Le virus de la Rougeole reste le plus meurtrier des virus contre lesquels il existe un vaccin, avec environ 350000 décès par an dans le monde. Ce virus appartient à la famille des Paramyxoviridae, qui sont des virus enveloppés de forme sphérique dont le génome est composé d’un seul brin d’ARN de polarité négative. L’élément central de la réplication et de la transcription du génome viral est le complexe, de forme hélicoïdale, entre l’ARN du virus et la nucléoprotéine. Cette association intime appelée nucléocapside a des propriétés étonnantes non encore élucidées. En effet, l’ARN des virus à ARN négatif a la particularité de n’être jamais nu, même lors des étapes de réplication/transcription nécessitant pourtant le passage de la polymérase virale. On suppose que l’interaction avec la phosphoprotéine, cofacteur de la polymérase, provoque un changement de la conformation de la nucléoprotéine pour rendre l’ARN viral accessible à la polymérase. Lorsque la nucléoprotéine est exprimée dans des cellules d’insectes, elle se fixe aux ARNs cellulaires et forme des nucléocapsides recombinantes. Les études précédentes sur d’autres virus à ARN négatif (Rage, Marbourg, Sendaï) ont montré que les nucléocapsides recombinantes sont semblables aux nucléocapsides virales. Au sein de la nucléocapside, le domaine C-terminal de la nucléoprotéine joue un rôle crucial en interagissant avec de nombreux partenaires viraux et cellulaires, notamment avec la phosphoprotéine dans les étapes de réplication/transcription du génome viral. Cependant, des observations en microscopie électronique à transmission avaient montré que les nucléocapsides recombinantes contenant la nucléoprotéine entière était trop flexibles pour envisager leur reconstruction tridimensionnelle par analyse d’image, ce qui avait conduit à les rigidifier par un traitement protéasique dont l’effet latéral est justement l’élimination du domaine C-terminal de la nucléoprotéine. Nous avons mis au point des conditions de préparation en coloration négative permettant de rigidifier la nucléocapside intacte, afin d’en calculer une reconstruction tridimensionnelle à basse résolution et de la comparer avec celle de la nucléocapside protéolysée. Nous avons ainsi montré que les nucléocapsides de la Rougeole changeaient radicalement de structure tridimensionnelle en réponse au traitement protéolytique, non seulement en terme de pas de l’hélice ou de nombre de sous-unités par tour, mais aussi au niveau de la conformation de la nucléoprotéine et de ses contacts avec les sous-unités adjacentes, ce qui n’avait encore jamais été observé aussi clairement. / Flexible helical protein polymers exemplified by actin filaments, microtubules and bacterial flagella areubiquitous in biology. Due to their size and intrinsic irregularities, the structure of these polymers cannot be solved by Xraycrystallography. Since half a century, three-dimensional (3D) reconstruction from two-dimensional (2D) ElectronMicroscopy (EM) images appears as a method of choice to solve the structure of large helical polymers. However,depending on the degree of flexibility of the analyzed helices, the 3D reconstruction process can still be a daunting task.For the most regular helices, the classical reciprocal space-based Fourier-Bessel approach can allow both to determinethe helical symmetry and to calculate 3D structures. For more flexible structures, recent “single-particle” approachesconsist in segmentation of long irregular helices into short (i.e. locally more regular) segments and their processing asasymmetrical objects with defined symmetry-imposed constraints (Egelman, 2000; Sachse et al., 2007). However, twomajor difficulties remain: the heterogeneous data must be sorted into homogeneous populations and the helical symmetryfor each population has to be determined. In the presented work, we explored various single-particle approaches,developed new analysis methods, and implemented most of them into a user-friendly processing pipeline. The targetbiological objects were helical nucleocapsids of two negative strand RNA viruses, Measles (MeV) and VesicularStomatitis Virus (VSV ; the prototype for Rabies), the latter being particularly flexible in terms of helical parameters(diameter, number of subunits per turn). Nucleocapsids are formed by the viral genomic RNA coated by thenucleoprotein and serve as a template for viral replication and transcription. To overcome the heterogeneity problem, weused 2D classification, described general processing protocols and applications for helical segments, and introduced anew classification method based on the power spectra of the images. The determination of helical symmetry(ies) wasaddressed by a novel approach relying on ab initio exhaustive search of helical parameters whereby we start from asingle 2D image, reconstruct as many 3D structures as parameters to test by cropping the image and assigning views tothe obtained segments, and calculate the cross-correlation (CC) of the reprojection of the 3D model with the initialimage. Applied to artificial data sets, the method was effectively able to detect a maximum of CC for the true symmetryparameters, but also showed intrinsic ambiguities of helical symmetry determination on which we extensively comment.Altogether, the result of this method-oriented work allowed us to address several biological questions. First, the 3Dreconstruction by negative stain EM of two forms of nucleocapsids of MeV coupled to a docking of a homologouscrystal structure enabled us to determine the orientation of the nucleoprotein and of the RNA in the nucleocapsids.Secondly, we assessed the structure of in vitro formed nucleocapsids of VSV and showed that assemblies close to thenative viral nucleocapsids can be formed in the absence of any other viral proteins, thus providing new insights into theassembly of this virus. As a perspective of this work, our pipeline of flexible helical analysis is being extended andsuccessfully used for other projects.

CD9 und andere Mitglieder der Tetraspaninfamilie sind an der strukturellen Organisation und der Plastizität der Plasmamembran beteiligt. Dabei inhibiert mAK K41, ein spezifischer CD9-Antikörper, die Hundestaupe-induzierte Zell-Zellfusion und die Virusfreisetzung, während die MV-induzierte Zell-Zellfusion nicht beeinflusst wird. So ist die extrazelluläre Domäne des Hämagglutinin-Proteins von CDV diejenige, die die Empfindlichkeit der Zell-Zellfusion gegenüber dem CD9-Antikörper verursacht, die aber selbst nicht an das CD9-Molekül bindet. Diese Erkenntnisse ließen vermuten, dass strukturelle Veränderungen an der Plasmamembran der Grund für die Hemmung sind bzw. räumliche Expressionsmuster des Rezeptors involviert sein könnten. In der vorliegenden Arbeit konnte gezeigt werden, dass mAK K41 das Konformationsepitop der großen extrazellulären Domäne (LEL) von CD9 erkennt, die nachweislich über ß1-Integrin mit verschiedenen Signalwegen im Innern in Kontakt steht. Die Bindung dieser Domäne induziert folglich eine schnelle Umlagerung und ein Clustern der CD9-Moleküle bis zur Bildung von netzähnlichen Strukturen an den Kontaktstellen zweier Zellen. Durch konfokale und rasterelektronenmikroskopische Untersuchungen konnten mikrovilli-ähnliche Ausstülpungen aufgedeckt werden, die von beiden Seiten aneinander liegender Zellen gebildet werden. Nach einer Zeitspanne von 2 h bis 20 h bildeten diese CD9-haltigen Ausstülpungen feine, mehrere µm lange Mikrovilli aus, die sich in einer Art Geflecht miteinander vernetzten und mikrovilli-artige Reißverschlussstrukturen bildeten. Weiterhin konnte eine starke Kolokalisierung des Ewi-F-Proteins vor und nach der Antikörperinkubation gezeigt werden, sowie eine partielle Kolokalisierung mit ß1-Integrin. Im Vergleich konnten MV-Proteine innerhalb der CD9-haltigen Netzstrukturen beobachtet werden, während CDV-Proteine komplett aus diesen ausgeschlossen wurden. Somit ist die Ausgrenzung der viralen Fusionsmaschinerie von CDV von den CD9-Clustern sowie die physikalische Trennung von den Zellkontakten wohl die Erklärung für die Inhibition der Virus-induzierten Zell-Zellfusion durch mAK K41. Da experimentell keine kausale Verbindung zwischen der Induktion der CD9-haltigen Cluster und bestimmten Signalwegen gezeigt werden und auch kein Beweis hervorgebracht werden konnte, dass die Grundlage der Netzstrukturen durch die Umlagerung des Zytoskeletts entsteht, scheint die Interaktion des Antikörper selbst die treibende Kraft für die Strukturbildung zu sein (Singethan et al., 2008). Zusammenfassend ist zu sagen, dass die Ergebnisse die Relevanz des CD9-Moleküls in gesunden und pathogenen Zell-Zellfusionsprozessen unterstreichen und eindeutig zeigen, dass CD9 die Zellfusion von CDV steuern kann, indem es den Zugang der Fusionsmaschinerie zu den Zellgrenzen reguliert. Das Nipah-Virus (NiV) ist ein hochpathogenes Paramyxovirus, das in Schweinen eine Erkrankung des Respirationstrakts und in Menschen eine schwere fiebrige Enzephalitis mit hohen Mortalitätsraten verursacht. Da es noch keine Vakzine bzw. antivirale Medikamente gegen dies Erkrankung gibt, war die Entwicklung kleiner inhibitorischer Moleküle notwendig. Für das Masern-Virus (MV) wurden bereits kleine Inhibitoren, wie Ox-1, AM-2 und AS-48 entwickelt, die in die Bindungstasche des MV F-Proteins passen und so die Membranfusion verhindern. Basierend auf struktureller Ähnlichkeiten der Paramyxovirus F-Proteine konnte ein Testsystem mit F- und H- oder G-exprimierenden Vektoren entwickelt werden, indem eine Gruppe von Chinolon-Derivaten sowie mehrere andere Substanzen auf ihre Fähigkeit untersucht wurden, die eine MV-, CDV- oder NiV-spezifische Fusion zu hemmen. Dazu wurde zuerst die Zytotoxizität aller Substanzen bewertet, um anschließend ihre Hemmungsaktivität in Zell-Zellfusion-Assays zu untersuchen. So inhibierten zwei Substanzen, QED15B - 12 und QED15A - 12, aus der Gruppe der Chinolon-Derivate die NiV-induzierte Synzytienbildung in Hüllprotein-Transfektions- und Infektions-Assays. Bei molekularen Untersuchungen der Bindungstasche des NiV F-Proteins wurde die hemmende Aktivität beider Chinolon-Derivate bestätigt. So konnte eine hervorragende Wechselwirkung und strukturelle Paßform für die Protein-Bindetasche identifiziert werden und deren Interaktion bewiesen werden. Somit konnte die Substanzklasse der Chinolone als Inhibitoren gegen die NiV-Fusion identifiziert und der Mechanismus der Interaktion mit der Bindetasche als Grund für die inhibierende Wirkung aufgeklärt werden (Niedermeier S. und Singethan K. et al., 2008 zur Veröffentlichung eingereicht). Dabei sind diese Chinolon-Derivate mit ihrer Struktur, die völlig verschieden von den meisten aktiven Molekülen gegen die Masern-induzierte Zellfusion sind, eine vielversprechende neue Verbindungsstruktur, auf der weitere Entwicklungen neuer Inhibitoren und antiviraler Agentien aufgebaut werden können. Letztlich sollte ein Testsystem für Untersuchungen der Dengue-Virus… / Members of the tetraspanin family including CD9 contribute to the structural organization and plasticity of the plasma membrane. K41, a CD9-specific mAb, inhibits canine distemper virus (CDV) induced cell-to-cell fusion and virus release, whereas measles virus (MV) induced cell-to-cell fusion is not affected. The extracellular domain of the viral haemagglutinin (H) of CDV determines the susceptibility of cell-cell fusion to certain CD9-antibodies, however does not itself bind to CD9. This suggested that structural alterations of the plasma membrane influencing the activity and/or spatial expression pattern of receptors are involved. In the present thesis we found that K41, which recognizes a conformational epitope on the large extracellular loop (LEL) of CD9, induces rapid relocation and clustering of CD9 in net-like structures especially at contact areas between two cells. The high resolution analyses by confocal and electron microscope revealed that CD9 clustering is accompanied by the formation of microvilli-like protrusions that are formed from both sides of adjacent cell surfaces. After about 2 h to 20 h the protrusions are forming more and more structures like microvilli zippers. While the cellular CD9-associated protein EWI-F is co-clustering with CD9 at cell contact areas after and before mAb K41 treatment, the ß1-integrin can only partially be found within the CD9 structures built at the cell interfaces. In contrast viral proteins in infected cells were differentially affected by the treatment of mAb K41. While MV envelope proteins were detected within the microvilli zippers, the CDV proteins were displaced and excluded from CD9 clusters. The exclusion of the viral fusion machinery and its physical separation from cell contact areas explains the inhibition of virus-induced cell-cell fusion by K41. Since we have no experimental support that signal transduction and remodeling of the cytoskeleton may drive the clustering, it is more likely that the antibody interaction on the cell surface is the driving force (Singethan et al., 2008). Although it is known that LEL is interacting with ß1-Integrin and inducing signaling pathways within the cell, it was not possible to find any evidence for the involvement of any signaling pathway.The findings underscore the relevance of the tetraspanin CD9 for healthy and pathogenic cell-to-cell fusion processes and clearly show that CD9 can regulate cell-cell fusion by controlling the access of the fusion machinery of CDV to cell contact areas. Nipah virus (NiV), a highly pathogenic paramyxovirus, causes a respiratory disease in pigs and severe febrile encephalitis in humans with high mortality rates. There is no vaccine and no antiviral treatment available until now. Small molecule inhibitors, like Ox-1, AM-2 or AS-48, fitting into a pocket of the measles virus (MV) F protein and preventing membrane fusion have been designed earlier. Based on the structural similarity of viral fusion (F) proteins within the family Paramyxoviridae, we tested a library of quinolone derivatives and several other small molecules in a NiV, CDV and MV envelope protein-based fusion assay. The cytotoxicity of all substances was evaluated and they were tested for their ability to inhibit cell-to-cell fusion induced by the three mentioned viruses. The most active molecules, QED15A-12 and QED15B-12, inhibiting the syncytium formation induced by transfection of pCz-CFG5-NiV-F and -G and infection with NiV, revealed aan active quinolone-type compound structure, which is different from the most active molecules against MV induced cell fusion. In conclusion, this study revealed a class of promising compounds fitting into a protein cavity of the NiV F protein and inhibiting NiV-induced cell-cell fusion (Niedermeier S. and Singethan K. et al., 2008 submitted for publication). Finally we had the aim to establish a test system for investigations of the Dengue-Virus envelope protein induced virus-cell fusion using DENV-E pseudotyped retroviral particles. This should open an experimental way to develop small inhibitory peptides and molecules against the Dengue E-protein, which mediates the fusion of the viral membrane with the cellular membrane, working under BSL-2 conditions. As there is not sufficient information about intermediate conformational stages of the class II fusion proteins during the fusion process, similarities to the class I proteins should help to develop highly active antiviral peptides inhibiting this process. Although cloning of DENV-3-E-cDNA in the pCG-eGFP vector lead to a high expression of the E-protein, the test system based on pseudotyped retroviral particles could not be established. Even though there were used several different modification strategies like exchanging 3´protein tails in order to get a protein with a tail of the envelope protein of MuLV, which should lead to a better packaging into the pseudotyped particle system based on MuLV…

No description available.

Introdução: Os vírus parainfluenza são importante causa de infecções respiratórias. Na população pediátrica a doença característica associada com HPIV-1 e 2 é a laringotraqueobronquite, enquanto que o HPIV-3 está associado com a pneumonia e bronquiolite. Objetivos: Realizar a análise molecular do fragmento do gene da HN. Métodos: Foram analisados aspirados nasofaríngeos coletados entre 1995 a 2005, de crianças com doença respiratória aguda. A detecção dos HPIV 1, 2 e 3 foi realizada por multiplex RT-PCR. Posteriormente, o fragmento de gene HN foi amplificado, sequenciado e, a análise molecular foi realizada. Resultados e discussão: Um total de 6% amostras foram positivas para HPIV, sendo o HPIV-3 o vírus mais prevalente durante todos os anos estudados, o que corresponde a 80% dos casos positivos, seguido por HPIV-1 (15%) e, HPIV-2 (7%). A maioria das mutações observadas foram silênciosas, no entanto, algumas alterações de aminoácidos foram verificadas em áreas conservadas dos HPIV-3 e HPIV-2, e alterações em sítios potencias de N-glicosilação também foram observados. / Introduction: In paediatric population the characteristic illness associated with HPIV-1 and -2 is laryngotracheobronchitis, whereas HPIV-3 is associated with pneumonia and bronchiolitis. There are 5 virus distributed in two distinct genus: Respirovirus (HPIVI-1 and HPIV-3) and Rubulavirus (HPIV-2, HPIVI-4A and HIVI-4B). Objectives: To carry out the molecular analysis of the fragment of the HN gene. Methods: Nasopharyngeal aspirates from infants with acute respiratory illness collected from 1995 to 2005, were analyzed. The detection of HPIV 1, 2 and 3 were performed by multiplex RT-PCR and the fragment of HN gene was amplified, sequenced and, the molecular analysis was carried out. Results and discussion: A total of 6% samples were positive for HPIV. The HPIV-3 was the most prevalent virus during, corresponding to 80% of positive cases, followed by HPIV-1 with 15% and HPIV-2 7%. Most mutations observed were silent in all PIVs, however, some amino acids alterations in conserved areas, verified in PIV-3 and PIV-2, and alterations in N-glicosilation sites were observed.

Introdução: Os vírus parainfluenza são importante causa de infecções respiratórias. Na população pediátrica a doença característica associada com HPIV-1 e 2 é a laringotraqueobronquite, enquanto que o HPIV-3 está associado com a pneumonia e bronquiolite. Objetivos: Realizar a análise molecular do fragmento do gene da HN. Métodos: Foram analisados aspirados nasofaríngeos coletados entre 1995 a 2005, de crianças com doença respiratória aguda. A detecção dos HPIV 1, 2 e 3 foi realizada por multiplex RT-PCR. Posteriormente, o fragmento de gene HN foi amplificado, sequenciado e, a análise molecular foi realizada. Resultados e discussão: Um total de 6% amostras foram positivas para HPIV, sendo o HPIV-3 o vírus mais prevalente durante todos os anos estudados, o que corresponde a 80% dos casos positivos, seguido por HPIV-1 (15%) e, HPIV-2 (7%). A maioria das mutações observadas foram silênciosas, no entanto, algumas alterações de aminoácidos foram verificadas em áreas conservadas dos HPIV-3 e HPIV-2, e alterações em sítios potencias de N-glicosilação também foram observados. / Introduction: In paediatric population the characteristic illness associated with HPIV-1 and -2 is laryngotracheobronchitis, whereas HPIV-3 is associated with pneumonia and bronchiolitis. There are 5 virus distributed in two distinct genus: Respirovirus (HPIVI-1 and HPIV-3) and Rubulavirus (HPIV-2, HPIVI-4A and HIVI-4B). Objectives: To carry out the molecular analysis of the fragment of the HN gene. Methods: Nasopharyngeal aspirates from infants with acute respiratory illness collected from 1995 to 2005, were analyzed. The detection of HPIV 1, 2 and 3 were performed by multiplex RT-PCR and the fragment of HN gene was amplified, sequenced and, the molecular analysis was carried out. Results and discussion: A total of 6% samples were positive for HPIV. The HPIV-3 was the most prevalent virus during, corresponding to 80% of positive cases, followed by HPIV-1 with 15% and HPIV-2 7%. Most mutations observed were silent in all PIVs, however, some amino acids alterations in conserved areas, verified in PIV-3 and PIV-2, and alterations in N-glicosilation sites were observed.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 6 uppsatser.

Peste des petits ruminants virus (PPRV) belongs to the family Paramyxoviridae and genus morbillivirus. It is a highly contagious, fatal and economically important viral disease of small ruminants that is still endemic and militate against the production of sheep and goats in Nigeria. It is a notifiable disease according to the World Organization for Animal Health (Office International des Epizooties). In this study, a molecular analysis of PPRV from sheep and goats from recent outbreaks across the different regions of Nigeria was carried out. The aim was to describe the viral strains and the movement of the virus within the country compared to other endemic areas of the world. This was carried out through tissue and swab samples collected from sheep and goats in various agro-ecological zones of Nigeria.The evolution and relationship of earlier PPRV strains/isolates and those circulating and causing recent outbreaks was determined by sequencing of the nucleoprotein (N)-gene. Twenty tissue and swab samples from apparently healthy and sick sheep and goats were collected randomly from each of three states of each of the six agro-ecological zones visited. A total of 360 samples were collected. A total of 35 samples of 360 (9.7 %) tested positive by RT-PCR, of which 25 were from oculo-nasal swabs and 10 were from tissue samples (Table 4.2). Phylogenetic analysis was carried out using the N-gene sequences of the PPRV amplicons. Alignment of the sequences and related sequences from GenBank and neighbor-joining phylogenetic analysis using PAUP identified four different lineages, i.e. lineages I, II, III and IV. Interestingly, the Nigerian strains described in this study grouped in two separate major lineages i.e. lineages II and IV. Strains from Sokoto, Oyo, Plateau and Ondo states grouped according to the historical distribution of PPRV together with the Nigerian 75/1 strain of lineage II, while other strains from Sokoto, Oyo, Plateau, Akwa-Ibom, Adamawa, Kaduna, Lagos, Bauchi, Niger and Kano states grouped together with the East-African and Asian strains of lineage IV. This finding suggests that both lineages II and IV strains of PPRVs are circulating presently in Nigeria, contrary to an earlier publication which indicated that only strains of lineage II were circulating in the country (Shamaki, 2002). / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

Baigiamojo darbo tikslas – ištirti Paramyxoviridae šeimos virusų nukleokapsidės baltymų sintezės mielėse Saccharomyces cerevisiae galimybes ir nustatyti ar mielėse susintetintus nukleokapsidės baltymus galima taikyti Paramyxoviridae šeimos virusų infekcijų serologinei diagnostikai. Tikslas pasiektas ištyrus Paramyxoviridae šeimos Sendai, žmogaus parainfluenza, žmogaus respiracinio sincitinio, Nipah, Hendra ir Menangle virusų nukleokapsidės baltymų sintezę mielių Saccharomyces cerevisiae kamiene 214∆pep4. Nustatyta, kad mielėse susintetinti nukleokapsidės baltymai pasižymi natyviems analogiškų virusų nukleokapsidės baltymams būdingomis savybėmis: yra tirpūs, formuoja virusų nukleokpsidę primenančias daleles, kurios nespecifiškai įjungia ribonukleorūgštis, pasižymi antigeninėmis savybėmis, būdingomis natyviems analogiškų virusų nukleokapsidės baltymams. Taip pat mielėse susintetinti nukleokapsidės baltymai išgryninti juos centrifuguojant per tankų sacharozės tirpalo sluoksnį ir CsCl tankio gradiente. Įsitikinta, kad mielėse Saccharomyces cerevisiae susintetinti Paramyxoviridae šeimos virusų nukleokapsidės baltymai reaguoja su atitinkamais šios šeimos virusais užsikrėtusių žmonių ir gyvūnų kraujo serumo antikūnais, todėl tinka šių virusų infekcijų serologinės diagnostikos sistemų kūrimui. Naudojant mielėse susintetintus nukleokapsidės baltymus buvo gauti specifiniai monokloniniai antikūnai reaguojantys su natyviais virusų nukleokapsidės baltymais. Įvertinos gautų monokloninių... [toliau žr. visą tekstą] / The aims of this study was to investigate synthesis of SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV nucleocapside (N) proteins in yeast Saccharomyces cerevisiae, to determine properties of recombinant N proteins and evaluate the feasibility to use them in diagnostics. In this study it was demonstrated, that yeast S.cerevisiae is an excellent host for a high-level production of proteins SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV N proteins as virus nukleokapsid-like particles (vNLP). The yeast-expressed hPIV1, hPIV3, hRSV, NiV, HeV and MenV vNLPs represent useful tools for the development of new virus detection systems and demonstrate the effectiveness of yeast as a host for generation of recombinant proteins organized in complex structures like human virus NLPs.

The aims of this study was to investigate synthesis of SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV nucleocapside (N) proteins in yeast Saccharomyces cerevisiae, to determine properties of recombinant N proteins and evaluate the feasibility to use them in diagnostics. In this study it was demonstrated, that yeast S.cerevisiae is an excellent host for a high-level production of proteins SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV N proteins as virus nukleokapsid-like particles (vNLP). The yeast-expressed hPIV1, hPIV3, hRSV, NiV, HeV and MenV vNLPs represent useful tools for the development of new virus detection systems and demonstrate the effectiveness of yeast as a host for generation of recombinant proteins organized in complex structures like human virus NLPs. / Baigiamojo darbo tikslas – ištirti Paramyxoviridae šeimos virusų nukleokapsidės baltymų sintezės mielėse Saccharomyces cerevisiae galimybes ir nustatyti ar mielėse susintetintus nukleokapsidės baltymus galima taikyti Paramyxoviridae šeimos virusų infekcijų serologinei diagnostikai. Tikslas pasiektas ištyrus Paramyxoviridae šeimos Sendai, žmogaus parainfluenza, žmogaus respiracinio sincitinio, Nipah, Hendra ir Menangle virusų nukleokapsidės baltymų sintezę mielių Saccharomyces cerevisiae kamiene 214∆pep4. Nustatyta, kad mielėse susintetinti nukleokapsidės baltymai pasižymi natyviems analogiškų virusų nukleokapsidės baltymams būdingomis savybėmis: yra tirpūs, formuoja virusų nukleokpsidę primenančias daleles, kurios nespecifiškai įjungia ribonukleorūgštis, pasižymi antigeninėmis savybėmis, būdingomis natyviems analogiškų virusų nukleokapsidės baltymams. Taip pat mielėse susintetinti nukleokapsidės baltymai išgryninti juos centrifuguojant per tankų sacharozės tirpalo sluoksnį ir CsCl tankio gradiente. Įsitikinta, kad mielėse Saccharomyces cerevisiae susintetinti Paramyxoviridae šeimos virusų nukleokapsidės baltymai reaguoja su atitinkamais šios šeimos virusais užsikrėtusių žmonių ir gyvūnų kraujo serumo antikūnais, todėl tinka šių virusų infekcijų serologinės diagnostikos sistemų kūrimui. Naudojant mielėse susintetintus nukleokapsidės baltymus buvo gauti specifiniai monokloniniai antikūnai reaguojantys su natyviais virusų nukleokapsidės baltymais. Įvertinos gautų monokloninių... [toliau žr. visą tekstą]

O Vírus Respiratório Sincicial humano (hRSV human Respiratory Syncytial Virus) está entre os principais causadores de doenças do trato respiratório. O hRSV pertence à família Paramyxoviridae. Os sintomas da infecção podem variar de simples estado gripal a doença respiratória grave, e eventualmente levar a óbito. Atualmente não há vacina licenciada ou droga eficaz contra esse vírus. Anteriormente foram obtidos, em nosso laboratório, vetores com genes otimizados. Com essas ferramentas a proteína M foi produzida e purificada tendo sido possível obter anticorpos policlonais específicos e eficientes na sua detecção. Com esses anticorpos confirmamos a interação da proteína M com as proteínas celulares tropomiosina e nucleofosmina. Também foi demonstrado por imunofluorescência e por western blotting que a proteína M quando expressa fora do contexto de infecção apresenta localização nuclear e citoplasmática. Foram feitos testes de imunização com a proteína M purificada ou com um vetor eucariótico que a expressa (DNA). A imunização com proteína M resultou apenas em resposta humoral, enquanto com a vacina de DNA obtivemos apenas resposta celular. Nenhum desses imunógenos, entretanto, foi capaz de conferir proteção contra hRSV. / The human Respiratory Syncytial Virus (hRSV) is among the main causes of respiratory tract diseases, hRSV belongs to the Paramyxoviridae family. The symptoms of the infection can range from simple flulike state to severe respiratory disease eventually leading to death. Currently there is no licensed vaccine or effective drug against this virus. Vectors with genes optimized for of the hRSV Matrix protein (M) expression in bacteria and in eukaryotic cells were previously obtained in our lab. With these tools the M protein was produced and purified. Specific and efficient polyclonal antibodies could then be obtained and used for its detection. Using these antibodies we confirmed M interaction with cellular proteins tropomyosin and nucleophosmin. It was also demonstrated by immunofluorescence and western blotting that protein M when expressed out of viral infection context, presents cytoplasmic and nuclear localization. Immunization tests were made with the purified M protein or with a eukaryotic vector that expresses it (DNA). Immunization with M protein resulted only in humoral response, while with the DNA vaccine only cellular response was obtained. None of these antigens, however, was able to confer protection against hRSV.