Universiẗat, Diss., 2004--Frankfurt (Main).

University, Diss., 2005--München.

The present study was aimed at the improvement of Chinese wheat cultivars for resistance to Soil-borne wheat mosaic virus (SBWMV) by transgenesis, and an examination of virus movement by studying the complementation of movement proteins (MPs) from Tobacco mosaic virus (TMV) and SBWMV using a TMV-based virus vector. A number of SBWMV coding sequences, including the functional coat protein (CP) gene, dysfunctional MP gene, and functional and dysfunctional replicase complex sequence, were cloned into an expression vector pUbi35S and the expression of those viral genes under the control of maize ubiquitin-1promoter was tested in transfected tobacco protoplasts. Parameters for the bombardment of immature embryos using the biolistic gun were optimised through transient expression of a chimeric pglucuronidase (<i>uid</i>A) gene. Transgenic wheat for the model variety Bob White and three Chinese semi-winter wheat cultivars were obtained by microprojectile bombardment of pre-cultured immature embryos using achimeric bar gene as the selectable marker. Plants were regenerated under phosphinothricin (PPT) selection. The transgenic nature of the regenerated wheat plants was demonstrated by Southern hybridization analysis. Expression of viral CP gene was confirmed from most CP-transgenic wheat by RT-PCR testing, while only one CP-transgenic plant produced CP at a detectable level. The meiotic stable transmission of transgenes in several transgenic lines was confirmed by polymerase chain reaction (PCR) analysis of R1 progenies. <i>ELISA </i>testing of several selected transgenic lines mechanically inoculated with <i>Chinese wheat mosaic virus</i> (CWMV), a new member of <i>Furovirus </i>genus, revealed that most of the lines were infected by the virus, while some plants from several lines appeared not to be infected. A new selection approach based on the phosphomannose isomerase (<i>pmi</i>) gene as the selectable marker, and mannose as the selective agent,was developed to produce transgenic wheat plants for both Bob White and Chinese wheat varieties. The introduction of SBWMV CP gene into Chinese wheat cultivars was accomplished using the mannose selection system, and Southern analysis confirmed the integration of transgenes in the genome of transgenic wheat. An expression vector was constructed by cloning the putative SBWMV MP coding sequence into a TMV-based vector in which the native TMV MP gene was rendered defective by open reading frame shift. When using this chimeric virus to infect tobacco plants, it was found that the 37 kDa protein (p37) encoded by SBWMV could be functionally complementary to the 30kDa of TMV MP, this also confirmed that the p37 is the MP of SBWMV.

Dissertação (mestrado) - Universidade de Brasilia. Departamento de Biologia Vegetal / Made available in DSpace on 2012-10-15T22:29:47Z (GMT). No. of bitstreams: 0

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The oncolytic virus field is in the midst of strong and sustained growth. The clinical utility of this class of therapeutics has been bolstered in recent years by the rise of immune checkpoint inhibition, which has the potential to work synergistically with oncolytic viruses to increase the scope of patients who respond favourably to therapy. This growth has been further driven by clear industry support with several pharmaceutical companies acquiring or developing oncolytic virus products following the 2015 FDA approval of Talimogene laherparepvec and the generally-accepted potential of immunotherapeutic approaches to cancer treatment. Vaccinia virus is a double-stranded DNA virus with an extensive history of vaccine use in humans and a desirable safety profile. It is a large virus with a complex lifecycle, and its history of use as a vaccine has resulted in the generation of dozens of unique strains. Although it has been studied extensively, much remains unknown about many vaccinia virus gene function(s) and the virus’ interactions with cellular hosts. Vaccinia virus-based oncolytic viruses have been developed, however clinical outcomes thus far have been unsatisfactory. A more complete understanding of vaccinia virus gene functions must therefore precede the effective design of a next-generation vaccinia virus-based oncolytic candidate. With this downstream goal, we sought to (1) understand the unique oncolytic virus-relevant phenotypic properties of five clinical candidate vaccinia virus strains, and (2) generate and characterize a library of single-gene mutants of the Copenhagen strain of vaccinia virus. These studies resulted in the selection of vaccinia virus-Copenhagen as the wild-type strain of choice that will be utilized for future oncolytic virus development. Furthermore, the generation and initial characterization of an 89-member clonal library of vaccinia-Copenhagen single-gene mutants will be an important tool as we seek to generate a next-generation oncolytic virus candidate. Completed characterization studies challenge the role that viral thymidine kinase should play in oncolytic virus design, demonstrate novel functions of the vaccinia virus gene A47L, and provide an understanding of the role of the vaccinia virus gene F15L. These studies also raise the concept of the personalized selection of oncolytic virotherapeutics. This virus library has the potential to increase the fundamental understanding of vaccinia virus biology in this field as well as in the study of vaccine development and pathogen-host interactions.

Approximately half of all adults diagnosed with acute leukemia (AL) relapse after standard chemotherapy, highlighting the need for alternative treatment options. We have previously shown that vaccination with irradiated autologous tumour cells infected with an oncolytic virus (OV) can elicit a durable, tumour specific, T cell- mediated response in a mouse model of AL. In the context of this AL infected cell vaccine (ICV) model, infection of autologous cells ex vivo with an OV is essential for stimulating a lasting immune response. While the murine AL line L1210 can be robustly infected with Maraba MG1, creating a potent infected cell culture, this ICV still has room for improvement as ICV-vaccinated mice with high tumour burden still die from leukemia. Therefore, we sought to utilize a genome wide CRISPR-Cas9 screen to identify genetic factors that mediate OV resistance in this model of AL. L1210 cells stably expressing Cas9 were transduced with the mouse GeCKOv2 library, which contains 130,209 gRNAs against 20,611 genes within the mouse genome. Following selection, cells were treated with Maraba MG1 and genomic DNA from resistant populations was sequenced to identify genes enriched in resistant cells relative to mock treated cells. Our screen identified several genes that mediate susceptibility to OV infection including those involved in viral entry (Ldlr), receptor-mediated endocytosis (Atp6v1g2), intracellular signaling (Cav1), the cytoskeleton (Filip1 and Tmod4), as well as autophagy and exosome production (Atg5). We aim to use the findings from this work to improve therapeutic efficacy in otherwise OV resistant tumour models as well as identify biomarkers, to determine the feasibility of administering an ICV using patient derived tumour cells.

Introdução: O Vírus da Hepatite B (HBV) é um vírus de DNA com tropismo por hepatócitos, que apresenta um genoma circular parcialmente dupla fita. Baseado na divergência de sequência do genoma completo, dez linhagens evolutivas, denominadas “genótipos” de HBV, foram descritas (A-J), sendo F e H considerados como “indígenas” da América. Os genótipos de HBV apresentam uma forte estruturação geográfica, o que pode refletir padrões das migrações humanas. Na América do Sul, áreas de alto endemismo incluem a região amazônica, e as maiores taxas de infecção têm sido observadas em populações Nativas Americanas. Embora a forte estruturação geográfica seja indicativa de uma origem antiga, a maioria das análises visando datar a origem dos genótipos “americanos” F e H resulta em datações extremamente recentes que não condizem com eventos históricos relacionados ao HBV. Objetivo: Os objetivos desse trabalho compreendem avaliar o impacto de diferentes taxas evolutivas e da seleção purificadora sobre as estimativas de datação molecular a fim de inferir quais taxas são mais condizentes com a origem do HBV na América; caracterizar o HBV circulante em uma amostra histórica de Nativos Americanos, e discutir os processos históricos que possam ser relevantes para entender os padrões observados. Material e Métodos: Nós realizamos análise Bayesiana utilizando sequências disponíveis dos genótipos F e H e diferentes taxas evolutivas previamente reportadas, e comparamos a ocorrência de mutações sinônimas e não-sinônimas em ramos da filogenia classificados como “antigos” ou “recentes” a fim de inferir a atuação da seleção purificadora ao longo do tempo. Para caracterização do HBV presente nas populações Nativas Americanas, detecção e amplificação do DNA viral foi obtida através de PCR seguido de sequenciamento e análise filogenética. Análise Bayesiana de Skyline Plot foi realizada para comparar a dinâmica populacional do subgenótipo A1 presente na amostra de Nativos Americanos e em outras cepas isoladas no Brasil. Resultados e conclusão: Nossos resultados mostram que as estimativas de datação molecular são fortemente influenciadas pelas taxas evolutivas utilizadas na análise. Além disso, foi observado excesso de mutações não-sinônimas nos ramos recentes da filogenia, o que é compatível com a ocorrência de seleção purificadora, e pode gerar um viés sobre as estimativas, produzindo datações recentes demais. Na amostra de Nativos Americanos, nós constatamos o predomínio do subgenótipo A1, relacionado com populações africanas. Análise de Skyline Plot mostrou que a expansão populacional nas cepas isoladas de Nativos Americanos é mais recente que aquela inferida para outras cepas brasileiras, sugerindo que os processos históricos que contribuíram para a formação do subgenótipo A1 dos Nativos Americanos são relacionados com ondas migratórias mais recentes em direção à região amazônica. / Introduction: Hepatitis B virus (HBV) is a hepatotropic DNA virus that presents a partially double-stranded circular genome. Based on sequence divergence of the complete genome, ten HBV evolutionary lineages, called “genotypes” have been described (A-J), with F and H being considered as indigenous from the Americas. HBV genotypes present a remarkable geographic structure which may reflect historic patterns of human migrations. In South America, areas of high endemism include the Amazon basin, and high prevalence rates have been observed in Native American populations. Although the strong geographical structure indicates an ancient origin, most analysis trying to date the origin of the “American” genotypes F and H result in extremely recent dates that disagree with historical events related with HBV. Objective: The aims of this study comprise evaluate the impact of different evolutionary rates and of the purifying selection on molecular dating estimates in order to infer which rates are in better agreement with the origin of HBV in the Americas; to characterize the HBV circulating in a historical sample of Native Americans, and discussing the historical processes that might be relevant to understand the observed patterns. Materials and Methods: We performed a Bayesian analysis using the available sequences of F and H genotypes and different evolutionary rates previously reported, and compared the occurrence of synonymous and non-synonymous mutations in branches of phylogeny classified as “old” or “young” in order to infer the effects of purifying selection over time. For the characterization of HBV from Native American populations, detection and amplification of viral DNA were obtained through PCR followed by sequencing and phylogenetic analysis. Bayesian Skyline Plot analysis was performed to compare the population dynamics of the A1 subgenotype present in the sample of Native American and in other strains isolated from Brazil. Results and Conclusion: Our results show that molecular dating estimates are strongly influenced by the evolutionary rate assumed in the analysis. In addition, we observed an excess of non-synonymous mutations in recent branches of phylogeny, which is compatible with the occurrence of purifying selection and may create a bias on the estimates, producing too recent datings. In the sample of Native Americans, we observed a predominance of the A1 subgenotype, related with African populations. Skyline Plot analysis showed that population expansion in strains isolated from Native Americans is more recent than that inferred from other Brazilian strains, suggesting that the historical processes that contributed to the presence of A1 subgenotype A1 Native Americans are related with more recent migratory waves towards the Amazon region.

La résistance au séchage des virus dépend de plusieurs facteurs : le type de support, la température, l'humidité et la composition de l'enveloppe ou de la capside. Un virus déposé sur une surface peut conserver son pouvoir infectieux pendant plusieurs jours voire plusieurs mois si les conditions sont réunies. Inactiver des virus présents sur différentes surfaces est un enjeu majeur dans la lutte contre la propagation des maladies nosocomiales. Cette problématique est importante et elle nécessite de réaliser des travaux pour connaître davantage la sensibilité des virus aux substances et produits utilisés dans ce but. [...] Dans le cadre de cette thèse des méthodes ont été développées pour comparer l'activité virucide de désinfectants sur des virus en suspension ou sur une surface (acier inoxydable ou support non tissé-fonctionnalisé). Deux approches ont été mises en oeuvre : la première afin de tester l'activité virucide de supports préalablement fonctionnalisés, la seconde afin de comparer l'activité virucide de produits sous forme liquide ou diffusés par voie aérienne à l'encontre de virus en suspension ou séchés sur un support solide. [...] / The resistance of viruses to drying relies on several factors : the type of surface, temperature, humidity and the compositon of the viral envelope or capsid. A virus adsorbed on a surface can remain infectious for several days or months if the conditions are met. [...] In this thesis, methods were developed to compare the virucidal activity of disinfectants towards viruses in suspension and on surfaces (stainless steel or non-woven fuctionalized textiles). Two approaches were implemented : the first to test the virudical activity of functionalized surfaces

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2014. / Made available in DSpace on 2015-04-29T21:01:48Z (GMT). No. of bitstreams: 1 328079.pdf: 2882148 bytes, checksum: 0d8f6b84f74603e020abbf3892564514 (MD5) Previous issue date: 2014 / Pacientes imunodeprimidos podem apresentar infecções virais com evolução rápida, sintomatologias atípicas graves e muitas vezes fatais, sendo fundamental um diagnóstico precoce para estabelecimento do tratamento efetivo, redução da toxicidade e da resistência aos antivirais. HSV-1, HSV-2 e poliomavírus BK são vírus de importância clínica para imunodeprimidos e podem levar a rejeição de órgãos em transplantados. Assim, o objetivo deste trabalho foi desenvolver um sistema celular repórter, utilizando a proteína fluorescente GFP, para HSV-1 e 2 e implantar uma qPCR utilizando amostras clínicas de pacientes transplantados renais para detecção de poliomavírus BK. O sistema celular repórter foi construído através da transfecção de células Vero com o vetor pZsGreen1-1 ligado ao promotor ICP10 (F3R3 e F4R3) da RR1 do HSV-2. A regulação da expressão da GFP via ICP10 é dependente da infecção viral e acontece por meio da proteína viral transativadora VP16 e de fatores celulares Oct-1 e HCF-1. A efetividade do sistema foi avaliada por infecção viral e pela aplicação de antivirais (Aciclovir, ácido gálico, convalotoxina e extrato de Uncaria sp.) e candidatos antivirais inativos (Extrato de Passiflora edulis e derivados de cardenolídeos). O sistema repórter F4R3 ZsGreen1-1 expressou GFP em função da infecção por HSV-1 e 2, a qual foi detectada por microscopia de fluorescência e/ou citometria de fluxo. Em análise por citometria de fluxo, a fluorescência do sistema repórter correlacionou-se diretamente com os títulos virais (MOI de 4,0 x10-3 a 3,3 x10-4, ou seja, 1 partícula viral a cada 250 a 3000 células), o sistema manteve a capacidade de expressão da GFP na presença de agentes sem propriedade antiviral e não expressou fluorescência quando tratado com antivirais. O sistema F4R3 ZsGreen1-1 mostrou-se um sistema funcional com possíveis aplicações para diagnóstico clínico, para elaboração de testes de resistência aos antivirais e para a pesquisa de novos medicamentos. A qPCR para poliomavírus BK foi implantada utilizando amostras de DNA cedidas pelo HEMOSC com iniciadores dirigidos para o antígeno T viral. O limite de detecção foi de 18 cópias genômicas/ reação com quantificações variando entre 9,8 x 105 a 6,7 x 107 cópias genômicas/ mL. A qPCR foi efetiva para análises de amostras clínicas e apresentou limite de detecção suficiente para avaliação de risco de nefropatia em transplantados renais.<br> / Abstract : Immunosuppressed patients can present viral infections with fast evolution, severe atypical symptomatologies and often fatal, being essential the early diagnosis for the establishment of effective treatments, reduction of toxicity and development of resistance to antiviral. HSV-1, HSV-2 and polyomavirus BK are virus of clinical importance for immunosuppresed and can lead to the rejection of transplanted organs. Therefore, the aim of this work was to develop a reporter cellular system, using the fluorescent protein GFP, for HSV-1 and 2, and deploy a qPCR using clinical samples from patients submitted to renal transplant, for the detection of polyomavirus BK. The reporter cellular system was constructed through the transfection of Vero cells with the vector pZsGreen1-1 connected to the promoter ICP10 (F3R3 and F4R3) of the RR1 of the HSV-2. The regulation of the expression of GFP via ICP10 is dependent of the viral infection and happens through the viral transactivating protein VP16, and the cellular factors Oct-1 and HCF-1. The effectivity of the system was evaluated by viral infection and through the application of antiviral (Acyclovir, gallic acid, convalotoxina and extract of Uncaria sp.) and inactive antiviral candidate (Extract of Passiflora edulis and derivatives cardenolide). The reporter system F4R3 ZsGreen1-1 expressed GFP as a function of the infection for HSV-1 and 2, which was detected by fluorescence microscopy and/or flow cytometry. In flow cytometry, the fluorescence of the reporter system was directly correlated with virus titers (MOI 4,0 x10-3 to 3,3 x10-4, that is, 1 viral particle to each 250 to 3000 cells), the system maintained the ability to GFP expression in the presence of agents without antiviral property and no expressed fluorescence when treated with antivirals. The system F4R3 ZsGreen1-1 revealed a functional system with possible applications for clinical diagnosis, elaboration of tests of resistance to the antiviral and for new drugs research. The qPCR to polyomavirus BK was deployed using DNA samples provided by HEMOSC with primers directed to the viral antigen T. The limit of detection was 18 genome copies /reaction with quantification ranging between 9.8 x 105 to 6.7 x 107 genome copies /mL. The qPCR was effective for the analyses of clinical samples and presented enough sensitivity for risk evaluation of nephropathy in renal transplant.