By the RT-PCR the isolates Pe-06, Pe-07, Pe-09, Pe-10... (Complete abstract, click electronic address below). / Mestre