Rozprawy doktorskie: „Orf virus”

1

Gemeniano, Maria Lourdes Charmaine. "Characterization of Orf-A of feline immunodeficiency virus /". For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2002. http://uclibs.org/PID/11984.

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Westphal, Dana, i n/a. "Characterisation of a novel inhibitor of apoptosis expressed by Orf virus". University of Otago. Department of Microbiology & Immunology, 2008. http://adt.otago.ac.nz./public/adt-NZDU20080922.162136.

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Apoptosis plays important roles in host defences against virus infection. It is therefore not surprising that viruses have developed a vast array of modulators that block this process at different stages within the apoptotic pathways. Intrestingly, Orf virus (ORFV), a member of the Parapoxvirus genus, did not reveal any of the known poxviral inhibitors of apoptosis, but was found to express a unique anti-apoptotic protein, ORFV125. The aim of this PhD project was to determine the subcellular localisation of this protein and to further characterise its anti-apoptotic activity. This included exploring its ability to inhibit early, intermediate and late events of apoptosis and identifying the mechanism by which this viral protein functions to prevent cell death. Experiments revealed that ORFV125 was localised to the mitochondria through a C-terminal mitochondrial-targeting motif, and this specific location was necessary for the protein�s anti-apoptotic function. Furthermore, the viral protein inhibited UV-induced apoptotic events at and downstream of the mitochondria such as cytochrome c release, caspase activation and DNA fragmentation. However, it was not able to prevent UV-induced activation of the c-Jun-NH₂ kinase (JNK), an event occurring upstream of the mitochondria, consistent with its localisation to this organelle. The ability to prevent apoptosis was comparable with that of the cellular anti-apoptotic protein Bcl-2, which belongs to a family of mitochondrial regulators of apoptosis. Although standard BLAST analysis failed to detect homology to anti-apoptotic members of the Bcl-2 family, a manual alignment of the primary sequence of ORFV125 with these proteins revealed characteristic residues of Bcl-2 homology (BH) domains within ORFV125. These motifs are conserved within the Bcl-2 proteins and important for their structure and function. In addition, mutating amino acids within the ORFV125 BH domains led to a loss of the anti-apoptotic function of the mutated proteins, indicating the functional importance of these residues for the viral protein. These observations suggest that ORFV125 might be classified as a viral Bcl-2-like protein. To provide evidence for this hypothesis, it was investigated if ORFV125 acts in a Bcl-2-like manner to inhibit apoptosis. The viral protein was able to entirely block the activation of the pro-apoptotic Bcl-2 family members Bak and Bax, although it did not directly bind to these proteins. Instead, ORFV125 interacted with a subset of the pro-apoptotic BH3-only proteins, which can trigger the activation of Bax and Bak. Furthermore, this study demonstrated that ORFV125 could inhibit apoptosis induced by BH3-only proteins to which the viral protein could bind. On the other hand, ORFV125 was not able to prevent the activity of pro-apoptotic proteins that it failed to interact with. This shows that ORFV125�s mechanism of action is to inhibit the activity of BH3-only proteins by binding and neutralising their function. Overall, these results provided evidence that ORFV125 is potent anti-apoptotic protein that can prevent UV-induced cell death without the participation of other ORFV proteins. Furthermore, the viral protein shared primary sequence and secondary structure similarities to Bcl-2 family members and acted in a Bcl-2-like manner to inhibit apoptosis.

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3

McKeever, Declan James. "Studies of the immunology and epidemiology of orf". Thesis, University of Edinburgh, 1987. http://hdl.handle.net/1842/30488.

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Mo, Min, i n/a. "Characterization of an Orf virus RING-H2 protein, B5L : a mimic of cellular anaphase promoting complex subunit 11". University of Otago. Department of Microbiology & Immunology, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090220.085825.

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The anaphase promoting complex (APC/C) is an ubiquitin ligase that is an essential regulator of multiple steps in the cell cycle. The complex consists of at least 12 subunits with a catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. The Parapoxvirus, Orf virus (OV), encodes a RING-H2 protein, B5L, with clear sequence similarities to APC11. The disruption of APC/C function leads to pre-mature entry into S phase and a delayed M phase exit and, potentially, apoptosis. This investigation explored the functional significance of the similarity between B5L and APC11 and specifically sought to determine if B5L manipulates cell cycle regulation by targeting APC/C function. Co-immunoprecipitation experiments from lysates of cells expressing a range of constructs revealed an interaction between B5L and APC2 in the same manner as seen with APC11. Furthermore, B5L was found to associate with endogenous APC/C. However, although APC11 promoted the formation of polyubiquitin chains in substrate-independent in vitro assays, B5L was inactive in this assay. Bioinformatics comparisons of APC11 and other known RING ubiquitin ligases with B5L and its poxviral homologues revealed some subtle differences. In particular a domain of APC11 (amino acids 61-74), that is essential for its ubiquitin ligase activity is not conserved in B5L or its homologues. When this APC11 domain was incorporated in place of the corresponding region of B5L (amino acids 59-67), the mutated B5L acquired ubiquitin ligase activity. On the other hand, APC11 protein in which the domain was replaced with that of B5L lost ubiquitin ligase activity. Stable cell lines expressing B5L showed an increased number of cells in G2/M phase (30�4%) compared with cell lines expressing APC11 (11�2%, n=3, p<0.05, ANOVA, Tukey�s), consistent with impaired APC/C function. APC/C substrates such as cyclin A, cyclin B and the thymidine kinase were stablized in B5L-expressing cells compared with control cells. Furthermore, transient hyper-expression of B5L induced apoptosis in 25�2% (n=3, p<0.05) of the cell population compared with only 6�1% apoptotic cells when APC11 was hyper-expressed. Analysis of the DNA content of OV-infected cells revealed enhanced DNA synthesis compared with cells infected with a B5L knockout OV. These observations indicate that B5L is a non-functional mimic of APC11. It associates with APC/C, but lacks ubiquitin ligase activity, and hence disrupts APC/C function. These abilities may enable OV to induce a cellular environment that enhances viral replication.

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Chand, Puran. "Molecular and immunological characterisation of a major envelope protein of capripoxvirus". Thesis, University of Surrey, 1992. http://epubs.surrey.ac.uk/2774/.

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Analysis of the proteins of capripoxvirus (KS-1) revealed a 32kd protein that is one of the major structural proteins of the virus and is localised in the virus envelope. Monospecific serum prepared against the 32kd envelope protein neutralised the virus indicating that this protein contains neutralising epitopes. Lymphocyte proliferation studies, using the 32kd protein and peripheral blood mononuclear cells from capripoxvirus (KS-i) vaccinated sheep, showed that this protein strongly induced cellmediated immune responses. The 32kd protein is capripoxvirus specific and induced antibodies in early stages of capripoxvirus infections. Immunoblot analysis of antibody responses against this protein has provided a basis for the differential diagnosis of capripoxvirus and orf virus infections. The 32kd protein bound to the surface of cultured lamb testis cells. The binding of the 32kd protein was completely inhibited by prior incubation of cells with purified capripoxvirus (KS-1) but not by bovine serum albumin. Trypsin treatment of capripoxvirus (KS-1) degraded the majority of the 32kd protein with a minimal effect on a few other virus proteins. Trypsin removed an external 10kd fragment from the 32kd protein, leaving a 22kd fragment associated with the virus. In addition, the trypsin treatment reduced the virus infectivity by at least ten fold, suggesting that the cell surface binding domain of the 32kd protein is located within the external 10kd fragment. The monospecific serum to the 32kd protein had no effect of the infectivity titre of the trypsin treated virus further supporting the concept that the external 10kd fragment of the 32kd protein is involved in binding of the virus particle to the cell surface. A degenerate oligonucleotide probe, based on an internal amino acid sequence obtained from V8 protease cleavage products of the 32kd protein, was used to identify the gene encoding the 32kd protein. The gene encoding the 32kd protein was identified within the 2.8kb HindI1l Q1 fragment of the capripoxvirus (KS-1) genome. The nucleotide sequence analysis of the Hindu Q1 fragment revealed five open reading frames (Q11L, Q12R, Q13L, Q14R and Q15L), one of these open reading frames, Q13L, is capable of encoding a 30.6kd protein and contains the complete internal amino acid sequence obtained from the V8 protease cleavage products of the 32kd protein, indicating that the Q13L encodes the 32kd envelope protein of capripoxvirus (KS-1). The deduced amino acid sequence of the Q13L shows a 34.1% identity and 61.3% similarity with that of H3L open reading frame of vaccinia virus.

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6

Dickinson, Victoria Jane. "The cloning and subcellular localisation of maize streak virus ORF V1". Thesis, University of Hull, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321050.

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Lear, Andrea. "The characterisation of the ovine skin response to orf virus infection". Thesis, University of Edinburgh, 1995. http://hdl.handle.net/1842/20625.

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Orf is a highly contagious, eruptive skin disease of sheep and goats caused by a parapox virus. The virus enters through abrasions in the skin, where it replicates in the regenerating epidermal keratinocytes. Despite the generation of a specific antiviral response, orf virus reinfections can be established easily, although the lesions are milder and generally regress more rapidly than after primary exposure. The cutaneous response to orf involves the formation of a dense network of MHC class II⁺ dendritic cells at the lesion. The primary aim of this project was to characterise these dendritic cells and to identify the cytokines produced by orf infected keratinocytes in vitro, that might be involved in the accumulation of the dendritic cells in vivo. In vivo studies of primary and secondary orf virus lesions identified the class II⁺ dendritic cells to be a population of CD1^- cells, which are also found within the dermis of normal ovine skin. A subpopulation of these cells also expressed the antigen, coagulation factor XIIIa. Factor XIIIa⁺ dendritic cells comprised over half the dendritic cells seen in the network of a primary orf lesion but were only observed in small numbers, transiently, in the secondary orf lesion. All the dendritic cells lacked the expression of ovine macrophage markers. The proliferative response of the primary and secondary orf lesions also differed. High proliferative activity was observed in the epidermis and dermis in the primary response to orf but not in the secondary response. A few of the proliferating cells were identified as dendritic cells but it would appear that the dendritic cell network in both primary and secondary orf lesions does not arise by local cell division.

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Cargnelutti, Juliana Felipetto. "Infecção experimental de coelhos e camundongos com o vírus do ectima contagioso". Universidade Federal de Santa Maria, 2010. http://repositorio.ufsm.br/handle/1/10060.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Contagious ecthyma (orf) is a cutaneous disease that affects sheep and goats, and may be occasionally transmitted to humans. The disease is caused by orf virus (ORFV). ORFV infection produces croustous and proliferative lesions, usually on the nostrils and labial commissures of lambs, and also in the udder, teat skin and coronary bands of adults animals. The pathogenesis of ORFV infection is poorly understood and a search for an adequate animal model is required, yet the disease has been already reproduced in sheep, goats and rabbits. This dissertation relates the clinical, virological and pathological aspects of ORFV infection in rabbits and mice experimental inoculated. Ten rabbits, ten mice and two lambs were inoculated intradermally after skin scarification with an hypodermic needle. A viral suspension of ORFV IA-82 strain (108.5TCID50/mL) was inoculated in the internal face of the ear, back skin and labial commissure of rabbits; internal face of the ear of mice. Lambs were inoculated in the labial commissures and in the internal face of hind limbs. All animals were monitored clinically, virologically, and pathologically for 21 days. All rabbits developed clinical signs in the inoculation sites, begining with mild hyperemia that evolved to macules, papules, vesicle, pustules and scabs. Lesions appeared at days 3 and 4 post-inoculation (pi) and lasted to 3 to 10 days. Viral shedding was detected from days 2 to 14pi. Histological examination of lesions revealed focal proliferative dermatitis with ballooning degeneration and eosinophilic intracytoplasmic inclusions in keratinocytes, histological hallmarks of contagious ecthyma in sheep. A similar, albeit much milder clinical course was observed in 5 out of 10 inoculated mice. All lambs presented characteristic contagious ecthyma clinical and histopathologycal lesions from days 3 to 18pi, and the virus was recovered from lesions between days 2 and 19pi. At day 28pi, seroneutralization test (SN) was unable to detect neutralizing antibodies in all inoculated animals. These findings show that ORFV replicates and produce local lesions in rabbits and mice. However, rabbits are more susceptible to infection and disease, and may be used as an animal model to study some aspects of ORFV pathogenesis.
O ectima contagioso (ou orf) é uma doença infecto-contagiosa de pele que afeta principalmente ovinos e caprinos, e que ocasionalmente pode acometer o homem. O seu agente etiológico é o vírus da orf (ORFV). O ORFV produz lesões proliferativas, geralmente na comissura labial e no plano naso-labial de cordeiros, e também na pele do úbere, nos tetos e no rodete coronário dos cascos de animais adultos. A patogenia da infecção pelo ORFV é pouco conhecida, embora a doença já tenha sido reproduzida em ovinos, caprinos e coelhos. Essa dissertação relata os achados clínicos, virológicos e histopatológicos da infecção experimental de coelhos e camundongos pelo ORFV. Para isso, coelhos, camundongos e cordeiros foram inoculados pela via intradérmica (ID), após escarificação da pele com agulha hipodérmica. A inoculação dos cordeiros serviu como controle positivo. Uma suspensão viral da cepa IA-82 do ORFV (108,5DICC50/mL) foi inoculada na face interna da orelha, na pele do dorso e na comissura labial dos coelhos; na face interna da orelha dos camundongos; e na comissura labial e face interna do membro pélvico dos cordeiros. Os animais foram monitorados por 21 dias nos aspectos clínicos, virológicos e patológicos. Todos os coelhos inoculados apresentaram lesões semelhantes nos locais de inoculação, iniciando com hiperemia, evoluindo para máculas, pápulas, vesículas, pústulas e crostas. Os sinais surgiram 3 a 4 dias pós inoculação (pi) e duraram por 3 a 10 dias. Excreção viral foi detectada entre os dias 2 e 14pi. A análise histológica das lesões revelou dermatite focal proliferativa, com degeneração balonosa e corpúsculos de inclusão intracitoplasmáticos eosinofílicos nos queratinócitos, semelhante às alterações histológicas observadas nos cordeiros. Lesões similares, mas de menor intensidade foram observadas em 5 de 10 camundongos. Os cordeiros, utilizados como controles positivos, apresentaram lesões clínicas e histopatológicas características de ectima contagioso entre os dias 3 e 18pi, sendo que o vírus foi recuperado das lesões entre os dias 2 e 19dpi. No dia 28pi, pelo teste de soroneutralização (SN), não foram detectados anticorpos neutralizantes no soro dos animais inoculados. Esses resultados demonstram que a inoculação de ORFV resulta em replicação viral e produção de lesões em coelhos e camundongos, porém a doença é reproduzida de forma mais consistente em coelhos. Portanto, sugere-se que coelhos possam ser utilizados como modelos para estudos in vivo com o ORFV.

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Coelho, João Nuno Santos. "Molecular characterization and functional analysis of ORF P1192R from African swine fever virus". Doctoral thesis, Universidade de Lisboa. Faculdade de Medicina Veterinária, 2016. http://hdl.handle.net/10400.5/10907.

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Tese de Doutoramento em Ciências Veterinárias. Especialidade de Ciências Biológicas e Biomédicas
African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA arbovirus and the single member of the family Asfarviridae. It infects soft ticks of the genus Ornithodoros as well as all members of the family Suidae, representing a global threat for pig husbandry for which there is currently no effective vaccine or treatment. Since the ASFV viral cycle is mainly cytoplasmic, it has been found/predicted to code for many components of the replicative and transcriptional machineries. Of these, and based in sequence homologies, a putative type II DNA topoisomerase-coding ORF (P1192R) was identified in the ASFV genome. DNA topoisomerases are enzymes that modulate the topological state of DNA molecules. They are ubiquitous and essential, participating in processes such as DNA replication, recombination and repair and also in transcription. Since ASFV has a large linear genome, with 170 to 190 kbp depending on the isolate, containing terminal inverted repeats and covalently closed ends, a type II topoisomerase may be indispensable for viral replication and/or transcriptional events. The main objectives of this work were to deepen the study on ORF P1192R and determine if it indeed codes for a type II DNA topoisomerase and, if so, to characterize its activity. Bioinformatics and phylogenetic analyses showed that ORF P1192R is highly conserved among the fourteen ASFV isolates analyzed and, although its amino acid sequence clearly diverges from other type II topoisomerases, the structural organization is preserved and conserved motifs and domains essential for activity are present. Transient expression of GFP-pP1192R in COS-7 cells revealed an exclusively cytoplasmic distribution of the protein, which remained unaltered by treatment with leptomycin B. Using Vero cells or swine macrophages infected with ASFV isolate Ba71V or L60, respectively, expression of pP1192R was observed in the late phase of infection, co-localizing with the viral factories, where the bulk of viral replication and transcription occurs. Heterologous expression of pP1192R in Saccharomyces cerevisiae demonstrated that it functionally complements a top2 thermo-sensitive mutation and that it exhibits ATP-dependent decatenation activity. The purified recombinant pP1192R was found to efficiently decatenate kDNA and to processively relax supercoiled plasmid DNA, which are characteristics of a type II topoisomerase. The optimal requirements in terms of pH, temperature and salt, divalent ions and ATP concentrations for pP1192R activity in vitro were determined and its sensitivity to a panel of topoisomerase poisons and inhibitors was tested. Our results indicate that P1192R may be a target for studying, and possibly controlling, ASFV transcription and replication.
RESUMO - O vírus da peste suína africana (VPSA) é um arbovírus icosaédrico núcleo-citoplasmático de DNA de cadeia dupla, classificado no género Asfivirus da família Asfarviridae, da qual é o único membro conhecido. Este vírus infecta carraças do género Ornithodoros assim como todos os membros da família Suidae, constituindo uma ameaça global para a suinicultura para a qual não existe actualmente qualquer vacina ou tratamento. A prevenção da peste suína africana é feita através de medidas que visam reduzir o risco de introdução de animais ou produtos de origem animal infectados em regiões livres da doença, enquanto o controlo de um surto se baseia exclusivamente em medidas que incluem o abate sanitário de todos os animais susceptíveis na área do foco e a proibição de movimentos e comercialização de animais. Embora o VPSA tenha sido inicialmente descrito como um vírus com replicação exclusivamente citoplasmática, actualmente sabe-se que o núcleo da célula hospedeira é indispensável na fase inicial da infecção. Contudo, a grande maioria do ciclo infeccioso ocorre no citoplasma da célula infectada, não sendo por isso surpreendente que, das 150 a 167 grelhas de leitura aberta (ORF, do inglês “open reading frame”) identificadas no genoma do VPSA, algumas codifiquem para componentes das maquinarias de replicação e de transcrição. Dentre estas, prevê-se, com base em homologia de sequências aminoacídicas, que a ORF P1192R codifique para uma topoisomerase de DNA do tipo II. As topoisomerases de DNA estão presentes em todas as células e são responsáveis pela modulação do estado topológico do DNA, estado esse que se altera durante processos como a replicação, a recombinação e a reparação do DNA, assim como a transcrição, e dos quais resultam torções das moléculas de DNA que, não sendo resolvidas, podem comprometer a integridade genómica e consequentemente a viabilidade celular. Todas as topoisomerases exercem a sua actividade através da criação de quebras no DNA devido ao ataque nucleofílico de um resíduo de tirosina catalítico ao esqueleto fosfodiéster da molécula de DNA, gerandose assim uma ligação fosfotirosina covalente. As topoisomerases são classificadas em dois tipos, tendo por base a forma como quebram a molécula de DNA: as topoisomerases do tipo I, cuja actividade é independente de ATP e que geram quebras em cadeia única no DNA, facilitando assim o desenrolamento; e as topoisomerases do tipo II, que necessitam de ATP para gerar uma quebra nas duas cadeias do DNA, através da qual fazem passar uma dupla cadeia intacta. Considerando que o VPSA tem um genoma linear de grandes dimensões, com 170 a 190 quilopares de bases dependendo do isolado, e que contém repetições terminais invertidas fechadas covalentemente, uma topoisomerase do tipo II pode efectivamente ser essencial para eventos de replicação e/ou transcrição virais. Os objectivos centrais deste trabalho foram os seguintes: (i) realização de um estudo bioinformático e filogenético aprofundado da ORF P1192R do VPSA; (ii) estudo da proteína codificada por esta ORF (pP1192R), através da sua clonagem, expressão em sistema heterólogo, purificação da proteína recombinante e caracterização in vitro da sua actividade; (iii) determinação do efeito sobre a actividade da proteína recombinante dum painel de compostos químicos descritos como sendo inibidores de topoisomerases; (iv) identificação dos níveis de expressão e da localização intracelular da pP1192R em células infectadas pelo VPSA, a diferentes tempos de infecção; (v) avaliação do efeito de mutações dirigidas em resíduos ou motivos identificados como reguladores da actividade enzimática ou localização subcelular da pP1192R, tendo por base a informação gerada nos estudos bioinformáticos acima mencionados. A ORF P1192R do isolado L60 do VPSA foi amplificada por PCR e clonada e a sua sequência nucleotídica foi determinada e utilizada em análises bioinformáticas e filogenéticas. Verificou-se que esta ORF é altamente conservada entre os catorze isolados do VPSA cujo genoma se encontrava disponível nas bases de dados e, embora a sua sequência aminoacídica seja claramente divergente das de outras topoisomerases do tipo II incluídas neste estudo, quer sejam elas de origem procariota, eucariota ou viral, a organização estrutural da proteína está preservada e estão presentes motivos e domínios conservados que são essenciais para a actividade enzimática. O estudo da localização celular da pP1192R iniciou-se com a construção de plasmídeos quiméricos para a expressão da pP1192R em fusão com a proteína verde fluorescente (GFP) ou com uma variante vermelha (RFP). Transfectaram-se transientemente células de linha COS-7 com estas construções tendo-se observado que a proteína de fusão se distribuía exclusivamente pelo citoplasma. Esta distribuição não foi alterada após tratamento com leptomicina B que bloqueia uma das vias de exportação de proteínas do núcleo. Já a infecção das células a expressarem GFP-pP1192R com um isolado do VPSA adaptado a células Vero (Ba71V) induziu uma redistribuição da proteína de fusão, deixando de estar homogeneamente distribuída pelo citoplasma para estar principalmente concentrada nas fábricas virais a partir das 8 horas pós-infecção. Utilizando células de linha Vero infectadas com o isolado Ba71V, utilizado como modelo de infecção, ou macrófagos derivados de monócitos de sangue periférico de suíno (células alvo do vírus na infecção natural) infectados com o isolado virulento L60, e utilizando um soro anti-pP1192R produzido no decurso destes trabalhos, foi possível constatar que a pP1192R viral é produzida na fase intermédia/tardia da infecção (observável a partir das 6/8 horas pós-infecção) e que acumula nas fábricas virais ao longo da infecção. A expressão em sistema heterólogo da pP1192R iniciou-se num sistema procariota, baseado em Escherichia coli, mas embora tenha sido possível obter proteína recombinante em grandes quantidades, a sua purificação só foi conseguida recorrendo a agentes desnaturantes, impedindo a obtenção de proteína activa. Assim, avançou-se para um novo sistema de expressão baseado na levedura Pichia pastoris que apresenta diversas vantagens sobre o anterior, nomeadamente o facto de ser um sistema eucariota e por isso mais semelhante ao contexto em que a pP1192R é expressa em condições naturais. Contudo, neste sistema não foi possível obter proteína recombinante e o sistema foi abandonado. Tentou-se por fim a expressão heteróloga na levedura Saccharomyces cerevisiae. Neste organismo, a utilização das estirpes JCW26 e SD117 que contêm uma mutação termo-sensível no gene que codifica para a topoisomerase do tipo II endógena, permitiu demonstrar, quer in vivo através da complementação da mutação termo-sensível, quer in vitro recorrendo a ensaios funcionais de decatenação, que a pP1192R é efectivamente uma topoisomerase do tipo II funcional. Utilizando ainda S. cerevisiae como sistema de expressão, foi possível obter e purificar pP1192R recombinante para caracterização da sua actividade em ensaios funcionais in vitro. Observou-se que a pP1192R é capaz de relaxar DNA superenrolado, de decatenar DNA catenado e, quando em elevadas concentrações, de catenar DNA plasmídico, não tendo sido detectada actividade de superenrolamento de DNA relaxado. Determinaram-se também as condições óptimas de funcionamento em termos de temperatura, pH e concentrações de sal (NaCl ou KCl), ATP ou iões divalentes (Mg2+, Mn2+, Zn2+, Cu2+ e Ca2+), que foram posteriormente utilizadas para avaliar a sensibilidade da pP1192R recombinante a um painel de inibidores de topoisomerases, entre os quais se incluem drogas frequentemente utilizadas como agentes antimicrobianos ou antitumorais. Dos compostos testados, aqueles para os quais foram obtidos resultados mais promissores, i.e., os que revelaram níveis de inibição mais elevados, foram a coumermicina A1, a doxorubicina, a amsacrina e a genisteína. Pelo contrário, as quinolonas, normalmente utilizadas como antibióticos visando infecções provocadas por organismos procariotas, foram dos compostos com menor eficácia. Em suma, os resultados deste trabalho indicam que a ORF P1192R é um alvo promissor para o estudo e, eventualmente, o controlo dos processos replicativos e transcricionais do vírus da peste suína africana.

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Housawi, Fadhel Mohammed Taher. "Studies on parapoxvirus antigens through the development of monoclonal antibodies to orf virus". Thesis, University of Edinburgh, 1998. http://hdl.handle.net/1842/30287.

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Twenty-five monoclonal antibodies (mabs) against orf virus, a parapoxvirus (ppv), were produced following the immunisation of mice with a lysate of cells infected with orf-11 virus. These mabs, together with 2 others recloned from an earlier fusion, were identified by ELISA and IFT and characterised. No neutralising activity was shown by any of the mabs. The size of orf proteins detected by the mabs was measured using western blotting and radioimmunoprecipitation (RIP). Western blotting was conducted with two types of orf-11 antigen preparations - gradient purified virus or a lysate of orf-11 infected cells. Five mabs detected a protein of approximately 40 kDa with both purified virus and infected lysate antigens. These mabs detected a protein approximately 65 kDa in size, but only with infected cell lysate antigen. In RIP studies, 21 mabs produced bands 13 of which were against the 65 kDa protein, 7 against that 40 kDa protein while one was against a 50 kDa protein. Twenty-one of the 27 mabs reacted with at least two of 18 vaccinia virus orf virus (VVOV) recombinants expressing a library of orf genome fragments of the NZ-2 virus strain. Four of the mabs which had identified the native 40kDa protein reacted with 2 overlapping recombinants (245 and 247). Seventeen of the mabs, 16 of which had identified the native 65 kDa protein recognised three recombinants 79, 285 and 286 all of which contain different inserts from the same region of the orf virus genome. Subsequent sequencing of the overlapping site between recombinants 245 and 247 by New Zealand collaborators has identified a new orf gene, designated FIL which has been shown to be analogous to the H3L vaccinia virus gene which codes for an immunodominant 35 kDa envelope protein. Cells infected with a new VVOV recombinant expressing only the FIL orf gene showed positive fluorescence with 3 or the 4 mabs which reacted with the 245 and 246 recombinants, confirming the target of these mabs is the product of the FIL gene.

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Durantel, David. "Etude des gènes pnk/pnl (ORF 86), lef-4 (ORF 90) et lef-5 (ORF 99) du virus de la polyédrose nucléaire de la tordeuse de la luzerne, "Autographa californica" (AcMNPV) : implication dans l'expression des gènes tardifs". Montpellier 2, 1997. http://www.theses.fr/1997MON20225.

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Les travaux presentes constituent une contribution a la connaissance des genes pnl/pnl (orf 86), lef-4 (orf 90) et lef-5 (orf 99), qui sont impliques directement ou indirectement dans les mecanismes de regulation de l'expression des genes tardifs et tres tardifs chez acmnpv. L'etude de leur expression d'une part et de la fonction des proteines codees d'autre part, a ete entreprise. Les cinetiques d'expression des transcrits au cours du cycle viral dans les cellules sf21, ainsi que les sites d'initiation et de terminaison de la transcription ont ete determines. Ces donnees transcriptionnelles, cumulees avec une analyse de l'activite des promoteurs en cellules non infectees, nous ont permis de montrer que pnk/pnl etait un gene precoce immediat, alors que lef-4 et lef-5 etaient du type precoce retarde. La suppression du gene pnk/pnl du genome d'acmnpv c6 (knock-out) a permis de montrer que ce gene n'etait pas indispensable pour l'execution du cycle viral. Par ailleurs, l'analyse de populations virales a permis de montrer que ce gene etait absent de certaines souches d'acmnpv et d'autres virus comme bmnpv et opmnpv. L'obtention d'un anticorps dirige contre des peptides internes a lef-4, a permis d'une part, d'etudier la cinetique d'expression de cette proteine, et d'autre part de determiner la localisation sub-cellulaire par des techniques immunologiques couplees a l'observation en microscopie confocale et electronique. Differents systemes d'expression ont ete utilises, afin de produire les proteines en quantite importante. En systeme bacterien, des problemes d'insolubilite ont ete rencontres. En complement, le systeme baculovirus a ete utilise pour l'hyper-production de lef-4 et lef-5. Deux virus recombinants, acoverlef-4 et acoverlef-5 ont ete construits. Le virus acoverlef-4 nous a permis de realiser les premieres experiences concernant la determination des domaines fonctionnels de lef-4.

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Onwuka, S. K. "Studies of orf virus replication and the cutaneous cellular response to infection in sheep". Thesis, University of Edinburgh, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.257195.

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Tan, Joanne Li-Ching, i n/a. "Development of Orf virus as a vaccine vector : manipulation of structural proteins for surface display of immunogenic peptides". University of Otago. Department of Microbiology & Immunology, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090427.144304.

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Orf virus (ORFV) has the potential to be developed as a vaccine vector. Its ability to stimulate non-specific as well as specific immune responses in permissive and non-permissive hosts stands it in good stead to be utilised as such a tool. The fusion of immunogenic peptides to vaccinia virus (VACV) structural proteins have been shown to improve their immunogenicity due to presentation of the foreign antigens in a particulate form that can stimulate both B and T cells. The aims of this study were to fuse foreign antigens to ORFV structural proteins to demonstrate proof-of-concept that such surface display could also render the foreign antigens more immunogenic. Little is known about ORFV structure and morphogenesis. When this study commenced, the ORFV genome had recently been sequenced and this revealed a large number of homologues in common with VACV. It was thus assumed that both viruses may share structural similarities and that ORFV also assumes the different morphological forms such as the mature virion (MV) and extracellular virion (EV) that are present in VACV. The MV and EV forms are both infectious, with the EV containing an additional membrane acquired from the trans-Golgi network during viral morphogenesis. Furthermore, specific viral proteins are associated with both the MV and EV membranes. Six ORFV structural proteins ORFV 089, 10 kDa, F1, that are homologues of structural membrane proteins A13, A27 and H3 of VACV MVs, together with ORFV 109, ORFV 110 and B2, that are homologues of structural membrane proteins A33, A34 and F13 of VACV EVs were selected as possible candidates for manipulation. At present, there is some information available only for 10 kDa, F1 and B2. The 10 kDa is required for virus assembly, F1 for mediating cell attachment while B2 has been shown to induce significant antibody responses in sheep. Indeed proteomic analyses predicted similarities in the topologies of all of these proteins with their VACV counterparts. Using this information, preliminary studies were conducted to generate recombinant ORFVs (rORFVs) which had FLAG fused to the terminus of the protein that was exposed on the surface of the virus particle. Three rORFVs 10 kDa, F1L and 110 were successfully generated. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG-10 kDa and FLAG-F1 were displayed on the surface of MV particles whereas FLAG-ORFV 110 could not be detected. Western blot analyses of solubilised recombinant ORFV 110-FLAG particles revealed that FLAG-ORFV 110 was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking whereas FLAG-10 kDa and FLAG-F1 did not appear to be subjected to post-translational modifications. Fluorescent microscopy confirmed the prediction that ORFV 110-FLAG localised to the Golgi in virus-infected cells and immunogold labelling of EVs showed that ORFV110-FLAG became exposed on the surface of EV-like particles as a result of egress from the cell, suggesting that the membranes had been acquired from the Golgi. These modifications also appeared to have minimal effect on the infectivity of these rORFVs. The study was extended by replacing the small FLAG peptide with an immunogenic protein (EG95), derived from the oncosphere of the zoonotic parasite Echinococcus granulosus. This protein is known to confer protection in immunised animals. Three rORFVs were generated in which a truncated version of the protein, EG95[Delta]TM, was fused to 10 kDa in the absence (rORFV 699) or presence (rORFV 700) of a linker, and also to F1 (rORFV 701). Western blot analyses of these solubilised particles demonstrated that the fusion proteins appeared to be post-translationally modified while immunogold labelling using anti-EG95 monoclonal antibodies successfully demonstrated the surface labelling on these rORFVs. In order to test the immunogenicity of these rORFVs, prime-boost experiments in sheep were conducted using rORFVs 699, 700 and 701 and a glutathione-S-transferase (GST-EG95) based vaccine. The results showed the production of EG95-specific antibodies. In particular, antibody production by group rORFV 701 compared favourably with a control group that was primed and boosted by GST-EG95 vaccine. This was despite the slightly slower growth rates of rORFVs 700 and 701 and the decreased infectivity of all three rORFVs discovered in in vitro experiments. In conclusion, these studies indicated the feasibility of this strategy to manipulate ORFV structural proteins for use as an agent for vaccine delivery.

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Deane, David Leslie. "The characterisation of a GM-CSF and IL-2 inhibitory protein encoded by Orf virus". Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23323.

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In this study, the gene encoding the GM-CSF inhibitory factor (GIF) was isolated and mapped to the right terminal quarter of the orf virus genome. The orf virus GIF cDNA was expressed as a secreted protein in Chinese hamster ovarian cells as detected by GM-CSF inhibition ELISA. Recombinant GIF was purified by ovine GM-CSF affinity chromatography and gel filtration. Sequence analysis of the 20 N-terminal amino acids was performed on the purified GIF. The GIF gene encodes a 28 kDa protein that exhibits 32% amino acid sequence similarity to the predicted sequence of the A41L gene product encoded by vaccinia virus. Although the vaccinia virus A41L protein has sequence similarity to the T1 secreted chemokine-binding proteins of leporipoxviruses, its function is not known. GIF did not share any homology with any cytokine receptor molecule identified to date. In contrast to other parapoxvirus immunomodulatory proteins that are products of early viral genes, GIF was found to be the product of an intermediate/late viral gene of orf virus infected cells. GIF formed homodimers and homotetramers in solution and bound ovine GM-GSF with a Kd of 369 pM. In addition GIF bound ovine IL-2 with a Kd of 1.04 nM. Although orf virus infects humans, GIF did not bind human GM-CSF or IL-2. GIF was shown to inhibit the binding of ovine GM-CSF labelled with ¹²⁵I to its receptor on isolated sheep neutrophils and it inhibits the haematopoietic activity of ovine GM-CSF in a soft agar bone marrow colony assay. GIF also inhibited the binding of ovine IL-2 labelled with ¹²⁵I to CD4⁺ T cells and inhibited the stimulatory activity of ovine IL-2 in a T cell proliferation assay. This inhibitory activity was neutralised by a rabbit antiserum raised against purified GIF. GIF was produced in vivo during orf virus reinfection. GIF was detected in skin, localised to the area of orf virus infected cells and in afferent lymph draining the skin site of infection. The presence of GIF, 3-7 days after virus infection was associated with reduced levels of GM-CSF in the lymph plasma and the period of maximum viral replication in the skin.

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Komar, Monica. "Potentiating the Oncolytic Efficacy of Poxviruses". Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/23114.

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Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

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Anziliero, Deniz. "Efeitos do parapoxvirus ovis inativado sobre eventos da resposta imune inata em camundongos". Universidade Federal de Santa Maria, 2013. http://repositorio.ufsm.br/handle/1/4090.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The immunostimulatory properties of inactivated parapoxvirus ovis (iPPVO) have been investigated in different animal species and experimental settings. This study investigated the effects of administration of iPPVO on selected events of the innate response in mice. Neutrophil activation, phagocytic activity of macrophages, serum bactericidal activity, induction and antiviral activity of interferon type I (IFN - I) and expression of several classes of cytokines were assayed following intraperitoneal inoculation of Mus musculus with iPPVO (107 TCID50). Serum from iPPVO-treated animals showed IFN-I activity against murine encephalomyocarditis virus (EMCV) between 6 and 12 hours post infection (hpi), as shown by plaque reduction. A significant activation of neutrophils at 6hpi was observed by NBT reduction test in animals treated with the iPPVO. Peritoneal macrophages from mice treated with iPPVO demonstrated a significant increase (p<0.01) in phagocytic activity against Candida albicans both in vivo (between 12 and 96 hpi) and in vitro (24 and 72 hpi). iPPVO treated mice showed increased serum bactericidal activity against Escherichia coli (p<0.05) at two periods (24 and 72 hpi). A second study evaluated the expression of cytokines in response to inoculation of iPPVO. For this, spleens and serum samples were collected from mice treated with iPPVO at different intervals after inoculation and subjected to quantification of messenger RNA (mRNA) by real time PCR (qRT-PCR) and detection/quantification of serum cytokines by ELISA. Quantification of mRNA identified a significant and transient increase in the expression of various cytokines, with variable magnitude and kinetics. mRNA expression of proinflammatory cytokines (IL-1β, TNF-α and IL-8) peaked at 24 hpi (5.4 times increase) and 48 hpi (3 and 10 times, respectively). A 15-fold increase in expression of INF-γ and 6-fold for IL-12 was observed at 48 and 24 hpi, respectively. An increase in the expression of self-regulatory cytokines (Th2) cells, especially IL-10 and IL-4 was detected at later periods (72 and 96 hpi) with peaks of 4.7 and 4.9 fold, respectively. The determination of the concentration of serum cytokines by ELISA showed an increase in IL-1β, TNF-α, IL- 12, IFN-γ and IL-10 with kinetics similar to that observed by qPCR, especially for IL-1 and INF-γ. In summary, these results demonstrate that inoculation with iPPVO stimulates transiently a number events associated with cellular and humoral innate immune responses. If taken together, these effects would likely contribute for the enhanced resistance to certain pathogens observed in animals treated with iPPVO.
As propriedades imunoestimulatórias do Parapoxvirus ovis inativado (iPPVO) têm sido verificadas em diferentes espécies animais e condições experimentais. No presente trabalho foram investigados os efeitos da administração do iPPVO sobre eventos da resposta inata de camundongos. Ativação de neutrófilos, atividade fagocítica de macrófagos, atividade bactericida do soro, indução e atividade antiviral do interferon tipo I (INF-I) e expressão de várias classes de citocinas foram investigados em diferentes intervalos após inoculação de Mus musculus pela via intraperitonial com iPPVO (dose 107 TCID50). O soro de animais tratados com iPPVO apresentou atividade de INF-I frente ao vírus da encefalomiocardite murina (EMCV) entre 6 e 12 horas pós inoculação (hpi), como demonstrado pela redução significativa de formação de placas virais. Uma significativa ativação dos neutrófilos circulantes foi observada pela técnica de redução do NBT em animais tratados com o iPPVO às 6 hpi. Macrófagos peritoneais de camundongos tratados com iPPVO demonstraram um aumento significativo (p<0,01) na atividade fagocítica frente a Candida albicans tanto in vivo (entre 12 e 96 hpi) quanto in vitro (24 e 72hpi). Camundongos tratados com iPPVO apresentaram aumento na atividade bactericida do soro frente à Escherichia coli (p<0,05) em dois períodos avaliados (24 e 72 hpi). Um segundo estudo avaliou a expressão de citocinas em resposta à inoculação do iPPVO. Para isso, amostras de baço e soro foram coletados de camundongos tratados com iPPVO em diferentes intervalos após a inoculação e submetidas a quantificação de RNA mensageiro (RNAm) por PCR em tempo real (qRT-PCR) e detecção/quantificação de citocinas no soro por ELISA. A quantificação de RNAm permitiu detectar um aumento significativo e transitório da expressão de várias citocinas, com magnitude e cinética variáveis. A expressão de RNAm das citocinas pró-inflamatórias (IL-1β, TNF-α e IL-8) atingiu o pico às 24 hpi (aumento de 5,4 vezes), 48 hpi (3 e 10 vezes, respectivamente). Um aumento de 15 vezes na expressão gênica do INF-γ, e de 6 vezes para a IL-12 foi observado às 48 e 24 hpi, respectivamente. Um incremento na expressão das citocinas auto-regulatórias (Th2), principalmente IL-10 e IL-4, foi detectado em períodos mais tardios (72 e 96 hpi) com picos de 4,7 e 4,9 vezes, respectivamente. A determinação da concentração das citocinas séricas por ELISA revelou um aumento nos níveis de IL-1β, TNF- α, IL-12, INF-γ e IL-10, com uma cinética similar à observada pela técnica de qPCR, especialmente para IL-1β e INF-γ. Em resumo, esses resultados demonstram que o tratamento com iPPVO estimula de forma significativa e transitória uma série de eventos celulares e humorais ligados à resposta imune inata. Esses efeitos, se considerados em conjunto, provavelmente contribuem para o aumento da magnitude da resposta imunológica a certos patógenos observada em animais tratados com o iPPVO.

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Rohde, Jörg [Verfasser], i Hans-Georg [Akademischer Betreuer] Rammensee. "Herstellung neuer Orf Virus- (Parapoxvirus) Rekombinanten und Analyse deren protektiven und immunrelevanten Eigenschaften / Jörg Rohde ; Betreuer: Hans-Georg Rammensee". Tübingen : Universitätsbibliothek Tübingen, 2017. http://d-nb.info/1199545694/34.

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Barros, Danielle Ribeiro de. "Clonagem e expressão in vitro da ORF AC5 do Tomato rugose mosaic virus (ToRMV) e produção de anti-soro". Universidade Federal de Viçosa, 2003. http://www.locus.ufv.br/handle/123456789/10116.

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Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-24T11:15:46Z No. of bitstreams: 1 texto completo.pdf: 389658 bytes, checksum: d01e4f8e75c1bc6e7531f77722009e37 (MD5)
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O Tomato rugose mosaic virus (ToRMV) é um begomovírus descrito como parte de um complexo viral causando mosaico dourado e deformação (rugosidade) foliar em tomateiro na região do Triângulo Mineiro, Minas Gerais. Os dois componentes genômicos encontram-se completamente sequenciados, e a análise das seqüências de nucleotídeos do DNA-A indicou a presença de uma ORF adicional denominada AC5, presente apenas em algumas espécies de begomovírus. Esta ORF está quase totalmente inserida na sequência do gene cp, porém em orientação inversa e em outra fase de leitura. A ORF AC5 do ToRMV tem o potencial de codificar uma proteína com aproximadamente 27 kDa. A função do produto potencial da ORF AC5 foi estudada apenas para o Watermelon chlorotic stunt virus (WmCSV), não sendo encontrado nenhum indício da expressão da ORF. Entretanto, a seqüência de aminoácidos da proteína potencialmente codificada pela ORF AC5 do ToRMV possui apenas 41% de identidade com a AC5 do WmCSV. É possível que a ORF AC5 do ToRMV seja expressa, e que a proteína desempenhe alguma função no ciclo de infecção viral. Este trabalho teve por objetivo a clonagem e a expressão in vitro da ORF AC5 do ToRMV e a producão de anti-soro específico. Oligonucleotídeos específicos foram utilizados para a amplificação da região codificadora da ORF AC5 via PCR. O fragmento amplificado foi clonado no vetor de expressão pRSET-C. Plasmídeos recombinantes foram utilizados para a expressão da proteína AC5 em E. coli BL21::DE3. A proteína foi purificada a partir das células de E. coli e utilizada para a produção de anti-soro policlonal em coelhos. A especificidade do anti-soro policlonal obtido foi confirmada por Western blot. Esse anti-soro poderá ser utilizado em ensaios de imunolocalização da proteína AC5 em tecidos infectados pelo ToRMV.
Tomato rugose mosaic virus (ToRMV) is a typical bipartite begomovirus described as part of a viral complex inducing golden mosaic and leaf distortion (rugosity) in tomatoes at Triângulo Mineiro, Minas Gerais. Both genomic components have been completely sequenced, and the DNA-A sequence analysis indicated the presence of an additional open ready frame (ORF), named AC5, present in only a number of begomovirus species. This ORF is almost fully inserted into the cp gene, but in opposite orientation and in a different reading frame. The AC5 ORF of ToRMV has the potential to encode for a 27 kDa protein. The function of a putative AC5 protein has only been studied for Watermelon chlorotic stunt virus (WmCSV), but no evidence of its expression was found. However, the amino acid sequence of the putative AC5 protein from ToRMV is only 41% identical to the WmCSV AC5. It is thus conceivable that the ToRMV AC5 is expressed and plays a role in the viral infection cycle. The objective of this work was to clone and express the ToRMV AC5 ORF in vitro, and to raise an AC5-specific antiserum. Specific primers were used to direct the PCR-based amplification of the ToRMV AC5 ORF. The amplified fragment was cloned into the expression vector pRSET-C. Recombinant plasmids were used for protein expression in E. coli BL21::DE3. The AC5 protein was purified from E. coli cells by affinity chromatography and used for the immunization of rabbits. The specificity of the polyclonal antiserum obtained was assayed by Western blot. This antiserum can now be used in immunolocalization studies attempting to detect the AC5 protein in ToRMV-infected tissues.
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LEH, VERONIQUE. "Etude des interactions entre proteines engageant les produits des orf iii et vi du virus de la mosaique du chou-fleur". Université Louis Pasteur (Strasbourg) (1971-2008), 1999. http://www.theses.fr/1999STR13180.

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Le virus de la mosaique du chou-fleur (camv) est le chef de file du groupe des caulimovirus. L'etude d'interactions entre proteines engagees par le produit de l'orf iii (p3) du camv par la technique de farwestern, a mis en evidence sa capacite a interagir avec le facteur assistant de la transmission, p2. Des tests de transmission ont montre que cette interaction est essentielle au mouvement du virus de plante a plante par l'intermediaire du puceron. La proteine p3 est donc un deuxieme facteur assistant de la transmission. Par ses interactions avec p2 et la proteine de capside (p4), p3 cree un pont entre ces deux composants et joue un role cle dans la formation du complexe transmissible. L'interaction p3-p4 est en outre requit pour la multiplication du virus dans la plante. Le produit de l'orf vi du camv (p6) joue egalement un role fondamental dans l'infectivite du camv. Nous avons demontre que son extremite n-terminale est impliquee dans des interactions intermoleculaires pouvant etre a la base de la formation des viroplasmes. Ceux-ci permettent une compartimentation du cycle viral dans la cellule. P6 est egalement necessaire a la traduction des autres orf du camv. Des experiences de farwestern ont montre que p6 interagit avec des proteines ribosomiques et notamment avec les proteines rpl13 et rpl18 de la sous-unite 60s du ribosome. Ces interactions pourraient avoir lieu au cours du mecanisme de trans-activation traductionnel realise par p6.

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Scholz, Kai. "Immunmodulation durch Parapocken-Viren: Identifikation und Analyse funktionaler Viruskomponenten". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2003. http://nbn-resolving.de/urn:nbn:de:swb:14-1061969873968-07930.

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Fusionspeptid-, Redox-, Viruscore- und sonstige Proteine. Alle analysierten Single ORF (SO)-VVOV Rekombinanten vermittelten einen signifikanten Schutz vor einer tödlichen Belastung mit Aujeszky-Virus. Zwei der Rekombinanten (SO 93-, SO 94-VVOV) enthalten ORFs, die für ATI/Fusionspeptid-Proteine kodieren. In SO 19- und SO 70-VVOV sind dagegen für Redoxproteine kodierende ORFs integriert. Weiterführende Untersuchungen zeigten, dass SO 94- und SO 19-VVOV in zwei weiteren Modellsystemen immunstimulatorisch aktiv sind. Im Baculo-Virussystem exprimierte Proteine waren nur in Kombination mit Vaccinia Lister-Virus (VV) wirksam. Dabei zeigten jeweils Virus-Protein-Gemische mit dem geringsten Proteinanteil den stärksten immunstimulatorischen Effekt. Proben in denen VV durch bovines Herpes-Virus-1 ersetzt wurde, sind dagegen nicht wirksam. Dies lässt auf eine Beteiligung VV-spezifischer Faktoren schließen. Übereinstimmend mit diesen Ergebnissen führte eine Frameshift-Mutation in ORF 94r von SO 94mut-VVOV nur zur Abschwächung und nicht zum vollständigen Verlust der immunstimulatorischen Wirkung. Beide in Schizosaccharomyces pombe exprimierten Proteine, sp-ORF19 und sp-ORF94r, induzierten keinen signifikanten Schutz im Aujeszky Maus Modell. Mit der Identifikation einzelner immunstimulatorisch aktiver PPVO-Komponenten ist es erstmals gelungen, den paramunisierenden Effekt von Parapox-Viren einzelnen viralen Genen zu zuordnen. Insbesondere stellen SO 94- und SO 19-VVOV viel versprechende Kandidaten für die prophylaktische bzw. therapeutische Anwendung in verschiedenen Indikationen als auch für weitere Untersuchungen des Wirkmechanismus dar.

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Li, Hua. "RNA SEQUENCE DETERMINANTS OF A COUPLED TERMINATION-REINITIATION STRATEGY FOR TRANSLATION OF DOWNSTREAM ORF IN HELMINTHOSPORIUM VICTORIAE VIRUS 190S AND OTHER VICTORIVIRUSES (FAMILY TOTIVIRIDAE)". UKnowledge, 2014. http://uknowledge.uky.edu/plantpath_etds/9.

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Double-stranded RNA fungal virus Helminthosporium victoriae virus 190S (genus Victorivirus, family Totiviridae) contains two large open reading frames (ORFs) that overlap in the tetranucleotide AUGA. Translation of the downstream ORF, which encodes the RNA-dependent RNA polymerase (RdRp), was previously proposed to depend on ribosomal reinitiation following termination of the upstream ORF, which encodes the capsid protein. In this study, I provided evidence to confirm that coupled termination-reinitiation (stop-restart) is indeed used. A dual-fluorescence method was established to define the RNA sequence determinants for RdRp translation. Stop-restart depends on a 32-nt stretch of RNA sequence immediately upstream of the AUGA motif, including a predicted pseudoknot structure. The presence of similar sequence motifs and predicted RNA structures in other victoriviruses suggest that they all share a related stop–restart strategy for RdRp translation. The close proximity of the secondary structure to the AUGA motif appears to be especially important for promoting translation of the downstream ORF. Normal strong preferences for AUG start codons and canonical sequence context for translation initiation of the downstream ORF appear somewhat relaxed. With dual-fluorescence system, reinitiation efficiency of the downstream ORF was determined to be ~3.9%. Pseudoknot swapping between the one in HvV190S and those predicted from other victoriviruses showed that reinitiation from the downstream ORF of HvV190S is quite tolerant to varying primary sequences of the various pseudoknots. Mutational analysis by introducing different combinations of nucleotide mutations into pseudoknot stems reproducibly confirmed the determinant role of pseudoknot on reinitiation using two different experimental systems. Together, these results provide the first example of coupled termination-reinitiation regulated by a simple pseudoknot stucture. These data expanded the understanding of coupled termination-reinitiation mechanism employed by RNA viruses and refined a new model for genus victorivirus, the largest genus in the family Totiviridae. The dual fluorescence system used in this study represented the first application of an efficient in vivo assay for recording low-frequency events in filamentous fungi.

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Bhatta, Ankit. "Role of a Mitochondrial Micropeptide in Regulating Innate Immune Responses". eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1108.

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Short ORF-encoded peptides (SEPs) are increasingly being identified as functional elements in various cellular processes. The current computational methods and experimental molecular biochemistry allow us to discover putative SEPs or micropeptides from proteogenomic datasets and experimentally validate them. Here, we identified a micropeptide produced from a putative long noncoding RNA (lncRNA) 1810058I24Rik which is downregulated in both human and murine myeloid cells exposed to lipopolysaccharide (LPS), as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed this transcript is localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 amino acid micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene ‘Mitochondrial micropeptide-47 (Mm47)’. Functional studies using siRNA and Cripsr-cas9-mediated deletion in primary cells, showed that the transcriptional response downstream of TLR4 was not affected by Mm47 loss of function. In contrast, both the Crispr-cas9- and siRNA-targeted BMDM cells were compromised for Nlrp3 inflammasome responses. However, the primary macrophages derived from the Mm47 knockout mice do not require Mm47 for Nlrp3 activation, likely due to basal downregulation of a negative regulator microRNA of Nlrp3 called Mir-223. Notably, the Mm47-deficient mice are susceptible to influenza virus infection and succumb despite comparable antiviral and inflammatory response to wildtype mice. We hypothesize that the Mm47 deficiency may affect the antiviral resilience of mice due to secondary mitochondria dependent immunometabolic defect or failure of recovery from immune pathology, which warrants further investigation. This study therefore identifies a novel mitochondrial micropeptide Mm47 that is required for activation of the Nlrp3 inflammasome in cells and resistance to influenza virus infection. Broadly, this work highlights the presence of translatable ORFs is annotated noncoding RNA transcripts and underscores their importance in innate immunity and virus infection.

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Bentley, Emma. "The study of highly pathogenic emerging zoonotic virus envelope proteins through pseudotyped virus generation". Thesis, University of Westminster, 2017. https://westminsterresearch.westminster.ac.uk/item/q4yzx/the-study-of-highly-pathogenic-emerging-zoonotic-virus-envelope-proteins-through-pseudotyped-virus-generation.

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Emerging zoonotic viruses pose an increasing threat, causing outbreaks with high rates of morbidity and mortality and frequently significant economic implications. Often, there is a lack or shortfall of effective prophylaxis and diagnostic capabilities. Research towards their development, together with improved surveillance activities are high priority activities to prepare and respond to outbreak threats. Yet handling these viruses commonly requires high containment levels. This can be circumvented by the use of replication defective pseudotyped viruses (PVs), incorporating the viral envelope protein of interest which constitutes the primary surface antigen. This permits the serological detection of neutralising antibodies without the need to handle live virus, as well as other viral entry studies. Hence, PVs are increasingly proving to be a valuable tool for emerging virus research. The aim of this study was to exploit novelties in the unique flexibility of the PV platform to allow the serological assessment of emerging viruses and evaluate technical aspects towards standardisation. Current prophylaxis provides robust protection against rabies virus, yet only confers limited protection against other lyssavirus species, which have a near 100% fatality rate. It is thought protection is afforded against isolates of phylogroup I rabies virus, yet there is limited biological data for the Arctic-like rabies virus (AL RABV) lineage which is endemic across the Middle East and Asia. Although other lyssaviruses pseudotype efficiently, titres of AL RABV PV were low. Within this study, high titre PV was produced by constructing chimeric envelope proteins, splicing the AL RABV ecto-transmembrane domain with the cytoplasmic domain of vesicular stomatitis virus. Comparisons showed this did not alter the serological profile of the AL RABV and they were effectively neutralised by vaccines and antivirals. It could therefore be concluded that they do not pose a significant public health risk. However it is recognised broadly neutralising prophylaxis needs to be developed to protect against more divergent lyssaviruses. In a further study, again utilising the flexibility to manipulate the envelope protein, PV was produced switching the five known antigenic sites of the envelope protein between a phylogroup I (rabies virus) and III (West Caucasian bat virus) isolate. Screening polyclonal sera via a neutralisation assay, the immunologically dominant sites for phylogroup I and III were identified as III and I respectively. This can act to inform future development of more broadly neutralising vaccines. The 2013-16 outbreak of Ebola virus focused global efforts towards the urgent need for effective vaccines and antivirals. To permit low containment level serology studies to assist their development, a panel of filovirus PVs were rapidly produced. Work was carried out to optimise their method of production; determining lentiviral core PV produced by transfecting HEK 293T/17 cells was most efficient. Efforts to repeat the use of chimeric envelope proteins to increase titre proved unsuccessful. The evaluation of target cell lines permissive to infection and appropriate for neutralisation assays identified that the CHO-K1 cell line produced the clearest data. The PV neutralisation assay was subsequently applied to a range of projects to assess candidate prophylaxis and demonstrated the value of the platform to respond to emerging virus outbreaks. Given the increasing prominence in the use of PV, work was undertaken to expand their utility and methods for standardisation. An assessment of new reporter genes found a red fluorescent protein, with a nuclear localisation signal, improved the clarity of data collection and output in additional spectrum to the current repertoire. To be able to correlate the disparate readout units of fluorescent and luminescent reporters, recorded as infectious units (IFU) and relative light units (RLU) respectively, a new construct was produced to integrate and equally express two reporters from cells transduced with PV. It was determined that approximately 1260 RLU equates to 1 IFU, although future work to determine how this fluctuates between cell lines is required. Finally, alternative methods to quantify PV were evaluated, measuring the number of particles, genome copies and reverse transcriptase (RT) activity, in addition to the currently used biological titre. It was found that measures of genome copies and RT activity, in combination with biological titre provides information on the quality of PV preparations and could be used to standardise assay input.

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Rintoul, Julia. "ORFV: A Novel Oncolytic and Immune Stimulating Parapoxvirus Therapeutic". Thèse, Université d'Ottawa / University of Ottawa, 2012. http://hdl.handle.net/10393/22925.

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Replicating viruses for the treatment of cancer have a number of advantages over traditional therapeutic modalities. They are highly targeted, self-amplifying, and have the added potential to act as both gene-therapy delivery vehicles and oncolytic agents. ORFV, (Parapoxvirus ovis, or Orf virus) is the prototypic species of the Parapoxvirus genus, causing a benign disease in its natural ungulate host. ORFV possesses a number of unique properties that make it an ideal viral backbone for the development of a cancer therapeutic: it is safe in humans, has the ability to cause repeat infections even in the presence of antibody, and it induces a potent Th-1 dominated immune response. Here I show for the first time that live replicating ORFV induces an anti-tumour immune response in multiple syngeneic mouse models of cancer that is mediated largely by the potent activation of both cytokine-secreting, and tumouricidal natural killer (NK) cells. I have also highlighted the clinical potential of the virus by demonstration of human cancer cell oncolysis including efficacy in an A549 xenograft model of cancer. The mechanism of ORFV-mediated activation of NK cells has been explored, where I have demonstrated activation via direct ex vivo infection of NK cells. I have also highlighted ORFV-mediated activation of dendritic cells (DCs), both in vivo and by direct infection ex vivo. An in vivo DC depletion study demonstrated an indirect mechanism for ORFV NK cell activation, where in the absence of DCs, NK cell activation was diminished, as was the ability of ORFV to clear lung metastases. The ORFV innate immune stimulatory profile has been harnessed for therapeutic application in an experimental surgery model of cancer, where ORFV therapy at the time of surgery reduces the number of cancer metastases. These data highlight the clinical potential of a live, immune stimulating Parapoxvirus therapeutic.

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Yadav, Kush Kumar. "Genotype 1 hepatitis E virus (HEV) ORF4 protein enhances genotype 3 HEV replication". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574781581580768.

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Bagdassarian, Eugénie. "Interaction du virus de l'hépatite E avec la réponse intérféron de l’hôte". Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS266/document.

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L’infection par le virus de l’hépatite E (VHE) peut entraîner une hépatite aiguë chez l’homme évoluant en hépatite fulminante dans 1-4% des cas, voire 20% chez les femmes enceintes dans les régions endémiques. Le VHE, transmis par voie entérique, est responsable de grandes épidémies d’origine hydrique dans les pays en voie de développement et de nombreux cas sporadiques d’origine zoonotique dans des pays industrialisés. La description récente de cas d’hépatites E chroniques ou d’atteintes neurologiques graves souligne l’importance de caractériser les interactions du VHE avec son hôte. L’objectif de ce projet de thèse était de caractériser les interactions entre le VHE et la réponse immune innée de l’hôte et en particulier avec le système interféron de type I (IFN-I).La première partie de ce projet a consisté en l’étude de la modulation des voies de signalisation de l’IFN-I par la polyprotéine non-structurale ORF1 du VHE. Celle-ci est constituée de plusieurs domaines fonctionnels putatifs tels qu’une methyltransférase (Met) ou une protéase à cystéine de type papaïne (PCP) dont les fonctions sur la signalisation IFN-I restent encore peu connues. Les résultats obtenus ont montré que le domaine MetPCP de l’ORF1 est capable d’inhiber l’activation du promoteur de l’IFN-β et de celui des gènes sous le contrôle de l’IFN contenant des éléments de réponse à l’IFN (ISRE), ainsi que l’expression de certains des gènes induits par l’IFN (ISGs). En recherchant le mécanisme impliqué dans l’inhibition du promoteur ISRE, nous avons montré que le domaine MetPCP inhibe la phosphorylation de STAT1 et sa relocalisation nucléaire. Nous avons également montré que le domaine MetPCP n’inhibe pas la phosphorylation de STAT2. Le mécanisme d’action du domaine MetPCP reste encore à préciser. La deuxième partie de ce projet a été de déterminer si l’infection par le VHE entraîne la production d’IFN-I par les cellules dendritiques plasmacytoïdes (pDCs). En effet, les pDCs sont la principale source d’IFN-I et jouent un rôle crucial dans la réponse immunitaire innée et adaptative. Les résultats obtenus suggèrent que les pDCs ne produisent que modérément de l’IFN-I lorsqu’elles sont co-cultivées avec des cellules infectées par le VHE. L’ensemble des résultats obtenus pendant ce travail de thèse suggère que le VHE utilise plusieurs mécanismes pour moduler la signalisation IFN-I de l’hôte
Hepatitis E virus (HEV) is the causative agent of acute hepatitis in humans that can lead to fulminant hepatitis in 1-4% of cases, and in 20% of pregnant women in endemic regions. HEV is an enterically-transmitted virus responsible for large waterborne epidemics in developing countries and numerous cases of zoonotic hepatitis E in industrialized countries. The recent description of cases of chronic hepatitis E and severe neurological disorders highlight the importance to characterize the interactions between HEV and the host. The objective of this PhD project was to characterize the interactions between HEV and the host innate immune response and particularly with the type I interferon system (IFN-I).The first part of this project aimed to study the ability of the ORF1 non-structural polyprotein of HEV to modulate the IFN-I signalling pathways. HEV ORF1 contains several putative functional domains including a methyltransferase (Met) and a papain-like cysteine protease (PCP) whose functions on the IFN-I signalling remain poorly understood. The results obtained showed that the MetPCP domain of ORF1 inhibits IFN-β and IFN-stimulated response element (ISRE) promoter activation and the expression of some IFN-stimulated genes (ISGs). We then investigated the mechanism involved in this inhibition of ISRE promoter activation. We showed that the MetPCP domain inhibits STAT1 phosphorylation and nuclear translocation. In contrast, we found that the MetPCP domain does not inhibit STAT2 phosphorylation. However, the mode of action of MetPCP remains to be fully characterised. The second part of this project aimed to determine the ability of plasmacytoid dendritic cells (pDCs) to produce IFN-I in response to HEV infection. Indeed, pDCs are the main IFN-I source and play a crucial role in innate and adaptive responses. The results obtained suggest that pDCs produce IFN-I moderately when co-cultured with HEV-infected cells. Taken together, the results obtained during this PhD project suggest that HEV has evolved different mechanisms to modulate the IFN-I host response

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Kannan, Harilakshmi. "Functional characterization of the interaction of hepatitis E virus ORF3 product with the cytoskeleton". College Park, Md.: University of Maryland, 2008. http://hdl.handle.net/1903/8969.

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Thesis (M.S.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Virginia-Maryland Regional College of Veterinary Medicine. Maryland Campus. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

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Barkley, Russell. "Investigation of an Oncolytic MeV Cell-Cell Fusion Phenomenon Induced by an siRNA". Thesis, Université d'Ottawa / University of Ottawa, 2020. http://hdl.handle.net/10393/41531.

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Oncolytic measles virus is a promising cancer therapeutic in clinical trials which possesses multiple characteristics that are advantageous over traditional therapies. Currently, clinical oncolytic measles virus vectors are unmodified or express reporter transgenes that benefit its therapeutic efficacy. The next phase in its development will see genetically engineered vectors encoding transgenes that enhance its antineoplastic effects. To this end, preclinical research has focused on studying novel transgenes which favour viral replication, cytotoxicity, and the anti-cancer immune response. We sought to encode artificial micoRNAs targeting RIG-I as a strategy to interfere with innate immunity. Silencing RIG-I with multiple siRNAs yielded one which promotes measles virus syncytia formation through a mechanism that appears to be independent of RIG-I. The mechanism caused by the siRNA leads to enhanced measles virus cell-cell fusion and has peculiar characteristics which are not fully understood.

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Heidari, Farshad. "In vitro modulation of host immune response by the Varicella zoster virus ORF1 gene product". Thesis, Kingston University, 2017. http://eprints.kingston.ac.uk/41971/.

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Varicella-zoster virus causes chicken pox (Varicella) primary infection, which becomes latent in the dorsal root ganglia and trigeminal ganglia, and may reactivate to cause shingles, the most serious complication of which is post-herpetic neuralgia occurring in 50% of individuals over 60 years. Severity of lesions depends on host's immune response. Like many viruses, varicella-zoster virus appears to have evolved escape mechanisms from host immune surveillance by downregulating cell surface major histocompatibility complex class 1 expression and may delay of resolution of infection. Major histocompatibility complex class 1 is processed and transported to the cell surface through the Golgi apparatus. Varicella-zoster virus genome encodes a membrane gene, open reading frame type 1, which is localised in the enoplasmic reticulum and Golgi apparatus. Given the cellular localisation of open reading frame type 1 in the Golgi apparatus, this study investigated the expression of major histocompatability complex class 1 in human immortalised keratinocytes transfected with only empty vector. As a control, the expression of major histocompatability complex class 1 human immortalised keratinocytes parental cells were also examined using current molecular biology techniques. The results of this thesis demonstrate that the expression of varicella-zoster virus-open reading frame type 1 prevents the transport of major histocompatibility complex class 1 complexes to the cell surface and causes its retention in the Golgi apparatus, which is compensated by treatment with IFN-[alpha]. Varicella-zoster virus (VZV)-open reading frame type 1 does not affect the synthesis of human leukocyte antigen class 1 heavy chains or the expression of the transporter associated with antigen processing. Additionally, we determined that varicella-zoster virus-open reading frame type 1 impedes the surface expression of human leukocyte antigen class-A and human leukocyte antigen class-B, which present viral peptides to major histocompatibility complex class I-restricted cytotoxic T lymphocytes, but not the natural killer cell inhibitory ligands human leukocyte antigen class-C and non-classical human leukocyte antigen class-E. This selective downregulation of cell surface human leukocyte antigen class 1 molecules may allow the virus to establish infection by avoiding immune clearance of virus-infected cells by both cytotoxic T lymphocytes and natural killer cells. However, it remains to be seen if open reading frame type 1 expressing cells evade cytotoxic T lymphocytes killing, through downregulation of classical major histocompatibility complex class I, and natural killer killing, through lack of downregulation of non-classical major histocompatibility complex class I. The study outcome will be a valuable attempt to elucidate factors involved in varicella-zoster virus-related lesion progression, contribute to existing knowledge, and importantly allude to further investigations on the pathogenesis of this virus on human disease. The data obtained may also offer novel means of therapeutic intervention.

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Ankavay, Maliki. "Étude des modifications post-traductionnelles de la protéine de capside ORF2 du virus de l'hépatite E (HEV)". Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S013.

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L’infection par le virus de l’hépatite E (HEV) est un problème majeur de santé publiquequi touche plus de 20 millions de personnes et tue environ 70 000 chaque année à travers lemonde. Le HEV est la cause majeure d’hépatite virale aiguë dans le monde. En France, laséroprévalence du HEV est de 22,4% dans la population générale. Ce virus se transmet parvoie féco-orale ou par consommation de viande contaminée mal cuite. Très récemment, nousavons décrit un système de culture cellulaire permettant d’amplifier efficacement le HEV. Cesystème représente un outil unique pour caractériser les différentes étapes du cycle infectieuxdu HEV qui sont très mal connues à ce jour. Dans le cadre de ma thèse, je me suis intéresséplus particulièrement à la protéine de capside ORF2 qui est l’unité structurale des particulesvirales et est donc un acteur central du cycle infectieux du HEV. La protéine ORF2 est uneprotéine de 660 acides aminés (aa) qui possède un peptide signal (PS) et trois sites potentielsde N-glycosylation (N1, N2 et N3). J’ai donc étudié le rôle de la N-glycosylation de cetteprotéine ORF2 dans le cycle infectieux du HEV. Pour atteindre cet objectif, la soucheinfectieuse HEV-p6 génotype 3 a été utilisée pour infecter les cellules de la lignéehépatocytaire PLC3, sous clone de la lignée PLC/PRF/5. Les résultats obtenus ont montré quela protéine ORF2 est N-glycosylée uniquement au niveau des sites N1 et N3 ; le site N2 estdéfavorable à la N-glycosylation. Nos résultats ont montré que la N-glycosylation de laprotéine ORF2 n’est pas importante pour sa stabilité, son oligomérisation, sa reconnaissancepar des anticorps, l’assemblage et l’infectiosité des particules virales. Nous avons aussimontré que la protéine ORF2 est importée dans le noyau des cellules hôtes au cours du cycleinfectieux du HEV de manière indépendante de la N-glycosylation. Nous avons identifié pourla première fois un signal de localisation nucléaire (NLS) fonctionnel en position N-terminalede la protéine ORF2 lui permettant d'interagir probablement avec l’importine alpha1. Demanière surprenante, ce NLS régule non seulement l’import nucléaire de cette protéine virale,mais aussi sa translocation réticulaire. Nous avons également démontré que la protéine ORF2est exportée du noyau des cellules hôtes en interagissant avec l’exportine1 et possède 3signaux d’export nucléaire (NES) en position C-terminale. Ces résultats améliorent laconnaissance du trafic intracellulaire de la protéine ORF2 et pourraient orienter les stratégiesantivirales dirigées contre le HEV
Hepatitis E virus (HEV) infection is a major public health problem that affects more than20 million people and kills approximately 70,000 worldwide each year. HEV is the leadingcause of acute viral hepatitis in the world. In France, the seroprevalance of HEV is 22.4% inthe general population. This virus is transmitted by fecal-oral route or by consumption ofundercooked contaminated meat. Very recently, we have described an efficient cell culturesystem to amplify HEV. This system represents a unique tool for characterizing the variousstages of the infectious HEV lifecycle that are very poorly understood to date. As part of mythesis, I focused on the ORF2 capsid protein which is the structural unit of viral particles andis therefore a central player in the HEV lifecycle. ORF2 is a 660 amino acids protein thatcontains a signal peptide and three potential N-glycosylation sites (N1, N2 and N3). My workaimed to identify the role of the ORF2 N-glycosylation in the HEV lifecycle. Our resultsrevealed that the ORF2 protein is N-glycosylated on the N1 and N3 sites, whereas the N2 siteis not N-glycosylated. Also, the N-glycosylation has no significant relevance neither for thestability, oligomerization, antibody recognition of the ORF2 protein nor for viral assemblyand viral infectivity. We also revealed that, the ORF2 protein is translocated into the nucleusof infected cells, independently of N-glycosylation. We identified for the first time afunctional nuclear localization signal (NLS) located at the N-terminus of the ORF2 proteinthat interacts probably with the importin alpha1. Surprisingly, this NLS regulates both nuclearimport and endoplasmic reticulum translocation of the ORF2 protein. Finally, wedemonstrated that, the ORF2 protein possesses three nuclear export signals (NES) located atits C-terminus. The ORF2 protein interacts with the exportin1 and is exported from the cellnuclei. This interaction drives the ORF2 protein from the nucleus to the cytoplasm of theinfected cells. Hence, this study led to new insights into the molecular mechanisms of theORF2 protein and could help to the design of novel antiviral strategies against HEV

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Blignaut, Marguerite. "The molecular and biological characterisation of ORF5 of three South African variants of Grapevine Vitivirus A". Thesis, Stellenbosch : University of Stellenbosch, 2009. http://hdl.handle.net/10019.1/2421.

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Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well characterised single-stranded RNA virus that has been implicated in the grapevine diseases, Kober stem grooving and Shiraz disease. The virus infects both its host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological studies performed on the virus in its herbaceous host, Nicotiana benthami- ana, revealed that many divergent variants of the virus exists in South Africa and can induce di erent symptoms in the model plant. Further molecular analysis divided the variants into three molecular groups based on molecular heterogeneity and nucleotide identity. The establishment of an infectious full-length cDNA clone of GVA contributed towards the elucidation of gene functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5 as the pathogenicity determinant within the genome. Further studies also showed that ORF5 encodes for a nucleic acid binding protein that exhibits suppression activity of a plants' natural virus silencing mechanism. Many proteins that have previously been identi ed as the pathogenicity determinant within a viral genome have been found to encode for suppression activity. Although suppression activity has been elucidated within the ORF5 of the Italian cDNA clone of GVA, IS 151, no such study has yet been performed on the divergent South African variants of GVA. Three variants, GTR1-1, GTR1- 2 and GTG11-1, which represent each of the molecular groups (Group III, II and I), were selected for this study. The aim of this study was to visually elucidate suppression activity of RNA transgene silencing by the ORF5's of GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic N. benthamiana (line 16c). Pathogenicity studies for these variants were also performed. The ORF5 of the infectious full-length clone, GVA118, which can also serve as an expression vector, was deleted and provided with restriction enzyme sites into which the respective ORF5s and the marker genes, GFP and GUS could be cloned directionally. Infectivity, symptom development and systemic movement were compared between the di erent full length clones after co-in ltration in N. benthamiana. Preliminary results obtained in this study failed to visually indicate any suppression activity encoded by the ORF5 of GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was successful and rendered the infectious full length clone asymptomatic. Directional cloning of the ORF5 of GTR1-1 into the unique restriction enzymes provided previously, resulted in much milder symptoms than those observe for GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected. This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118 ORF5-1-1-pA, that can possibly induce much milder symptoms in the herbaceous host, N. benthamiana. This construct can be further characterised as a possible expression vector of foreign proteins in herbaceous hosts and grapevine.

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Guo, Hailong. "Antigenic epitope composition and protectivity of avian hepatitis E virus (avian HEV) ORF2 protein and vertical transmission of avian HEV". [Ames, Iowa : Iowa State University], 2006.

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Martinez-Soto, Eduan E. "Understanding the Role of Health Care Workers in a Trade-off Model between Contact and Transmission for Ebola Virus Disease". The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1467993935.

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Brusini, Jérémie. "Conséquences de l'incompatibilité végétative et de l'infection virale sur l'écologie et l'évolution de l'interaction Cryphonectria parasitica X Cryphonectria Hypovirus". Thesis, Bordeaux 1, 2009. http://www.theses.fr/2009BOR13826/document.

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Le système d'incompatibilité végétative a été décrit chez tous des champignons (Eumycètes) comme intervenant dans la limitation des fusions somatiques entre conspécifiques. Chez les champignons la fusion somatique est uniquement possible entre individus de même GCV (Groupe de Compatibilité Végétative). Comme tous les systèmes de reconnaissance du soi, le fonctionnement du système d'incompatibilité végétative des champignons est basé sur une grande diversité allélique. Cette thèse propose d'étudier la relation qui semble exister entre cette diversité des gènes impliqués dans l’incompatibilité végétative des champignons et la pression parasitaire exercée par des éléments cytoplasmiques délétères (ou DCE) transmis lors des fusions somatiques. Trois problématiques ont été abordées, avec trois approches différentes : (1) une approche conceptuelle générale portant sur l’évolution des systèmes de reconnaissance du soi, (2) une approche de modélisation sur le maintien de la diversité en GCV de la population de champignon par un DCE et (3) une approche expérimentale, pour étudier d’une part la perméabilité de la barrière d‘incompatibilité végétative et d’autre part l’interaction C. parasitica/CHV et les liens existant entre transmission et virulence du CHV. Ces études ont permis de montrer l'importance de la perméabilité de la barrière d'incompatibilité végétative à la fois au niveau du maintien de la diversité génétique de la population d'hôte et au niveau de la prévalence des DCE. Il semblerait donc que les DCE évoluent vers des niveaux de virulence faible du fait de la limitation de leur transmission par le système d'incompatibilité végétative de leur hôte. Nos résultats expérimentaux suggèrent que lorsque la diversité en GCV de la population d'hôte est faible, la virulence des DCE pourrait évoluer suivant le modèle du trade-off impliquant une évolution vers un niveau de virulence intermédiaire optimal. Ces travaux permettent donc de mieux comprendre les mécanismes agissant sur l'écologie et l'évolution des interactions champignon/DCE qui, au vu de cette étude, apparaissent comme de bon modèles pour l’étude des systèmes hôtes/parasites
Vegetative incompatibility systems have been described in Fungi as controlling somatic fusion between conspecifics. For fungi, only fungi of the same vc type can fuse together. As other self recognition systems, this system involved high allelic diversity at specific genes. The issue of this work is to study the cause and effect relationship between the evolution of vegetative incompatibility systems and the selective pressure drove by cytoplasmic deleterious elements, transmitted during somatic fusion. Three problematics with three different approach were done : (1) a conceptual general framework on the evolution of self recognition systems (2) a theoretical work on the maintenance of vc type diversity by DCE and (3) an experimental work on the study of relationship between transmission and virulence in the C. parasitica/CHV host-parasite system. Ours results showed the key role of the permeability of the vegetative incompatibility barrier both for vc type diversity maintenance and on DCE prevalence. DCE would evolve toward avirulence in response to the transmission limitation by host incompatibility systems. Experimental work suggested a positive link between virulence and transmission in some population of CHV when host present a low vc type diversity, which could allow the evolution of the DCE toward an intermediate optimal virulence. This study would shed some light on mechanisms acting on the ecology and the evolution of fungi/DCE interaction which, according to our results, would be good study models for works on host-parasite systems

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Mohamed, Mahgoub Mohamed Ahmed Mohamed. "Sporadic on/off switching of HTLV-1 Tax expression is crucial to maintain the whole population of virus-induced leukemic cells". Kyoto University, 2018. http://hdl.handle.net/2433/232139.

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Alsafi, Radi Taha M. "Generation of complex recombinant fowlpox virus 9 (FP9) encoding simian immunodeficiency virus (SIVmac239) sequences as a model HIV vaccine candidate". Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/generation-of-complex-recombinant-fowlpox-virus-9-fp9-encoding-simian-immunodeficiency-virus-sivmac239-sequences-as-a-model-hiv-vaccine-candidate(1a015762-8dc2-4153-a586-d7fab88b9658).html.

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The development of a safe and effective HIV vaccine remains challenging due to its high antigenic variability. Poxviruses are large, stable, and have a track record of use as human vaccine candidates. Recombinant fowlpox virus 9 (rFP9), a highly attenuated host range-restricted poxvirus strain, has been safely administered to humans with no ill effects, and is known to be immunogenic. This thesis describes the construction of complex rFP9 encoding various sequences of SIVmac239. The SIVmac239/macaque model is widely used for HIV vaccine development. The ultimate aim of this work was to combine the advantages of FP9 with those of live attenuated SIV to produce a safe yet hopefully effective model HIV vaccine candidate. Transfer plasmids for five different insertion sites within the FP9 genome were designed and constructed. Homologous recombination (HR) of adjacent FP9 sequences was employed to facilitate the integration of SIVmac239 sequences into the FP9 genome. Positive rFP9 were identified by blue colouration in presence of X-gal using a transient colour selection (TCS) technique, and the final markerless pure recombinants were confirmed by PCR. Expression of the target SIV proteins in the presence of T7 polymerase has been demonstrated by immunocytochemical (ICC) staining and Western blotting (WB) assays. Expression was also quantified by enzyme-linked immunosorbent assay (ELISA) in various cell lines at multiple time points. Five different unique rFP9 have been constructed through this project. All SIVmac239 open reading frames (ORFs) save nef have been integrated into the FP9 genome, and protein expression demonstrated where possible. Moreover, a single rFP9 vector expressing the defective SIVmac239 genome driven by T7 RNA polymerase has been successfully constructed and validated using a green fluorescent protein marker.rFP9 showed appropriate transgene expression in both avian and mammalian cells, although at different levels. The expression efficiency of rFP9 was finally compared to another attenuated poxvirus vector, modified vaccinia Ankara (MVA). Comparing the protein expression levels between rFP9 and rMVA was quite difficult because different poxvirus promoters (early/late in rFP9; intermediate in rMVA) were used to direct the transcription of the T7 RNA gene. Given this limitation, although generally higher levels of expression were seen with rFP9, this cannot be attributed to the FP9 with any certainty.

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Bardelli, Martino. "Strategies of viral multi-functional regulator proteins : adeno-associated virus Rep". Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/strategies-of-viral-multifunctional-regulator-proteins(6154fd50-eacd-4dbe-b456-bd08d2b13849).html.

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Adeno-associated virus (AAV) is a small non-pathogenic human DNA parvovirus. The AAV life cycle, which includes transcriptional regulation, DNA replication, assembly and site-specific integration, is orchestrated by AAV’s four Rep proteins. Structurally, these proteins share a AAA+ domain characteristic of the SF3 family of helicases, with the larger Rep68 and Rep78 additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains is the basis for the remarkable multi-functionality displayed by Rep68 and Rep78. To date, structural studies of Rep68 and Rep78 have been limited by the tendency of these proteins to aggregate when purified. Here, we describe a fully functional Rep mutant that does not aggregate even at high concentrations and use this mutant to investigate the structural requirements for Rep functions. We demonstrate that one of the determinants regulating the oligomerisation of the Rep proteins lies in the linker connecting the helicase domain and OBD. We also identify a series of key residues at the interface between Rep monomers and show that mutating them has drastic effects both on the oligomerisation and functionality of the Rep proteins. Importantly, these oligomerisation-deficient mutants do not support the AAV life-cycle and fail to bind DNA efficiently, an important Rep function necessary for DNA nicking, transcriptional regulation, viral DNA replication and site-specific integration. Finally, understanding the molecular details of Rep and its functions will contribute to the development of new AAV-based vectors that exploit the Rep- mediated integration mechanism and potentially have a lower risk of insertional mutagenesis than retroviral vectors. In the last chapter, we describe an AAV vector for testing the safety and feasibility of AAV-mediated targeted gene addition in induced pluripotent stem (iPS) cells within the therapeutic context of SCID-X1, an immunodeficiency caused by mutations in the common gamma chain gene.

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Key, Kijona Farthing. "Molecular characterization of the major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) and evaluation of its use for a diagnostic assay, vaccine development, and the examination of quasispecies evolution". Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27282.

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Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has devastated the global swine industry since the mid 1980s. Although modified live vaccines (MLVs) are typically used for the prevention of clinical disease, they are not always fully effective. Additionally, acute PRRS outbreaks, characterized by more severe clinical signs, have appeared in herds that were previously vaccinated. In this dissertation, we further analyzed the pathogenesis of PRRSV through genetic characterization, assay development, and quasispecies evaluation using the PRRSV ORF5 gene while also attempting to develop an improved PRRS vaccine. To explore the possible mechanism for the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. Sequence and phylogenetic analyses revealed that seven of the acute PRRS virus (PRRSV) isolates were related to other N. American PRRSV isolates while one isolate, 98-37120-2, was very closely related to and may have been derived from the MLV, RespPRRS. We also developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV field isolates with significant nucleotide sequence identities (â d98%) with the MLVs based on the amplification, denaturation, and reannealing of the ORF5 gene of the field isolates with those of MLV reference strains. All of the field isolates that were highly related to RespPRRS (â T2% nucleotide sequence divergence) were identified by the HMA to form homoduplexes with the reference RespPRRS MLV. We also developed a unique strategy for infecting pigs with PRRSV, known as in vivo transfection, by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrated that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. Using this method, we also examined the quasispecies populations of PRRSV. Finally, we evaluated the ability of Salmonella choleraesuis to express the PRRSV GP5, and tested its immunogenicity in mice. Based on our data, there was no indication of Salmonella replication in the mice or any evidence of antibody production against S. choleraesuis or PRRSV GP5.
Ph. D.

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Flight, William George. "The role of respiratory viruses in exacerbations of cystic fibrosis in adults". Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-respiratory-viruses-in-exacerbations-of-cystic-fibrosis-in-adults(fb64b48e-1b68-48c1-9095-5fdf5e96f9b4).html.

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Viral respiratory infections (VRI) are common in children with cystic fibrosis (CF) and are associated with significant clinical deterioration. Little previous research has been conducted on VRI in adults with CF. This thesis describes a prospective study to determine the epidemiology and clinical impact of VRI among 100 adults with CF.The incidence of identifiable VRI was 1.66 cases/patient-year. Rhinovirus accounted for 72.5% of viruses. Identifiable VRI was associated with increased risk of pulmonary exacerbation, increased respiratory symptoms and higher C-reactive protein levels. Changes in the climate and seasons affected the incidence of identifiable VRI. Rhinovirus was most common in autumn and other viruses predominated during winter. Warmer ambient temperatures were associated with increased risk of rhinovirus infection while other viruses were more common in colder temperatures. Genetic sequencing of a subset of 42 rhinoviruses identified during the study showed that rhinovirus A accounted for 69% of cases and was associated with more severe respiratory symptoms and higher C-reactive protein levels than rhinovirus B.The impact of identifiable VRI on changes to bacterial communities within the lungs of patients with CF was investigated. Ribosomal intergenic spacer analysis (RISA) was developed as a tool to profile the bacterial diversity of CF sputum and was compared with standard culture and 16S rRNA gene pyrosequencing. No consistent effect of identifiable VRI on the microbial diversity of CF sputum was detected with any of these methods in longitudinal analysis of a subset of 18 patients.

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Alosaimi, Bandar. "Studies on potential co-operativity between different types of tumour virus". Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/studies-on-potential-cooperativity-between-different-types-of-tumour-virus(5dea86d5-418d-46a0-943c-da7d53653423).html.

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Background: Although subclinical persistent infections with the human polyomaviruses are ubiquitous worldwide, they are known to vary in relation to geographical location, diseases present and may associate with different human tumours, especially in immunocompromised patients. The current study hypothesised that there may be co-operativity between HPV and polyomaviruses, particularly in HIV positive women, that could influence the rate of progression to invasive cervical carcinoma. Patients and Methods: Novel PCR methods were developed for the detection of SV40, MCV, JCV and BKV polyomavirus DNA. These were used to test DNAs extracted from 220 cervical smears and 77 invasive cervical carcinomas (ICCs) from HIV positive and negative Kenyan women of known HPV status. An expression plasmid was constructed containing JCV Large T (LT) antigen and this, in addition to empty vector control, used to stably transfect HPV16 E6/E7 immortalised human keratinocytes. Expression of LT was analysed in transfected cell lines by PCR, immunocytology and Western blotting. These cells were then used to test for changes in Cell contact growth inhibition; Growth rate and Epithelial to Mesenchymal Transition (EMT). Screening of full transcriptome microarrays was carried out on vector and LT transfected cells and their sensitivity to the drug mefloquine tested by comparison of growth rates and live/dead cell assays. Results: PCR accurately detected ~18 copies of SV40, MCV, JCV and BKV DNA in addition to simultaneous detection of JCV and BKV. None of the clinical samples tested were positive for SV40, MCV, or BKV DNA. However, JCV DNA was detected in 24/297 (8%) of cervical specimens. Comparison of the incidence of JCV in cervical smears and ICCs showed a ~3-fold increase in samples from HIV positive women with ICC (P=0.025) whereas no significant difference was found between smears and ICCs from HIV negative women (P=0.553). Analysis of the consequences of ectopic expression of JCV LT in E6/E7 immortalised human keratinocytes showed no difference in either growth rates or contact inhibition and changes in the EMT marker vimentin were found to be related to cellular clonality. Microarray analysis showed LT related alterations in gene expression which could have bearing on its carcinogenic potential in addition to changes related to clonality. JCV LT expressing monoclonal cell were the most sensitive to mefloquine treatment. Conclusion: The simultaneous JCV/BKV detection method, described herein, is unique and has been evaluated by the WHO for this purpose. The results indicate the prevalence JCV and BKV with respect to the African geographical location and suggest that JCV may combine with high-risk HPV in a sub-set of HIV positive women to influence the rate of progression to invasive cervical carcinoma. In vitro JCV LT was found not to be an overt oncogene in the cell system used although cell cloning procedures clearly affected the assays. LT induced changes in total gene expression were consistent with neoplastic progression although a high proportion of genes with unknown function were dsyregulated with respect to clonality. The anti JCV drug mefloquine showed some selectivity for LT expressing cells and further investigation of this indication is warranted.

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Maurin, Cédric. "Synthèse d'inhibiteurs potentiels de l'intégrase du VIH-1". Lille 1, 2004. https://ori-nuxeo.univ-lille1.fr/nuxeo/site/esupversions/ce7057c5-a24c-410e-a5fa-31a72a812588.

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Le virus d'immunodéficience humaine (VIH) est le rétrovirus responsable du syndrome d'immunodéficience acquise (SIDA), qui, en une vingtaine d'années, a causé la mort de plus de trente millions de personnes dans le monde. Les traitements actuels contre cette maladie sont composés essentiellement d'inhibiteurs de deux des trois enzymes constitutives du VIH: la transcriptase inverse et la protéase. La combinaison de ces médicaments constitue ce que l'on appelle communément la trithérapie. Malheureusement l'apparition de résistances impose la recherche de composés actifs sur d'autres étapes du cycle réplicatif du virus. La troisième enzyme du virus, l'intégrase, constituant notamment une cible intéressante, ce travail relate la synthèse de nouveaux inhibiteurs potentiels de cette enzyme. Par l'intermédiaire de plusieurs procédés de synthèse (dimérisation, couplage de Suzuki,. . . ), différents composés de type polyphénol et de type dicétoacide ont été obtenus, dont vingt et un démontrent lors de tests In vitro une activité importante illustrée par des CI50 inférieures ou égales à 10 ưM. Le résultat le plus prometteur concerne un 2-phénylnaphtalène hexahydroxylé qui affiche une activité submicromolaire. La synthèse et l'évaluation de l'activité de molécules dérivées du L-708,906 a mis en avant l'importance de l'intégrité de la fonction α,γ-dicétoacide. De plus, l'étude en solution d'un composé de structure simple portant cette fonction (l'acide benzoylpyruvique) a démontré la capacité de ce composé à complexer deux métaux divalents, renforçant les hypothèses concernant l'interaction de ce type d'inhibiteur avec l'intégrase.

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Adomavicius, Tomas. "Evaluation of the hepatitis B virus particle as a malaria vaccine carrier". Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/evaluation-of-the-hepatitis-b-virus-particle-as-a-malaria-vaccine-carrier(bb9e157f-dc5c-4b81-9731-5ced1cbef8fe).html.

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Malaria is a major health problem and an effective vaccine is essential for the eradication of the disease. Despite extensive efforts, a malaria vaccine remains elusive due to the parasite's complex life cycle, diverse morphology, and immune system evasion mechanisms. Antibodies against C terminal domain of merozoite surface protein 1 (MSP1-19), a highly conserved protein and the main vaccine candidate for blood-stage malaria, can inhibit erythrocyte invasion by the parasite and alleviate the disease symptoms. However, MSP1-19 is poorly immunogenic and classic protein-in-adjuvant MSP1-19-based vaccine formulations failed to induce strong immune responses due to low immunogenicity and generation of ineffective antibodies. The aim of this study was to use hepatitis B virus core (HBc) particles to increase the immunogenicity of MSP1-19. HBc forms particles with protruding spikes and induces a strong and specific immune response against foreign epitopes inserted at the tips of the spikes. In addition, positioning of MSP1-19 on the particle can influence the accessibility of certain antibody binding sites, possibly altering elicited antibody fine specificity and vaccine efficiency. MSP1-19 domain was inserted into the middle of the HBc sequence so that it is displayed at the tips of the HBc particle. Two HBc-MSP1-19 constructs, having different insert flanking linkers, displayed soluble particle formation after bacterial expression and lysis optimization. The particles were purified and the suitability of these two constructs as malaria vaccine candidates was assessed. Firstly, binding of the conformational anti-MSP1-19 antibodies indicated that MSP1-19 domain in the chimeric proteins has the correct disulphide bond pattern which is crucial for the protective properties of an MSP1-19-based vaccine. Furthermore, electron microscopy imaging and determination of initial 3D structures confirmed that both HBc MSP1-19 constructs form particles resembling the wild-type HBc particles, meaning the insertion of MSP1-19 did not heavily distort the overall HBc particle structure. In addition, it was shown that MSP1-19 domains are displayed at the tips of the particle spikes. Particle formation and foreign epitope display are important for the epitope's immunogenicity improvement. The immunogenicity of the chimeric particles was then assessed in mice. Both constructs elicited similar high antibody titres without the use of additional adjuvants, but no difference was observed between the particulate constructs and a non-particulate control (an MSP1-19-based protein). Interestingly, although both HBc-MSP1-19 and non-particulate MSP1-19-elicited antibodies recognized native malarial parasite, only the particulate construct antibodies demonstrated a moderate parasite growth inhibition while the antibodies from the control group did not show parasite inhibition above the background levels. In conclusion, it was shown that MSP1-19 can be expressed in bacteria as a soluble correctly folded protein fused to HBc. More importantly, the fusion protein is capable of forming immunogenic particles which generate antibodies that recognize native MSP1 and inhibit parasite growth more effectively than the protein without the HBc. Therefore, this work lays grounds and supports further chimeric HBc-MSP1-19 research and development.

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Schultz, Patrick. "Etudes structurales du minichromosome du virus sv40 et de la chromatine cellulaire : approches en microscopie electronique". Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13082.

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Auburn, Helen. "Evaluation of the role of Epstein-Barr virus and cellular gene expression in paediatric and adult transplant recipients". Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/evaluation-of-the-role-of-epsteinbarr-virus-and-cellular-gene-expression-in-paediatric-and-adult-transplant-recipients(d0044c58-3c67-48dd-9a78-f67821a2245a).html.

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Post-transplant lymphoproliferative disease (PTLD) is a life-threatening complication after solid organ, bone-marrow and haematopoietic stem cell transplantation. While early diagnosis is known to predict encouraging outcomes, no specific markers exist for the early detection of PTLD. Epstein-Barr virus (EBV) infection is considered to be an important risk factor for PTLD. However, the correlations between EBV DNA loads and the onset of PTLD are inconclusive. The aim of this study was to identify potential biomarkers for the early onset of PTLD. To accomplish this, we examined EBV and cellular gene expression patterns in (i) the context of EBV lytic induction in a lymphoblastoid cell line (LCL) model system, (ii) PTLD versus non-PTLD transplant patients. In addition we examined the contributions of lytic virus replication vs. latent infection to the onset of PTLD. We used real-time two-step RT-PCR assays with SYBR green detection to analyse EBV gene expression (76 lytic, 12 latent [n = 88 genes]), following 12-O-tetradecanoyl-13-phorbol acetate/sodium butyrate (TPA/NaB) induction of Raji cells. We identified 22 highly induced EBV genes (>90-fold) at 24 hrs post-induction. Using genome-wide Affymetrix microarrays, we identified cellular genes that were highly differentially expressed and regulated during the EBV lytic induction phase, 112 of which were regulated specifically by EBV, and 14 chosen for further study. We developed and validated real-time two-step TaqMan RT-PCR assays for clinical validation. Altogether, we evaluated 17 cellular and 14 EBV candidate genes in whole blood from paediatric solid organ transplant (SOT) recipients and adult umbilical cord transplant (UBCT) recipients, with and without PTLD. We detected a higher number and level of expression of EBV latent and lytic genes in PTLD patients, notably, BALF5, EBNA-LP and LMP-1, and in association with viral loads. Further, we detected latency III and more varied EBV latent gene expression patterns in PTLD. We also detected four cellular genes (CXCL9, CXCL10, CDC2, and CHI3L1) that were differentially expressed in PTLD vs. non-PTLD patients. To conclude, these findings suggest a basis for the likely microenvironment in which PTLD could develop. Further evaluation of candidate EBV and cellular markers could facilitate a more specific diagnosis of PTLD.

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Islam, Ayesha. "Interactions of human immunodeficiency virus type 1 with mucosal epithelial surfaces and Candida albicans". Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/interactions-of-human-immunodeficiency-virus-type-1-with-mucosal-epithelial-surfaces-and-candida-albicans(c8f83c82-f3af-469e-9790-8626f4b96d0f).html.

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Despite the magnitude of the HIV pandemic, the events involved in the initial HIV-1 entry into the body are not yet fully understood. Although the principle mode of HIV-1 transmission is through mucosal surfaces, the oral epithelium appears to be less susceptible to HIV-1 infection than vaginal epithelium. In addition, infections with co-pathogens that elicit immune activation, such as Candida albicans, may also promote HIV-1 infection. The project objectives are to determine whether (i) HIV-1 is able to bind and integrate into oral and vaginal epithelial cell lines, (ii) epithelial cell lines are able to transfer viable virus from their surface to permissive cells, (iii) epithelial cells are responding to HIV-1 with changes in intracellular signalling or gene expression profiling, and (iv) Candida albicans can affect epithelial susceptibility to HIV-1 or whether it can bind and/or transfer HIV-1 to permissive cells. We demonstrate that oral, oro-pharyngeal and vaginal epithelial cell lines do not express canonical receptors for HIV-1 but they do express other receptors known to promote HIV-1 binding, including GalCer and syndecan-1. Oral and vaginal epithelial cell models can capture HIV-1, which subsequently does not appear to integrate into the epithelial genome. Therefore, viral replication is not supported. Notably, HIV-1 captured on the epithelial surface remains infectious and can be transferred to permissive cells. Furthermore, like epithelial cell lines, C. albicans can also directly bind and transfer HIV-1 to permissive cells. The carbohydrate moieties chitin and β-glucan appear to play a role in mediating viral binding. Notably, transfer of HIV-1 to permissive cells occurs from chitin but minimally from p-glucan. This indicates that fungal-viral interactions may occur at mucosal surfaces that potentially promote HIV-1 infection.

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Goka, Edward Anthony Chilongo. "Influenza A viruses dual and multiple infections with other respiratory viruses and risk of hospitalization and mortality". Thesis, University of Manchester, 2014. https://www.research.manchester.ac.uk/portal/en/theses/influenza-a-viruses-dual-and-multiple-infections-with-other-respiratory-viruses-and-risk-of-hospitalization-and-mortality(256eb122-a52a-4276-8dc1-28b5a2cc6662).html.

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Introduction: Epidemiological studies have indicated that 5-38% of influenza like illnesses (ILI) develop into severe disease due to, among others, factors such as; underlying chronic diseases, age, pregnancy, and viral mutations. There are suggestions that dual or multiple virus infections may affect disease severity. This study investigated the association between co-infection between influenza A viruses and other respiratory viruses and disease severity. Methodology: Datum for samples from North West England tested between January 2007 and June 2012 was analysed for patterns of co-infection between influenza A viruses and ten respiratory viruses. Risk of hospitalization to a general ward ICU or death in single versus mixed infections was assessed using multiple logistic regression models. Results: One or more viruses were identified in 37.8% (11,715/30,975) of samples, of which 10.4% (1,214) were mixed infections and 89.6% (10,501) were single infections. Among patients with influenza A(H1N1)pdm09, co-infections occurred in 4.7% (137⁄2,879) vs. 6.5% (59⁄902) in those with seasonal influenza A virus infection. In general, patients with mixed respiratory virus infections had a higher risk of admission to a general ward (OR: 1.43, 95% CI: 1.2 – 1.7, p = <0.0001) than those with a single infection. Co-infection between seasonal influenza A viruses and influenza B virus was associated with a significant increase in the risk of admission to ICU/ death (OR: 22.0, 95% CI: 2.21 – 219.8 p = 0.008). RSV/seasonal influenza A viruses co-infection also associated with increased risk but this was not statistically significant. For the pandemic influenza A(H1N1)pdm09 virus, RSV and AdV co-infection increased risk of hospitalization to a general ward, whereas Flu B increased risk of admission to ICU/ death, but none of these were statistically significant. Considering only single infections, RSV and hPIV1-3 increased risk of admission to a general ward (OR: 1.49, 95% CI: 1.28 – 1.73, p = <0.0001 and OR: 1.34, 95% CI: 1.003 – 1.8, p = 0.05) and admission to ICU/ death (OR: 1.5, 95% CI: 1.20 – 2.0, p = <0.0001 and OR: 1.60, 95% CI: 1.02 – 2.40, p = 0.04). Conclusion: Co-infection is a significant predictor of disease outcome; there is insufficient public health data on this subject as not all samples sent for investigation of respiratory virus infection are tested for all respiratory viruses. Integration of testing for respiratory viruses’ co-infections into routine clinical practice and R&D on integrated drugs and vaccines for influenza A&B, RSV, and AdV, and development of multi-target diagnostic tests is encouraged.

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Estmer, Nilsson Camilla. "Viral Control of SR Protein Activity". Doctoral thesis, Uppsala : Acta Universatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5124-1/.

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Wong, Kiing Aik. "Age related seroepidemiological survey of measles, mumps, rubella, varicella zoster, herpes simplex type 1 and 2 viruses". Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/age-related-seroepidemiological-survey-of-measles-mumps-rubella-varicella-zoster-herpes-simplex-type-1-and-2-viruses(7188f535-e90e-4e18-a5cb-9b5ed1a75c68).html.

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Age stratified seroepidemiological studies play a crucial role in the design and assessment of vaccination strategies. An existing multiplex bead immunoassay for measles, mumps, rubella and varicella zoster virus antibodies together with a newly developed multiplex bead immunoassay for herpes simplex virus type 1 and type 2 antibodies were used to investigate the age-related seroepidemiology of these viruses in England during 2012.To develop the HSV-1 and HSV-2 antibody assay, attempts were made to produce full length of HSV-1 and HSV-2 glycoprotein G using a baculovirus vector expression system. While HSV-1 gG protein was produced, the proteins were extensively aggregated. Native glycoprotein G molecules undergo partial removal of HSV-1 signal sequence and HSV-1 short membrane anchor sequence during post translational modification. It is possible that such post translational modification is not performed when protein is processed in insect cell culture. Attempts to produce an HSV-2 glycoprotein G were not successful. It is possible that the high GC-content of HSV-2 glycoprotein G led to poor fidelity of copying the PCR amplification sequence. Commercially available truncated HSV-1 gG and HSV-2 gG were therefore used to develop a duplex microbead immunoassay for the simultaneous detection of specific HSV antibodies in human sera. The resultant assays performed with low sensitivity and specificity (HSV-1 of 89% and 66%, respectively and for HSV-2 of 79% and 85%, respectively) compared to the reference HerpeSelect ELISA.The MMRV multiplex bead immunoassay proved rapid, and required minimal sample volume to semi-quantify MMRV specific antibodies. The seroepidemiology of MMR results was compared with previous seroepidemiological studies performed in 1996 in England. The comparison showed an increase in the proportion of individuals who were positive for mumps and measles antibodies in the 2012 survey. The proportion of individuals positive for rubella was essentially unchanged. The increase in the proportion of individuals positive for mumps and measles antibodies in 2012 show the effectiveness of the change in MMR vaccination policy for England from 1996 onward. For VZV, the proportion of individuals who were positive for varicella antibodies between the 1996 and 2012 serological surveys were essentially unchanged. The comparison showed that most young children are susceptible to VZV. At this level of immunity, it can be expected that varicella will continue to produce epidemics of infection in the population, unless varicella vaccination is implemented as a part of routine childhood vaccination.

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Nizard, Mevyn. "Optimisation d'un vaccin thérapeutique dans les tumeurs des voies aérodigestives supérieures associées aux papillomavirus : rôle de l'induction d'une immunité muqueuse et de la combinaison à la radiothérapie". Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015PA05T027/document.

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Le cancer est la seconde cause de mortalité dans le monde et les cancers de localisation muqueuse (poumon, estomac, colorectal, du col de l’utérus, …) représentent la première cause de mortalité due au cancer dans le monde. La majorité des vaccins contre les cancers muqueux n’ont à ce jour, pas montré de résultats cliniques significatifs. Au cours de ce travail, nous avons développé une immunothérapie efficace basée sur la sous-unité B non toxique de la toxine de Shiga et montré pour la première fois dans le domaine de la cancérologie que la localisation de l’immunisation était cruciale pour induire des réponses immunitaires anti-tumorales. En effet, dans un modèle préclinique, une immunisation systémique intramusculaire n’a pas permis d’induire de protection thérapeutique efficace contre le développement de tumeurs muqueuses de la langue, alors que la voie d’immunisation intranasale a induit une réponse clinique complète. Nous avons identifié les lymphocytes T CD8+ comme les cellules nécessaires à cette protection et plus précisément la population de lymphocytes T résidents mémoires (Trm). Ces Trm présentent le phénotype classique CD103+ mais expriment également l’intégrine CD49a qui joue un rôle dans la migration/rétention au sein des tumeurs mais également dans la survie à long terme des Trm. Par ailleurs nous avons montré que les cellules dendritiques muqueuses pulmonaires permettaient d’induire ce phénotype CD49a sur les lymphocytes T CD8+ alors que les cellules dendritiques de la rate non. Notre travail montre que l’aspect quantitatif de ces Trm joue un rôle dans la protection anti-tumorale, en effet nous avons pu pour la première fois moduler in vivo le nombre de Trm en traitant les souris par un anticorps anti-TGF-β. La diminution du nombre des Trm est corrélée à la diminution de la protection anti-tumorale. Les patients atteints de cancers des voies aérodigestives supérieures sont majoritairement traités par radiothérapie. Dans l’optique d’essais cliniques à court terme, nous avons montré que la radiothérapie localisée associée à notre immunothérapie permet une protection plus efficace que le traitement seul de l’un ou de l’autre notamment en provoquant un remodelage du microenvironnement tumoral associé à une normalisation vasculaire. Nos résultats ouvrent de nouvelles perspectives dans le développement d’immunothérapies thérapeutiques efficaces contre les cancers muqueux et pourront mener rapidement à des essais cliniques
Cancer is the second mortality cause worldwide while mucosal cancers (lung, stomac, …) is the first mortality cause from. The majority of cancer vaccines against mucosal tumors have not given rise yet to significant clinical results. In this work we developed a strong immunotherapy based on the nontoxic subunit B from shiga toxin and showed for the first time that the localization of the immunization is crucial to induce potent and effective anti-tumoral responses. In a preclinical model a systemic immunization failed to induce a therapeutical protection against mucosal tumor challenge while intranasal immunization completely succeed. We identified a CD8 T lymphocyte population as a required cells in this protection and more precisely the T resident memory (Trm) cells. This Trm showed the classical CD103 phenotype as well as the CD49a which can play a specific role in the retention or the migration of this cells in the tumor tissue and might play a role in the survival. We also demonstrate that dendritic cells from the mucosal parenchyma was required to induce the CD49a expression on CD8 T cells while dendritic cells from the spleen was not. Our work shows that the Trm number as an impact in the anti-tumoral protection. We were able to reduce the Trm number in vivo using an anti-TGF-β antibody. This number diminution was correlated with a less efficient anti-tumoral protection. Patients with head and neck cancers are treated with radiotherapy. In this situation we showed that the combination of radiotherapy and our immunotherapy was associated with a better protection than radiotherapy alone or immunotherapy alone thanks to a vascular normalization. These results might rapidly lead to clinical trials and might open new ways to work with immunotherapies

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Lingen-Stallard, Andrew. "Testing HIV positive in pregnancy : a study of women's experience and personal testimony following a positive human immunodeficiency virus (HIV) antibody test result during pregnancy". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/testing-hiv-positive-in-pregnancy-a-study-of-womens-experience-and-personal-testimony-following-a-positive-human-immunodeficiency-virus-hiv-antibody-test-result-during-pregnancy(b7f9438d-eda0-44fd-9191-15c87d3b4b85).html.

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Midwives recommend antenatal HIV testing in pregnancy for all women. However,limited information is available on the experience of testing HIV positive in pregnancy.This thesis explored women’s experiences of receiving a positive HIV test resultfollowing antenatal screening in United Kingdom (UK). Black Africa women have highlevels of HIV infection in the UK and notably all participants were African in origin.The theoretical basis for the study was hermeneutic phenomenology, proposed byHeidegger (1962) and further guided by van Manen (1990), exploring essence andmeaning of this lived experience. Thirteen women were recruited and participated in asemi-structured interview. Participants were recruited from two NHS sites, several HIVsupport organisations and a national advert, in order to obtain diversity of this livedexperience.The emergent phenomenon is transition and transformation of “being,” as womenintegrated HIV into their lives. As women transformed with the HIV diagnosis theybalanced major themes. The major themes consisted of shock and disbelief; anger andturmoil; loss of old self; stigma and confidentiality issues and acceptance and resilience.Primary and secondary themes included: extreme reaction on being given a diagnosiswith a cultural belief that they would die; disbelief as the result was unexpected;sadness and loss of their old self; turmoil wanting to terminate the pregnancy; isolationfrom significant others; breakdown of their relationship and considering suicide and selfharm. Most reported the pervasiveness of stigma, and how they managed both thisstigma and HIV in their lives; growing resilience was apparent with time. Copingstrategies included keeping HIV “secret” and their child or children becoming the primefocus of life, with less importance on self.This study gives midwives a unique understanding of the complexities for womentesting HIV positive and supports Bonanno (2009) and Kübler Ross’ (1969 & 2005)findings on personal loss. Additionally this study provides a unique insight into thephenomenon of transition and transformation for women who tested positive inpregnancy and explores the factors and impact of testing HIV positive. The impact of anHIV diagnosis is culturally difficult for African women and had major implications andchallenges for their future life. Midwives are crucial in supporting and improving theexperience of women when they test HIV positive.

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