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Contributions au développement de modèles petit animal et cellulaire pour les études de pathogenèse des morbillivirus / Contributions to the development of a small animal and a cell model for morbillivirus pathogenesis studiesComerlato, Juliana 16 November 2016 (has links)
Morbillivirus est un genre de virus à ARN simple brin de polarité négative de la famille des Paramyxoviridae. Actuellement, il est composé de sept espèces responsables de maladies hautement contagieuses. Les infections par morbillivirus causent une forte mortalité chez l’Homme, les petits ruminants, les carnivores et chez certains mammifères marins. Les vaccins disponibles contre les morbillivirus exigent généralement 7-10 jours pour développer une immunité protectrice. Après la Peste Bovine, première maladie animale éradiquée avec succès, la peste des petits ruminants (PPR) est la prochaine cible pour l'éradication au niveau mondial d’ici 2030. Le vaccin actuel basé sur la souche PPR Nigeria 75/1 est adéquat pour la campagne d'éradication. Cependant, certaines améliorations peuvent être envisagées pour accroître son efficacité, raccourcir le temps nécessaire pour l’éradication et réduire les coûts. Par exemple, l’introduction d’un vaccin marqué positif/négatif permettrait la différenciation entre animaux infectés et vaccinés (DIVA stratégie), permettant ainsi en temps réel la surveillance de l'infection, la vaccination et l’élimination rapide des animaux infectés. Une autre amélioration pourrait être la modification du vaccin actuel par génétique inverse pour insérer une cassette exprimant des ARN interférents capables de cibler les souches virulentes PPRV. Ce vaccin thérapeutique serait utile en situation d'urgence pour contrôler l’infection le temps que l’immunité protectrice se mette en place après vaccination. Afin de développer et tester ces nouveaux vaccins et outils thérapeutiques, il est nécessaire d’utiliser des modèles petit animal et cellulaire pour limiter les expérimentations avec les animaux d'élevage Dans ce travail, nous avons contribué au développement d’un modèle cellulaire in vitro et d’un modèle murin in vivo pour étudier la pathogenèse des morbillivirus. Le présent document est divisé en trois principaux chapitres : « Identification d’un modèle cellulaire pour les études de pathogenèse du PPRV » ; « Construction d’un clone PPR marqué le gène luciférase rapporteur » ; et « Évaluation in vivo d’un petit ARN interférent (siRNA) contre les morbillivirus ». Dans le premier chapitre, la ligne cellulaire murine 10T1/2 a été éprouvée avec des souches atténuées et virulentes du PPRV dans des conditions différentes d’expression du récepteur SLAM de la chèvre et de la réponse interféron type I. Les résultats ont montré une permissivité des cellules 10T1/2 limitée aux souches virulentes du PPRV, laquelle est indépendante du récepteur SLAM de la chèvre et de la réponse interféron type I. Le deuxième chapitre visait à développer un PPRV recombinant capable d’exprimer une luciférase par la génétique inverse. Diverses stratégies d’assemblage du génome entier du PPRV ont été établies pour l’obtention du clone d’ADNc avec le génome complet du PPRV. Cependant, le rescue a été impossible à réaliser et les raisons en sont discutées. Le dernier chapitre englobait l’évaluation des siRNA comme outils thérapeutiques contre un autre morbillivirus recombinant capable d’exprimer la luciférase, le virus de la rougeole (MV). Alors que sur les lignées cellulaires nous avons observé 100% d’activité antivirale des siRNA contre le rMV-luc, la validation in vivo, utilisant le modèle souris transgénique CD46 humain sensible à la rougeole, a échoué. Pour conclure, ce travail apporte des avancées sur le développement d’outils pour étudier la pathogenèse non seulement du PPRV mais aussi d’autres morbillivirus. / Morbillivirus genus, a non-segmented negative single-strand RNA (ssRNA) group of viruses, belongs to the Paramyxoviridae family and is currently composed of seven species responsible to highly contagious diseases. Morbillivirus infections cause significant mortality in humans, small ruminants, carnivores and in some marine mammals. The available vaccines against morbillivirus infections require usually 7-10 days to induce a protective immunity. After Rinderpest, the first animal disease successfully eradicated, peste des petits ruminants (PPR) is the next target for global eradication by 2030.The current vaccine based on the Nigeria 75/1 is fit for purpose for the eradication campaign. However, some improvements can be envisaged to increase efficacy, shorten the time to complete the eradication and reduce costs. For instance, the introduction of a positive/negative marker in the vaccine could allow the Discrimination between Infected and Vaccinated Animals (DIVA strategy), thus enabling the real-time parallel monitoring of infection and vaccination, and rapid elimination of infected animals. Another improvement could be the modification of the current vaccine by reverse genetics to insert a cassette expressing interfering RNA targeting virulent strains of PPR. This therapeutic vaccine would be useful in emergency situations to control the infection over the delay necessary for the acquisition of an efficient vaccine protection. In order to develop and test these new vaccine tools, there is a need for new cell or small animal models to limit experiments with farm animals. In this work, we contributed in the development of in vitro and in vivo murine models to study morbillivirus pathogenesis. The present document is divided in three main chapters: “Identification of a cell model to PPRV pathogenesis studies”; “Construction of a full-length cDNA clone of PPRV with a luciferase reporter gene” and “In vivo evaluation of small interfering RNA (siRNA) against morbilliviruses”. In the first chapter the mouse cell line 10T1/2 was challenged with attenuated and wild type PPRV strains using different conditions of goat SLAM expression and type I IFN response. The results showed a restricted permissiveness of 10T1/2 to wild type PPRV, which was independent of goat SLAM receptor and type I IFN response. The second chapter aimed to develop a recombinant PPRV expressing luciferase through reverse genetics methodology. Various strategies to assembling the PPRV genome were established reaching up to the full-length cDNA PPRV-luciferase construction. However, the rescue could not be achieved and the reasons for that are discussed. The last chapter encompassed the evaluation of siRNA as a prophylactic tool against another luciferase recombinant morbillivirus, the measles virus. In vitro and in vivo studies were performed with the recombinant MV (rMV-luc). Whereas in cell lines siRNA showed 100% of antiviral activity against rMV-luc, the validation in vivo, using a human CD46 transgenic mouse model susceptible to measles, failed. In conclusion, this work provides advancements in the development of tools for the study of the pathogenesis of PPRV and other morbilliviruses.
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Molecular characterisation of peste des petits ruminants viruses in sheep and goats from NigeriaMantip, Samuel Elias Lashat January 2013 (has links)
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
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Dynamique de l’émergence in vitro des mutants d’échappement du virus de la peste des petits ruminants (PPRV) face à l’activité ARN interférente ciblant le gène de la nucléoprotéine : implications pour les stratégies thérapeutiques / Dynamics of the in vitro emergence of escape mutants of the peste des petits ruminants virus (PPRV) to interfering RNAs targeting the nucleoprotein gene : implications for therapeuticsHolz Correia, Carine Lidiane 04 November 2011 (has links)
Les membres du genre Morbillivirus, famille Paramyxoviridae sont responsables de graves maladies chez l'homme et les animaux, comme la rougeole, la peste bovine (RP) et la peste des petits ruminants (PPR). Malgré l'existence de vaccins efficaces contre ces maladies, des traitements spécifiques sont souhaitables. L'inhibition de la réplication de ces virus peut-être acquise par interférence ARN (ARNi), un mécanisme d'inhibition post-transcriptionnel déclenché par des séquences courtes d'ARN double-brin (siARN). Le CIRAD a précédemment identifié 3 siARNs ciblant des régions conservées du gène de la nucléoprotéine virale capables d'inhiber au moins 80% de la réplication in vitro des virus de la rougeole, de la RP et de la PPR. Cependant, un problème majeur dans la stratégie d'ARNi est le risque d'apparition de virus résistants. Dans cette étude, nous avons évalué le risque d'apparition de mutants d'échappement du virus de la PPR sous pression de sélection de 3 siARNs appliqués seul ou en association après plusieurs transfections successives in vitro. Excepté pour la combinaison des 3 siARNs, le virus a échappé à l'ARNi après 3 à 20 passages consécutifs, avec des mutations simples ou multiples (synonymes ou pas) ou une délétion de 6 nucléotides dans la zone cible des siARN. Ces résultats mettent en évidence une plasticité génomique inattendue des morbillivirus surtout illustrée par cette délétion non-délétère d'une partie significative d'un gène viral essentiel, qui devrait être considérée comme un obstacle à l'utilisation de l'ARNi comme thérapie antivirale. Cependant, l'utilisation combinée de 3 siARNs peut être proposée pour diminuer le risque d'échappement aux siARNs. / Viruses in the genus Morbillivirus, within the family Paramyxoviridae are responsible for severe humans and animal diseases, including measles, rinderpest (RP) and peste des petits ruminants (PPR). In spite of the existence of efficient vaccines against these diseases, specific treatments to be applied when the infection is already present are desirable. Inhibition of morbillivirus replication can be achieved by RNA interference (RNAi), a mechanism of post-transcriptional gene silencing triggered by small double-stranded RNA (siRNA). The CIRAD previously identified three siRNAs that target conserved regions of the essential gene encoding the viral nucleoprotein and are able to prevent in vitro at least 80% of the replication of measles, RP and PPR viruses . However, a major problem in RNAi is the important risk of emergence of escape mutants. In this study, we investigated the ability of PPR virus to escape the inhibition conferred by single or multiple siRNAs after several consecutive transfections in vitro. Except with the combination of the three different siRNAs, the virus systematically escaped RNAi after 3 to 20 consecutive passages. The mutations were characterized by either single or multiple punctual nucleotide mutations (synonymous or not) or a deletion of a stretch of 6 nucleotides into the siRNA target. These results demonstrate that the genomic plasticity of morbilliviruses, illustrated maily by this significant and no-deleterious deletion in an essential viral gene, should be considered as an obstacle to the use of RNAi in antiviral therapy. However, the combined use of three siRNAs can be proposed to prevent treatment failure with siRNAs.
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Détermination de la structure de protéines à l'aide de données faiblement résoluesPiuzzi, Marc 03 December 2010 (has links) (PDF)
La connaissance des structures tridimensionnelles des macromolécules biologiques est indispensable pour mieux comprendre leur rôle et pour la conception de nouvelles molécules thérapeutiques. Les techniques utilisées actuellement offrent une grande variété d'approches qui utilisent à la fois des informations spécifiques à la protéine étudiée et des informations génériques communes { l'ensemble des protéines. Il est possible de classer ces méthodes en fonction de la quantité d'information utilisée dans chacune de ces deux catégories avec d'un côté des méthodes utilisant le plus possible de données spécifiques { la protéine étudiée et de l'autre les méthodes utilisant le plus possibles de données génériques présentes dans les bases de données. Le travail présenté dans cette thèse aborde deux utilisations de techniques mixtes, présentant une autre combinaison entre données spécifiques et données génériques. En particulier nous avons cherché à obtenir la structure de protéines composée d'un ou deux domaines en ne disposant que d'un nombre restreint de données spécifiques. Pour déterminer la structure d'une protéine de grande taille composée de deux domaines { l'aide de données de diffusion des rayons X et de modèles obtenus par de la modélisation par homologie, nous avons adapté puis optimisé un programme récemment développé au laboratoire. Nous avons ensuite modélisé la structure d'un domaine d'une protéine de virus en incorporant un faible nombre de contraintes issues des données obtenues par RMN dans une méthode de prédiction de structure " ab initio ". Enfin, nous avons étudié l'intérêt d'intégrer les courants de cycle, une composante du déplacement chimique, dans un programme d'arrimage moléculaire pour la résolution de complexes protéine-ADN.
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Characterization of an In Vitro Transcription System for Peste Des Petits Ruminants Virus and Functional Characterization of RNA Triphosphatase Activity of RNA Dependent RNA Polymerase Protein LAnsari, Mohammad Yunus January 2012 (has links) (PDF)
Peste des petits ruminants virus (PPRV) belongs to the family paramyxoviridae which comprises non segmented negative sense RNA viruses including measles and rinderpest virus. PPRV is the causative agent of peste des petits rumaninats disease (also known as sheep or goat plague disease) in small ruminants. The viral genome contains a non segmented negative sense RNA encapsidated by viral encoded nucleocapsid protein (N-RNA). Viral transcription is carried out by the virus encoded RNA dependent RNA polymerase complex represented by the large protein L and phosphoprotein P. Viral transcription begins at the 3’ end of the genome synthesising all the viral transcripts (3’-N-P-M-F-HN-L-5’). A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end due to attenuation of polymerase transcription at each gene junction.
The objectives of the present study are characterization of peste des petits ruminants virus transcription and the associated activities required for post transcriptional modification of viral mRNA. In addition, an attempt has been made to develop in vitro transcription with heterologous combination of PPRV and RPV polymerase proteins. The first reaction in capping involves removal of γ-phosphate from triphosphate ended precursor mRNA by RNA triphosphatase. The domain having RNA triphosphatase activity within the L protein has been identified and expressed independently in E. coli. The details of the objectives are presented below.
1. Development of in vitro transcription system for PPRV mRNA synthesis
In order to develop an in vitro transcription reconstitution system for PPRV, the viral RNP complex comprising large (L), phospho (P) and N protein encapsidating viral genomic RNA was purified from virus infected Vero cells. The in vitro transcription reconstituted system with RNP complex was able to synthesise all the viral mRNA as analysed by RT-PCR. As a control, total RNA from virus infected cells was isolated and analysed by RT-PCR. In order to refine the in vitro transcription system, separately expressed recombinant polymerase complex was used to reconstitute transcriptional activity in vitro. For this,viral genomic RNA (N-RNA) was purified from PPRV infected cells using CsCl density gradient centrifugation. The recombinant baculovirus for PPRV P protein was earlier generated in the lab. A recombinant baculovirus harbouring the L gene of PPRV was generated in the present study (described in part one). The viral RNA polymerase consisting of L-P complex was expressed in Sf21 insect cells and partially purified by ultra centrifugation on 5-20% glycerol gradient. Glycerol gradient fraction containing the L-P complex was found to be active in the in vitro transcription reconstitution system. Further quantitation of transcripts made in vitro and in infected cells has been carried out by real time PCR. Notably, the gradient of polarity of transcription of viral mRNA observed in vitro with the partially purified recombinant L-P complex was similar to the gradient observed in infected cells. Host proteins have been shown to modulate the transcription of many paramyxoviruses. In order to test the role of host factors, uninfected cell lysate of Vero cells was added to the in vitro transcription reaction and the transcript level was measured by real time PCR. The result showed an increase in the transcription by addition of host proteins suggesting the involvement of host factors in viral transcription. Further, the newly developed in vitro reconstitution system was used to test if recombinant L and P proteins of RPV can functionally replace PPRV L and P protein in the in vitro transcription complementation assay. The result presented in part one indicates that the L or P protein of PPRV can be replaced by RPV L and P protein in heterologous transcription reconstitution system ,with a reduced efficiency. However, the homologous polymerase complex of RPV failed to recognise the N-RNA genomic template of PPRV.
2. RNA triphosphatase activity of PPRV L protein and identification of RNA triphosphatase domain
Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if PPRV RNA polymerase possesses any of the capping activities, the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from virion. Using the purified RNP complex, the first activity required for mRNA capping, RNA triphosphatase was tested and the results are described in part two.
RNP complex purified from virion showed both RNA triphosphatase (RTPase) activity. The RNA triphosphatase from viruses, fungi and other eukaryotes have been classified into two groups, metal dependent and metal independent. The cleavage of the γ-phosphate from triphosphate ended precursor mRNA by L protein of PPRV was found to be metal dependent. So, by the metal dependency of the RTPase reaction, PPRV L protein was assigned to the metal dependent RTPase tunnel family. One of the key features of metal dependent RTPase group members is the ability to hydrolyse γ-β phosphoanhydride bond of NTPs. PPRV L protein associated with RNP complex also was also able to cleave γ-β phosphoanhydride bond of NTPs.
Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to mRNA synthesis and post transcriptional modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Domain II has the catalytic motif for viral RNA dependent RNA polymerase. Multiple sequence alignment of PPRV L protein with known RNA triphosphatases predicted a two hundred amino acid long region on L protein comprising the C terminus of domain II and N terminus of DIII as a possible candidate for RNA triphosphatase domain. The above predicted domain was cloned and expressed in E. coli. The ability of the purified recombinant RTPase domain to cleave γ-β phosphoanhydride bond of RNA was tested. The results described in part two suggest that the predicted RTPase domain has RNA triphosphatase activity. In addition to RNA triphosphatase, the RTPase domain also has the NTPase activity.
The RNA triphosphatase of DNA viruses, yeasts and other fungi have three motifs essential for enzyme activity. Motif A and motif C are rich in glutamate and are involved in metal binding. Motif B is rich in basic amino acids and forms the centre for catalysis. The glutamate residue (E1647) of motif A of PPRV L protein RTPase domain was converted to alanine and the loss of RTPase activity was assessed. The results summarised in appendix 1 shows that the E1647A mutant has reduced RNA triphosphatase and NTPase activity.
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