O Semliki Forest Vírus recombinante (rSFV) vem sendo apontado como um dos vetores virais mais promissores, tanto para a expressão de proteínas de interesse biotecnológico, como para a utilização como vetor vacinal. Neste trabalho obtivemos um rSFV carregando o gene da proteína verde fluorescente GFP (rSFV-GFP), que foi utilizado para a padronização de métodos de obtenção do vetor viral e de sua titulação, duas etapas importantes para os trabalhos que utilizam rSFVs. Por meio da análise da expressão da GFP em células BHK-21 infectadas por lotes de vírus obtidos por um reagente de transfecção lipídico ou por eletroporação, estabelecemos as melhores condições de obtenção dos rSFV e de sua utilização para a infecção e produção de proteínas recombinantes de interesse. Além disso, padronizamos criteriosamente uma metodologia de RT-PCR quantitativa (qRT-PCR) absoluta, baseada em uma curva padrão, para realizar a titulação de partículas rSFV, independentemente do gene heterólogo clonado. A análise direta da quantidade de células infectadas, por citometria de fluxo e por microscopia de fluorescência, fez do rSFV-GFP a ferramenta adequada para nossos estudos de titulação viral por qRT-PCR. Foi demonstrada a associação entre o número de cópias do RNA viral utilizado para a infecção das células e a quantidade de células expressando GFP. Essa abordagem levou ao desenvolvimento de competências técnica e tecnológica (construção, avaliação e titulação) que facilitarão a obtenção de outros rSFV de interesse. Concluímos que a eletroporação é o melhor método de obtenção das partículas e que o título viral, determinado por qRT-PCR, pode ser utilizado para calcular o volume de um lote de rSFV necessário para obter a multiplicidade de infeção desejada. / The recombinant Semliki Forest Virus (rSFV) has been considered as one of the most promising viral vectors, both for the expression of proteins of biotechnological interest or as a vector for immunizations. In this study, an rSFV carrying the gene of the green fluorescent protein (GFP) was obtained (rSFV-GFP) and used for the standardization of rSFV production and of a novel titration methodology. Through the analysis of the amounts of GFP expressed in BHK-21 cells infected with virus stocks obtained by transfection with a lipid reagent or electroporation, we established the best conditions for rSFV production based on the production of this recombinant protein. In addition, a standardized method of absolute quantitative RT-PCR (qRT-PCR), based in a standard curve and applicable to virtually all rSFV particles, regardless of the heterologous gene cloned, was validated. The direct analysis of the quantity of infected cells, by flow cytometry and fluorescence microscopy, confirmed that the rSFV-GFP was an appropriated tool to support ours studies of viral titration by qRT-PCR. It was demonstrated the association between the amount of copies of viral RNA used for culture infection and the amount of GFP expression. This approach led to the development of technical and technological competence (construction, evaluation and titration) in the laboratory, that shall help the establishment of other rSFV of interest. We concluded that electroporation is the best methodology to obtain rSFV particles and the viral titre, as determined by qRT-PCR, can be used to calculate the volume of an rSFV stock necessary to obtain the desired multiplicity of infection.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

O Semliki Forest Vírus recombinante (rSFV) vem sendo apontado como um dos vetores virais mais promissores, tanto para a expressão de proteínas de interesse biotecnológico, como para a utilização como vetor vacinal. Neste trabalho obtivemos um rSFV carregando o gene da proteína verde fluorescente GFP (rSFV-GFP), que foi utilizado para a padronização de métodos de obtenção do vetor viral e de sua titulação, duas etapas importantes para os trabalhos que utilizam rSFVs. Por meio da análise da expressão da GFP em células BHK-21 infectadas por lotes de vírus obtidos por um reagente de transfecção lipídico ou por eletroporação, estabelecemos as melhores condições de obtenção dos rSFV e de sua utilização para a infecção e produção de proteínas recombinantes de interesse. Além disso, padronizamos criteriosamente uma metodologia de RT-PCR quantitativa (qRT-PCR) absoluta, baseada em uma curva padrão, para realizar a titulação de partículas rSFV, independentemente do gene heterólogo clonado. A análise direta da quantidade de células infectadas, por citometria de fluxo e por microscopia de fluorescência, fez do rSFV-GFP a ferramenta adequada para nossos estudos de titulação viral por qRT-PCR. Foi demonstrada a associação entre o número de cópias do RNA viral utilizado para a infecção das células e a quantidade de células expressando GFP. Essa abordagem levou ao desenvolvimento de competências técnica e tecnológica (construção, avaliação e titulação) que facilitarão a obtenção de outros rSFV de interesse. Concluímos que a eletroporação é o melhor método de obtenção das partículas e que o título viral, determinado por qRT-PCR, pode ser utilizado para calcular o volume de um lote de rSFV necessário para obter a multiplicidade de infeção desejada. / The recombinant Semliki Forest Virus (rSFV) has been considered as one of the most promising viral vectors, both for the expression of proteins of biotechnological interest or as a vector for immunizations. In this study, an rSFV carrying the gene of the green fluorescent protein (GFP) was obtained (rSFV-GFP) and used for the standardization of rSFV production and of a novel titration methodology. Through the analysis of the amounts of GFP expressed in BHK-21 cells infected with virus stocks obtained by transfection with a lipid reagent or electroporation, we established the best conditions for rSFV production based on the production of this recombinant protein. In addition, a standardized method of absolute quantitative RT-PCR (qRT-PCR), based in a standard curve and applicable to virtually all rSFV particles, regardless of the heterologous gene cloned, was validated. The direct analysis of the quantity of infected cells, by flow cytometry and fluorescence microscopy, confirmed that the rSFV-GFP was an appropriated tool to support ours studies of viral titration by qRT-PCR. It was demonstrated the association between the amount of copies of viral RNA used for culture infection and the amount of GFP expression. This approach led to the development of technical and technological competence (construction, evaluation and titration) in the laboratory, that shall help the establishment of other rSFV of interest. We concluded that electroporation is the best methodology to obtain rSFV particles and the viral titre, as determined by qRT-PCR, can be used to calculate the volume of an rSFV stock necessary to obtain the desired multiplicity of infection.

Glioblastoma is the most common primary brain tumor in humans and has a poor prognosis. Current therapies are not curative. Oncolytic viruses (OVs) are being investigated as tools to induce immunogenic cell death (ICD), cell death capable of activating the immune system. Semliki Forest Virus (SFV) strain 4 is an OV being investigated to treat glioblastoma. Previous studies in our lab have shown that SFV4 can induce ICD in human osteosarcoma (HOS) cells and ongoing in vivo studies show that SFV4 infected GL261 cell vaccination providesprotective immunity in mouse models. This study aimed to characterize the ICD induced by SFV4in glioblastoma cell lines, namely GL261, SB28 and CT2A, and to explain some of our in vivoobservations, namely why vaccination with SFV infected GL261 provides protective immunity but vaccination with infected CT2A and SB28 does not. Our in vitro studies found that GL261 is resistant to SFV4 while SB28 and CT2A are susceptible. We show that the virus can replicate in all three cell lines as seen by the presence of dsRNA, but that viral translation is delayed or inhibited in GL261 cells as not all cells positive for dsRNA were positive for SFV4 protein. Additionally, the type I interferon (IFN) pathway, responsible for antiviral defense, was highly upregulated in CT2A and SB28 but not as much in GL261 after infectionas seen by surveying IFIT1, IFITM3 and IFN-beta genes. Interferon stimulated genes (ISG) like CXCL10, a chemoattractant, was highly upregulated in GL261 after infection and might account for the protective immunity seen in vivo after vaccination. PDL1, an interferon stimulated gene responsible for self-tolerance, was highly upregulated in CT2A after infection. The IFN-beta ELISA revealed that both infected and uninfected GL261 cells produce IFN-beta suggesting a constitutively active pathway. Our DC phagocytosis assay showed that SFV4 infection of CT2A and SB28 cells induced a significant increase in DC phagocytosis and SFV4 infection of all three cell lines significantly increased DC maturation. We conclude that SFV4 infection of GL261 cells may induce ICD in vivo through a persistent viral infection and increased expression of CXCL10.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 4 uppsatser.

<p>Abstract</p><p>Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.</p>

Abstract Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.

Myelin plays an essential function in the behaviour of higher organisms by increasing the speed of axonal conduction. Indeed, myelin deficiency or damage can lead to serious motor and sensory dysfunction. In the central nervous system, myelin is produced by a specialized macroglial cell known as the oligodendrocyte. Although much is known about oligodendrocytes with respect to their role in myelin production, many details regarding their development and maintenance still remain unclear. These details may be of great importance for fully understanding the fundamental properties of oligodendrocytes and for devising strategies for treating myelin-related diseases. In this thesis, we report the development of a system using organotypic hippocampal slices, viral gene delivery methods, immunostaining techniques and confocal imaging that can be used to study the properties of oligodendrocytes and myelin. This system preserves many features of the intact brain and can be used to investigate cells of the oligodendrocyte lineage at different developmental stages. Individual oligodendrocytes were targeted using this system and we showed that they can be induced to express various proteins, such as proteolipid protein and neurofascin-155, that localized to specific compartments of oligodendrocytes. This system was then used to address the importance of glutamate receptor signalling on myelin. Studies were performed at the light microscopic level using agonists and antagonists of ionotropic glutamate receptors. Myelin showed progressive pathology over the course of hours following exposure to glutamate receptor agonists and, interestingly, glutamate signalling was not essential for myelin maintenance over a 48 hour period. This thesis work thus describes the use of a novel system that can help analyze both the cellular and molecular aspects of oligodendrocyte development and maintenance.

No description available.

Emerging arthropod-borne viruses, such as alphaviruses and bunyaviruses, represent a serious threat to human and animal health worldwide, and for most of them, vaccines and specific treatments are unavailable. Viral host cell entry can be divided into several entry checkpoints, and the most important checkpoints for low pH-dependent enveloped viruses, such as bunyaviruses and alphaviruses, include receptor binding at the cell surface and, followed by endocytosis, low pH dependent membrane fusion from within intracellular compartments. A more thorough understanding of the detailed mechanisms allowing the viruses to pass these checkpoints is a pre-requisite for the design of viral entry inhibitors. This thesis reports the in vitro analysis of native alphavirus-receptor interactions, with the help of electron cryo-microscopy and icosahedral reconstruction of virus-recaptor complexes, using the prototypic alphavirus Semliki Forest virus (SFV) and the C-type lectin DC-SIGN. Together with results from collaborative work on SFV glycosylation, this study provides progress in defining the binding sites of DC-SIGN at the surface of SFV. Second, an in vitro system for phlebovirus fusion was developed using standard fluorometry, and has been characterized with the help of electron cryo-microscopy. It was discovered that negatively charged phospholipids with a conical shape, including the late endosomal phospholipid BMP, allow efficient phlebovirus fusion in vitro, thereby providing a possible rationale for phlebovirus fusion in late endosomes. Furthermore, electron cryo-microscopy of phlebovirus-liposome complexes allowed the capture of early stage fusion intermediates and laid the basis for possible future higher resolution studies of these fusion intermediates.