Дисертації: "Virus de la peste équine"

1

Zientara, Stéphan. "Epidémiologie moléculaire du virus de la peste équine : étude de la diversité génomique des souches par amplification génique, séquencage et comparaison de la séquence du fragment 10." Nancy 1, 1995. http://docnum.univ-lorraine.fr/public/SCD_T_1995_0466_ZIENTARA.pdf.

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2

Zientara, Stéphan. "Epidémiologie moléculaire du virus de la peste équine : étude de la diversité génomique des souches par amplification génique, séquencage et comparaison de la séquence du fragment 10." Nancy 1, 1995. http://docnum.univ-lorraine.fr/public/SCD_T_1995_0466_ZIENTARA.pdf.

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3

Sailleau, Corinne. "Typage moléculaire du virus de la peste équine par amplification génique. Etude de la protéine non-structurale NS3 et application au diagnostic sérologique." Paris, EPHE, 2000. http://www.theses.fr/2000EPHE3025.

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La peste équine est une arbovirose qui affecte les équidés et plus particulièrement les chevaux dont elle provoque très fréquemment la mort. Les conséquences dues au caractère meurtrier de la maladie mais surtout aux mesures de prophylaxie qu'elle nécessite sont préjudiciables pour l'économie de la filière chevaline des pays touchés. Nous avons développé des tests d'amplification en chaîne par polymérase spécifiques de type qui permettent, en 24 heures, la détection et le typage du virus équipestique (qui compte 9 sérotypes). Cette technique a été validée sur 54 échantillons codés. Les résultats obtenus sur 7 de ces échantillons ont permis de mettre en évidence la difficulté du typage (classique et moléculaire) lorsque plus d'un type viral est présent dans l'échantillon. Le typage moléculaire par RT-PCR a été appliqué avec succès à des prélèvements nécropsiques. Dans le but de différencier les anticorps induits par une vaccination et par une infection, deux tests ELISA, utilisant comme antigènes deux domaines de la protéine NS3, ont été développés. Les deux ELISA ont présenté une sensibilité et spécificité satisfaisantes, mais leur capacité à discriminer les sérums d'animaux infectés de ceux d'animaux vaccinés reste à confirmer. Parallèlement, des sérums de lapins produits contre les deux domaines de la protéine NS3, précédemment utilisés comme antigène dans l'ELISA, ont permis de préciser partiellement la topologie de NS3 au niveau de la membrane cellulaire. Ainsi, l'immunofluorescence sur cellules infectées a confirmé la localisation extra-cellulaire (prédite par certains auteurs) du domaine N-terminal de NS3. Enfin, la détermination des séquences nucléotidiques du segment génomique 10 (qui code pour les protéines NS3/NS3A) des sérotypes 2, 4, 5, 6 et 7 a permis de compléter les données existantes sur ce gène et de confirmer l'importante variabilité génétique de ce segment et de la protéine NS3 par rapport à celle des autres Ortivirus.

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4

Dash, Pradyot. "Development of reverse genetics for Peste des Petits Ruminants Virus." Thesis, Royal Veterinary College (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522189.

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5

Bailey, Dalan. "Development of reverse genetics for peste des petets ruminants virus (PPRV)." Thesis, University of Reading, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494788.

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Peste des petits ruminants virus (PPRV), a member of the Morbillivims genus in the family Paramyxoviridae, is the cause of a serious and emerging plague of small ruminants, mostly affecting sheep and goats. It is endemic in many developing countries in Africa and southern Asia and is highly contagious, with the mortality rate approaching 90% in some outbreaks. Development of reverse genetics systems for other morbilliviruses such as rinderpest and measles has allowed more focused research into replication, pathogenicity and marker vaccines.

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6

Miszczak, Fabien. "Artérite virale équine : détection moléculaire, épidémiologie, émergence et impact virologique d'une vaccination anti-GnRH." Caen, 2012. http://www.theses.fr/2012CAEN3003.

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Le virus de l’artérite virale équine (EAV) est l’agent pathogène responsable de l’artérite virale équine, une maladie observée uniquement chez les équidés. Au cours d’infections naturelles, l’EAV peut entraîner des avortements et des infections chroniques asymptomatiques chez les étalons. Les étalons porteurs chroniques représentent le réservoir naturel du virus et favorisent la persistance et l’émergence de nouveaux variants potentiellement virulents. Les virus sont excrétés dans le sperme, parfois tout au long de la vie de l’étalon. Une méthode de détection des ARN de l’EAV par rRT-PCR dans le sperme des étalons a été optimisée. Elle a présenté une sensibilité de détection supérieure à la technique de référence d’isolement viral par culture cellulaire (méthode OIE : Office International des Epizooties). La caractérisation moléculaire des souches d’EAV a montré que l’épizootie d’AVE, survenue en France durant l’été 2007, trouvait son origine chez un étalon porteur chronique. Cette étude a mis en évidence une distribution en quasi-espèces de l’EAV chez cet étalon et l’émergence d’un variant pathogène qui a par la suite été transmis lors de l’insémination artificielle d’une jument. L’impact virologique et hormonal d’un vaccin anti-GnRH correspondant à un traitement de type "castration chimique" a également été évalué chez des étalons porteurs chroniques. Ce traitement a permis l’élimination du virus chez la totalité des étalons vaccinés. Cette dernière étude offre des perspectives encourageantes pour le traitement des étalons
Equine arteritis virus (EAV) is the causative agent of equine viral arteritis, a disease of equids. During natural outbreaks of the disease, EAV may cause abortion and persistent subclinical infection in stallions. Persistently infected stallions represent the natural reservoir of the virus, ensuring its persistence and making possible the emergence of new pathogenic variants. Stallions shed the virus in the semen for years, or even lifetime. The method for EAV nucleic acids detection by rRT-PCR in equine semen has been optimised. RRT-PCR showed higher sensitivity for EAV diagnosis than virus isolation, which is currently the OIE-approved gold standard for EAV detection in semen. The origin of the 2007 French EAV outbreak was determined by molecular analyses and revealed a persistently infected stallion being the source of the outbreak. Viral population carried by this stallion revealed a quasi-species organisation, with emergence of a new pathogenic variant lately transmitted to a mare via artificial insemination. The virological and hormonal impact of an anti-GnRH vaccine has also been evaluated on persistently EAV-infected stallions. This treatment induced a virus clearance in all vaccinated stallions. This study then suggests potentially promising perspectives for stallion treatments

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7

Pradier, Sophie. "Circulation enzootique du virus West Nile en population équine : identification de facteurs de risque environnementaux en Camargue, France." Phd thesis, AgroParisTech, 2010. http://pastel.archives-ouvertes.fr/pastel-00605812.

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L'objectif était d'évaluer le risque de circulation enzootique du virus West Nile (WN) chez le cheval en Camargue, région dans laquelle ce virus a déjà causé plusieurs épizooties. L'épidémiologie de la maladie de WN est très complexe, du fait de l'implication potentielle d'un grand nombre d'espèces de vecteurs et d'hôtes. De par sa transmission principalement vectorielle, la circulation du virus WN est fortement influencée par des facteurs environnementaux. L'espèce équine a été choisie comme témoin de la circulation du virus WN, car le cheval est particulièrement sensible à l'infection par ce virus. La méthode appliquée est basée sur l'utilisation du lien direct existant entre l'environnement et la circulation enzootique du virus WN, par l'étude de la séropositivité (IgG) chez le cheval. Dans les deux premières études présentées, certains facteurs de risque environnementaux ont été identifiés, comme des classes d'occupation du sol (zones agricoles hétérogènes, végétation inondée) ou des indices de paysage (Indice d'Imbrication et de Juxtaposition), ayant conduit à l'élaboration d'une carte de risque pour cette circulation dans le bassin méditerranéen français. Des facteurs de risque individuels, comme la race, l'âge et l'activité du cheval, ont également été identifiés. Dans la troisième étude présentée, des hypothèses de transmission du virus en Camargue ont été testées. Dans la région d'étude, le virus serait introduit par les oiseaux migrateurs et amplifié par Culex modestus et plusieurs espèces d'oiseaux compétentes. L'effet de dilution n'aurait pas d'impact sur l'amplification du virus en Camargue. Le virus serait transmis au cheval par C. modestus et C. pipiens.

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8

Bodjo, Sanne Charles. "Etude de la nucleocapside des virus de la peste bovine et peste des petits ruminants : caractérisation moléculaire des interactions protéiques et des sites antigénique." Montpellier 2, 2007. http://www.theses.fr/2007MON20073.

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L'étude des structures antigéniques des nucléoprotéines (N) des virus de la peste bovine et de la peste des petits ruminants a montré que la région N-terminale est immunodominante. Les études de cartographie d’anticorps monoclonaux anti-N ont permis de localiser dans cette région des épitopes communs mais aussi des épitopes spécifiques à chacun des deux virus. Les tests de compétition ELISA (cELISA), développés avec certains de ces monoclonaux spécifiques à chaque virus détectent malheureusement aussi bien des sérums anti-PPR et qu'anti-peste bovine. Ce croisement serait probablement la résultante d’encombrement stérique engendré par des anticorps fixés sur les épitopes communs aux deux virus mais proches des sites spécifiques. Une partie de nos travaux a porté sur l'analyse des interactions protéine-protéine du virus PPR. Ils ont permis de localiser le domaine d'interaction N-N dans la zone couverte par les 240 premiers aminoacides de N. Quatre régions capables de se lier à la protéine M ont été identifiées sur la protéine N. Les séquences de ces zones d’interaction N-M sont conservées au sein groupe Morbillivirus

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9

Gopilo, Abraham. "Epidemiology of peste des petits ruminants virus in Ethiopia and molecular studies on virulence." Phd thesis, Toulouse, INPT, 2005. http://oatao.univ-toulouse.fr/7414/1/gopilo.pdf.

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Peste des petits ruminants (PPR) is an acute and highly contagious viral disease of small ruminants, which is characterised by high fever, ocular and nasal discharge, pneumonia, necrosis and ulceration of the mucuous membrane and inflammation of the gastro-intestinal tract leading to severe diarrhoea and high mortality. In Africa, goats are severely affected while sheep undergo a mild form or rarely suffer clinical disease. PPR is one of the most important economical diseases in Ethiopia. Clinical PPR is confirmed in Ethiopian goats, however, its circulation in other animals has never been described. In the present work, we showed that the antibody seroprevalence in camel, cattle, goat and sheep confirmed natural transmission in these animals without clinical disease. The apparent absence of pathogenicity in these animals may have been due to host resistance or loss of virulence of the virus strain. We have further investigated the latter point by in vitro studies on PPRV comparing strains from Ethiopia and other countries with the vaccine strain which has been attenuated after several cell culture passages. In a first approach, virulence of PPRV was monitored in cell culture system and the use of virus specific monoclonal antibodies enabled to detect differences in virulence between PPRV and RPV. Vero (primate origin) and 293T (human) cell lines supported virus replication permitting the in vitro growth of both PPRV and RPV. In contrast to RPV, B95a (marmoset B) cells infected with PPRV were non permissive. The capability of cells to support active virus replication, which may result in intercellular spread and induce damages in infected cells, has implications on the pathogenesis and epidemiology. Cellular receptors are major determinants of host range and tissue tropism of a virus. The difference in infectivity of PPRV and RPV may have depended on the H protein epitopes and their cellular receptors. Therefore, we decided to compare the amino acid epitope of H protein of PPRV with that of other morbilliviruses. As part of our investigation of virulence factors, we have sequenced and compared genome and antigenome promoters of a vaccine strain with field strains of PPRV. The promoters contain the polymerase binding sites to initiate and generate the positive-strand replication and transcription of mRNAs. Nucleotide base change differences between vaccine strain and field strains would provide molecular basis for attenuation. Alignment of the genome promoter sequences revealed seven nucleotide mutations at certain positions. Our finding on nucleotide mutation on PPRV are in agreement with the nucleotide changes in rinderpest virus and other morbillivirus promoter regions between vaccine strain and wild type virus. Certain mutations were specific to PPRV. The promoter sequences were clustered around the geographic origin of the viruses and were lineage specific. Phylogenetic analysis of PPRV promoters was used for PPR phylogeograhy, and for comparison with other paramyxoviruses. The thesis is divided in 6 chapters. The first chapter deals with the natural history of PPR including the virus, the genome, epidemiology, transmission, clinical signs, immunology, diagnosis, control and its economic cost in the low income subsistence farming systems in sub-Saharan Africa. The second chapter is about comparative biology of PPRV with regard to other groups of morbillivirus genus in the Paramyxoviridae family. The third chapter deals with field study and observations on epidemiology of PPR in Ethiopia. In chapter four, PPRV virulence was monitored in cell culture system and comparison of H protein epitopes. In chapter five, sequence analysis of genome and antigenome promoters of PPRV was described In chapter six, general discussion and recommendations were forwarded.

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10

Sanz, Bernardo Beatriz. "Control of host innate immune (interferon) responses by peste des petits ruminants virus (PPRV)." Thesis, St George's, University of London, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.703281.

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Peste des petits ruminants virus (PPRV) produces clinical disease in goats and sheep. PPRV is a morbillivirus, others of which are known to affect the activation of the innate immune response by the actions of their accessory proteins (V&C). A crucial part of normal activation is the induction of interferon β (IFNβ) after pathogen recognition by pattern recognition receptors (PRRs). The presence of viral RNA can be sensed by the cytosolic proteins MDA-5 and RIG-I, starting a signalling cascade that leads to the activation of the IFNβ promoter and the synthesis of IFNβ. The production of IFNβ is a defence mechanism to control the spread of infection to neighbouring cells. Many viruses have evolved to antagonize this cell response. In this thesis I present a study of the induction of IFNβ following PPRV infection in both goat and primate cells, and the effects of infection with PPRV on the induction of IFNβ following MDA-5 and RIG-I mediated activation. Using both reporter assays and direct measurement of IFNβ mRNA, I found that PPRV infection does not induce IFNβ and can block the activation of expression of IFNβ. A study of the interaction of the PPRV accessory proteins with MDA-5 and RIG-I was carried out, including the cloning of goat RIG-I and LGP2. I also generated mutant viruses that lack expression of either accessory protein to characterize the role of these proteins in IFNβ induction during virus infection. Overexpression of V blocks MDA-5 mediated induction of IFNβ, but PPRV lacking V can still block MDA-5 mediated activation of IFNβ. PPRV C bound to neither MDA-5 nor RIG-I, but PPRV lacking C lost the ability to block MDA-5 and RIG-I mediated activation of IFNβ. These results shed new light on the inhibition of the induction of IFNβ by PPRV.

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11

Gopilo, Abraham Picavet Dominique-Pierre. "Epidemiology of peste des petits ruminants virus in ethiopia and molecular studies on virulence." Toulouse : INP Toulouse, 2006. http://ethesis.inp-toulouse.fr/archive/00000226.

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12

Woma, Timothy Yusufu. "Epidemiology of peste-des-petits-ruminants virus in sheep goats and camels in Nigeria." Thesis, University of Pretoria, 2015. http://hdl.handle.net/2263/53316.

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Peste-des-petits-ruminants (PPR), caused by the peste-des-petits ruminants virus (PPRV) is endemic in Nigeria and is a major constraint to the improvement of small ruminant production, thereby affecting the rural poor who depends on these livestock species as their source of livelihoods. The major objectives of this work were: 1) to conduct a seroprevalence of PPRV in camels, sheep and goats in Nigeria; 2) to isolate PPRV from naturally infected animals; and 3) to characterize the isolates using molecular techniques and compare the profile of the current isolates with the worldwide vaccine (Nig75/1) and other existing strains from other parts of the world. The major contribution of this work to current knowledge were: finding that more than one lineage of PPRV was circulating in Nigeria, the emerging Asian lineage IV gradually replacing the traditional lineage II in the country; the isolation in cell cultures and full genome sequencing of current field viruses which was last done in 1976; the unique amino acid residues in the individual proteins of the various lineages of PPRV were identified and used to develop a lineage-specific diagnostic test for the virus; and the current seroprevalence report of PPR in camels, sheep and goats from all the agro-ecological zones of the country. This work has also increased the sequences of PPRV available in GenBank. A total of 6,065 field serum samples from camels (1,517), goats (3,489) and sheep (1,059) were collected from all the six agro-ecological zones of Nigeria. The study subjects cut across all ages and sexes. The samples were subjected to both the N and H protein based c-ELISA. In addition, 140 clinical samples from 16 sheep and 63 goats with symptoms suggestive of PPRV infection were collected from different (15) states of Nigeria during a four year period (2010 2013). Published primers were used to amplify a 351 bp segment of the PPRV nucleoprotein (N) gene and a 370 bp segment of the fusion (F) gene. Monkey CV 1 cell line expressing the sheep-goat SLAM protein was used to isolate PPRV from RT-PCR positive samples. Thirty primer pairs were used to amplify and sequence two full genome isolates of the current circulating PPRV in Nigeria. Primer express software was used to develop a lineage-specific real time PCR for PPRV. The overall prevalence estimate of serum positive results for PPRV in sheep and goats was 23.16 % (n = 1,018/4,548, 95 % confidence interval [CI]: 21.79 - 24.57. There were significant differences in prevalences between the states (p = 0.001), zones (p = 0.058) and age (p = 0.032). Taraba State had the highest seroprevalence rate of 27.97 % while the lowest rate of 14.76 % was observed in Cross River State. There were no significant differences in the PPRV prevalences between male and female animals (p = 0.571) and between species (p = 0.639). The overall prevalence in camels was 3.36 % (51/1517, 95 % CI: 2.51 4.39). There were no significant differences in prevalences between states (p = 0.892) and between male and female camels (p = 0.742). The prevalence differed significantly (p < 0.001) by body condition scores - camels with poor body condition score have a higher (16.67 %) antibody seroprevalences to PPRV compared to those with fair and good body condition scores. There was a statistically significant difference between camels aged ? 5 years and those > 5 years (p = 0.004). The clinical samples subjected to RT-PCR showed that 33 (42 %) animals were positive for PPRV nucleic acid, which included two sheep (13 %) and 31 goats (49 %). The amplicons were sequenced and phylogenetic analysis using the neighbour joining, maximum parsimony and Bayesian analysis of the sequences with those available in GenBank showed that isolates from the current study belonged to lineage II and lineage IV. The lineage II viruses from this study grouped into two clades, one closely related to the vaccine virus (Nigeria75/1) and the other clade group with other wild-type viruses from Mali, Senegal and Sierra Leone. The lineage IV isolates also grouped into two sub-clades, one closely related to a 2011 strain from Gabon and the other closely related to a 1997 strain from Cameroon. Analysis of two full genomes of PPRV from Nigeria representing the two lineages of the virus circulating currently in the country showed that the lineage II representative isolated in 2012 has an identity of 96 % at the nucleotide level with the lineage II Nigeria75/1 virus isolated in 1975 and used as a vaccine. The lineage IV representative isolated in 2013 revealed an identity of 96 % with Sungri/96 from India. The unique amino acid residues in the individual proteins of the various lineages of PPRV were identified and used to develop a one-step TaqMan® real-time RT-PCR assay targeting the H gene of PPRV. This study reports for the first time, the presence of PPRV lineage IV, the Asian lineage in Nigeria. As part of the study, a lineage-specific assay was also developed, which once validated, could be included in the panel of assays recommended by the World Organization for Animal Health (OIE). The seroprevalence studies showed occasional transient PPRV infection of camels and the current antibody seroprevalence to PPRV in the small ruminants population in Nigeria. There is the need to include camels among species to be studied in elucidating the epidemiology of the disease in sheep and goats. The seroprevalence studies may be helpful in reaching a decision on the vaccination strategy for the control of the disease in the country.
Thesis (PhD)--University of Pretoria, 2015.
tm2016
Veterinary Tropical Diseases
PhD

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13

Fougerolle, Stéphanie. "La grippe équine : détection moléculaire et caractérisation des souches de virus influenza : caractérisation de la réponse immunitaire après vaccination." Caen, 2016. http://www.theses.fr/2016CAEN2067.

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Le virus influenza équin (EIV) appartient à la famille des Orthomyxoviridae et est l’un des pathogènes respiratoires les plus importants des équidés notamment en raison des pertes économiques considérables que ces épizooties peuvent entrainer. Chez le cheval, l’infection par le virus grippal entraîne une morbidité importante et de rare cas de mortalité sont décrits, le plus souvent dus à une infection secondaire par des bactéries. Bien qu’il existe des vaccins depuis les années soixante, des épidémies, causées par le sous-type H3N8, sont enregistrées un peu partout dans le monde, Suède, Japon, Australie et France. Parmi les problématiques actuelles liées au virus de la grippe équine, ce travail de thèse s’intéresse, d’une part, au virus lui-même en étudiant la diversité moléculaire des souches et un éventuel lien avec la virulence et, d’autre part, à l’hôte en étudiant l’absence de réponse vaccinale optimale chez certains chevaux. La problématique des faibles répondeurs est un phénomène admis mais mal connu chez les chevaux. Le premier point a permis de renforcer la surveillance des souches d’EIV circulant en France et d’apporter de nouvelles notions sur les mécanismes de virulence. Lors du second point, un suivi, par la réalisation de tests SRH, de la réponse immunitaire humorale stimulée au cours de la primo-vaccination contre la grippe équine a été réalisé chez 202 poulains. Celui-ci a permis de définir, la fréquence des individus n’ayant pas développé une réponse immunitaire adéquate et d’identifier deux facteurs indépendants jouant un rôle majeur dans l’établissement de cette réponse : l’âge du poulain et la présence d’anticorps maternels ; L’évaluation, au cours d’une étude préliminaire, des niveaux d’expression (ARNm) des cytokines induites après immunisation contre l’EIV n’a pas permis d’identifier de facteurs intrinsèques qui seraient associés à la faible réponse vaccinale
The equine influenza virus (EIV) belongs to the family Orthomyxoviridae and is one of the most important equine respiratory pathogens, especially due to the economic losses associated with outbreaks. In horses, infection with the influenza virus causes significant morbidity. Mortality is uncommon and mostly associated with complications, such as secondary bacterial infections. Although there are vaccines since the 60’s, outbreaks, caused by the H3N8 subtype, are recorded around the world, including Sweden, Japan, Australia and France. Among the current issues related to EIV, this thesis focuses on both the pathogen and the host. , The molecular diversity of EIV strains and a possible link with virulence was investigated. This work involved a monitoring of EIV strains circulating in France and brought new notions about virulence mechanisms. The problem of low responder is a phenomenon accepted but not well understood in horses. The second aspect of this thesis was to study sub-optimal response to immunisation observed in some horses. The humoral immune response monitored, through the performance of SRH tests, in 202 foals during the primary course of vaccination against equine influenza. Results allowed to define the frequency of individuals that did not develop an adequate immune response and to highlight two independent factors playing a major role in the establishment of this sub-optimal response: the age of the foal and the presence of maternal antibodies at the time of first immunisation. In a preliminary study, evaluation of mRNA cytokines expression levels induced after EI immunization did not allow identification of intrinsic factors associated with low vaccination response

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14

Buczkowski, Hubert. "Development of marker vaccines for rinderpest (RPV) and peste des petits ruminants (PPRV) viruses." Thesis, Royal Veterinary College (University of London), 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.558958.

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15

Keita, Djénéba. "Utilisation de l’interférence ARN pour l’inactivation post-transcriptionnelle de gènes viraux et le contrôle de la réplication de deux virus animaux in vitro : Morbillivirus (ARN) et Peste Porcine Africaine (ADN)." Montpellier 2, 2008. http://www.theses.fr/2008MON20163.

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Ce travail avait pour objectif d'utiliser le mécanisme de l'interférence ARN pour le contrôle in vitro de la réplication de virus à ARN (Morbillivirus) et à ADN (Asfivirus). Par une approche bioinformatique et biologique par mesure de l'inhibition de la réplication de ces virus en culture cellulaire, des gènes cibles et des siARN actifs ont été mis en évidence. Le genre Morbillivirus inclus d'importants pathogènes de l'homme et de l'animal, comme le virus de la rougeole, de la peste des petits ruminants et celui de la peste bovine. La nucléoprotéine (N) joue un rôle central dans la transcription et la réplication des Morbillivirus, aussi, nous avons définis des siARN dirigés contre les séquences conservées déterminées par alignement multiple du gène N de ces virus. Dans le cas du virus à ADN étudié, le virus de la peste porcine africaine, l'objectif consistait à déterminer le rôle dans la réplication virale, de quatre gènes présents dans une région devant être délétée pour l'atténuation raisonnée du virus. Pour les pays confrontés à ces maladies virales extrêmement contagieuse, le développement de vaccins thérapeutiques reposant sur l'interférence ARN est un progrès majeur en santé animale, spécialement pour la peste porcine africaine contre laquelle il n'y a pas encore de vaccin et pouvant déboucher sur de nouvelles stratégies de lutte
This work aimed at using the mechanism of RNA interference for the in vitro control of the replication of RNA (Morbillivirus) and DNA viruses (Asfivirus). By bioinformatics and in vitro biological approaches measuring the inhibition of the replication of these viruses in cell culture, target genes and active siRNA were identified. Morbillivirus genus includes important pathogens of human and animals. They include measles virus, peste des petits ruminants virus and rinderpest virus. Nucleoprotein (N) plays an essential role in transcription and replication of Morbilliviruses, therefore we defined siRNA targeting the conserved sequences as defined by multiple alignment of the N gene of these viruses. In the case of the DNA virus studied, African swine fever virus, the objective was determining the role in the viral replication, of four genes present in an area having to be deleted for the carefully thought out attenuation of the virus. . For countries facing these extremely contagious viral diseases, the development of therapeutic vaccines based on siRNA interference is a major progress for animal health, especially for African swine fever against which there is not yet vaccine and having the potential to open the door on new control strategies

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16

Faverjon, Céline. "Risk based surveillance for vector-borne diseases in horses : combining multiple sources of evidence to improve decision making." Thesis, Clermont-Ferrand 2, 2015. http://www.theses.fr/2015CLF22604/document.

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Les maladies émergentes à transmission vectorielle sont une préoccupation croissante et particulièrement lorsqu’elles affectent les chevaux, une population spécifiquement à risque vis-à-vis de la propagation de maladies. En effet, les chevaux voyagent fréquemment et, malgré l’impact sanitaire et économique des maladies équines, les règlementations sanitaires et les principes de biosécurité et de traçabilité censés assurer la sécurité des mouvements d'équidés ne sont pas toujours en place. Notre travail propose d'améliorer la surveillance des maladies à transmission vectorielle chez les chevaux en utilisant différentes méthodes pour estimer la probabilité d'émergence d'une maladie. Tout d'abord, nous avons développé un modèle quantitatif et spatio-temporel combinant différentes probabilités pour estimer les risques d'introduction de la peste équine et de l’encéphalose équine. Ces combinaisons permettent d’obtenir une image plus détaillée du risque posé par ces agents pathogènes. Nous avons ensuite évalué des systèmes de surveillance syndromique par deux approches méthodologiques: l'approche classique avec un seuil d'alarme basé sur un multiple de l'erreur standard de prédiction, et l'approche bayésienne basée sur le rapport de vraisemblance. Nous avons travaillé ici principalement sur la détection précoce du virus West Nile en utilisant les symptômes nerveux des chevaux. Les deux approches ont fourni des résultats prometteurs, mais l’approche bayésienne était particulièrement intéressante pour obtenir un résultat quantitatif et pour combiner différentes informations épidémiologiques. Pour finir, l'approche bayésienne a été utilisée pour combiner quantitativement différentes sources d'estimation du risque : surveillance syndromique multivariée, et combinaison de la surveillance syndromique avec les résultats d’analyses de risques. Ces combinaisons ont données des résultats prometteurs. Ce travail, basé sur des estimations de risque, contribue à améliorer la surveillance des maladies à transmission vectorielle chez les chevaux et facilite la prise de décision. Les principales perspectives de ce travail sont d'améliorer la collecte et le partage de données, de mettre en oeuvre une évaluation complète des performances des systèmes de surveillance multivariés, et de favoriser l'adoption de ce genre d’approche par les décideurs en utilisant une interface conviviale et en mettant en place un transfert de connaissance
Emerging vector-borne diseases are a growing concern, especially for horse populations, which are at particular risk for disease spread. In general, horses travel widely and frequently and, despite the health and economic impacts of equine diseases, effective health regulations and biosecurity systems to ensure safe equine movements are not always in place. The present work proposes to improve the surveillance of vector-borne diseases in horses through the use of different approaches that assess the probability of occurrence of a newly introduced epidemic. First, we developed a spatiotemporal quantitative model which combined various probabilities in order to estimate the risk of introduction of African horse sickness and equine encephalosis. Such combinations of risk provided more a detailed picture of the true risk posed by these pathogens. Second, we assessed syndromic surveillance systems using two approaches: a classical approach with the alarm threshold based on the standard error of prediction, and a Bayesian approach based on a likelihood ratio. We focused particularly on the early detection of West Nile virus using reports of nervous symptoms in horses. Both approaches provided interesting results but Bayes’ rule was especially useful as it provided a quantitative output and was able to combine different epidemiological information. Finally, a Bayesian approach was also used to quantitatively combine various sources of risk estimation in a multivariate syndromic surveillance system, as well as a combination of quantitative risk assessment with syndromic surveillance (applied to West Nile virus and equine encephalosis, respectively). Combining evidence provided promising results. This work, based on risk estimations, strengthens the surveillance of VBDs in horses and can support public health decision making. It also, however, highlights the need to improve data collection and data sharing, to implement full performance assessments of complex surveillance systems, and to use effective communication and training to promote the adoption of these approaches

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17

Pereira, de Oliveira Rémi. "Mécanismes de transmission vectorielle du virus de la Peste Porcine Africaine et facteurs influençant cette transmission : étude de différentes associations tique-virus." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTG013.

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Aucun vaccin n’est actuellement disponible pour lutter contre la Peste Porcine Africaine (PPA), une des maladies les plus graves des suidés, faisant des ravages en Afrique, en Europe et en Asie. Pour combattre cette maladie infectieuse causée par le virus de la PPA (vPPA), la compréhension des différents modes de transmission est essentielle pour appliquer des mesures sanitaires adéquates. Un des modes de transmission est la piqûre de tiques infectées. L’objectif de ma thèse était de comprendre les mécanismes et les facteurs qui déterminent la compétence vectorielle des tiques molles Ornithodoros pour le vPPA. Ce travail de thèse a tout d’abord permis de montrer que les tiques présentes en Europe n’étaient pas compétentes pour les souches circulant actuellement en Eurasie, mais qu’elles pouvaient maintenir le virus infectieux pendant plusieurs mois et être infectantes pour les porcs par ingestion. Cette étude a également permis de mettre en évidence, qu’outre la dissémination du vPPA chez la tique jusqu’aux organes de transmission, le niveau de réplication virale était un facteur essentiel à la transmission. Toutefois, nos résultats suggèrent d’autres déterminants, encore inconnus, qui tendent à moduler chacune de ces étapes. Une analyse comparative de deux génomes de vPPA aux profils de transmission différents a ainsi montré quelques différences génétiques qui peut représenter une piste pour la compréhension des facteurs génétiques contribuant à déterminer la compétence vectorielle. En outre, une étude préliminaire menée au cours de cette thèse a montré que l’infection de la tique par le vPPA induisait une modulation de certains peptides antimicrobiens, mettant en lumière l’existence d’interactions moléculaires entre la tique et le vPPA. Les résultats obtenus ont été discuté au regard des risques potentiels à l’établissement d’un cycle de transmission entre tique et suidés et de la mise en place de mesures sanitaires adaptées à ces zones au contexte épidémiologique particulier
There is currently no vaccine available to control African Swine Fever (ASF), one of the most important swine diseases that ravages Africa, Europe and Asia. To fight the ASF virus (ASFV) that induces infectious disease, understanding the different modes of transmission is essential to apply adequate sanitary measures. One mode of transmission is through the bite of an infected tick. The main objective of my thesis was to understand the mechanisms and factors that determine the vectorial competence of the Ornithodoros soft ticks for ASFV. First, this thesis project showed that the ticks present in Europe are not competent for the strains currently circulating in Eurasia, but can maintain the virus for several months and be infectious to pigs, at least by ingestion. This study also showed that dissemination of ASFV inside ticks towards transmission organs is not enough and must be completed by a sufficient level of viral replication to allow transmission. However, our results also suggest the existence of other factors, partially unknown, that modulate each of these stages. A comparative analysis of two ASFV genomes with different vectorial transmission patterns showed several genetic differences, which may contribute to determining vector competence. In addition, a preliminary study conducted in this PhD project demonstrated that the infection of ticks with ASFV induced modulation of some antimicrobial peptides, highlighting that there is an interaction at the molecular level between the tick and the ASFV. All these results were discussed in regard to potential risks for the establishment of a tick-suid transmission cycle and the implementation of appropriate sanitary measures in these peculiar areas

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Renson, Patricia. "Caractérisation des mécanismes pathogéniques associés à la virulence du virus de la Peste Porcine Classique (PPC)." Rennes 1, 2009. http://www.theses.fr/2009REN1S119.

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Le virus de la Peste Porcine Classique est responsable d'une maladie hémorragique dont les symptômes varient en fonction de la virulence des souches. L'infection provoque une diminution du nombre de lymphocytes circulants permettant de différencier les souches sur des critères d'intensité et de cinétique, dont le mode d'induction implique un processus d'apoptose. Ce phénomène étant difficile à reproduire in vitro, des porcs ont été infectés par une souche hyper ou moyennement virulente afin d'étudier les différences observées lors de la mise en place de la lymphopénie. L'analyse transcriptomique a révélé une induction plus immédiate et intense des gènes stimulés par l'interféron et associés à un processus de mort cellulaire avec la souche hyper-virulente. Pour chacune des souches, cette induction est corrélée à la présence d'interféron alpha dans le sérum ainsi qu'à l'induction de la lymphopénie. Nos résultats suggèrent que le virus exacerbe la réponse antivirale de l'hôte, conduisant à la mort des lymphocytes, et que la virulence des souches influe sur les capacités de l'hôte à maintenir un contrôle sur la régulation de cette réponse interféron
Classical Swine Fever Virus is the causal agent of an hemorrhagic disease which clinical signs depend on the strains virulence. The infection induces a blood lymphocyte depletion that however allows the strains differentiation based on intensity and kinetics measures. Its way of induction is not well described but an apoptotic process is involved. Because of the difficulty to reproduce in vitro the virus-mediated apoptosis induction in lymphocytes, we infected pigs by either a highly virulent strain, or a moderately one, in order to decipher differences when the lymphocyte depletion takes place. Microarray analysis revealed a more sudden and acute induction of cell death-related interferon stimulated genes with the highly virulent strain. For each strain, this induction is correlated to the interferon alpha presence in serum and the lymphopenia induction. Our results suggest that the virus exacerbate host's antiviral response, leading to the death of lymphocytes, and that the strains virulence affect the host's capacities to keep control on this interferon response regulation

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19

Mantip, Samuel Elias Lashat. "Molecular characterisation of peste des petits ruminants viruses in sheep and goats from Nigeria." Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/40708.

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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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20

Ahmed, Osman Elhag Nussieba [Verfasser]. "Expression studies of peste des petits ruminants virus (PPRV) haemagglutinin and fusion envelope glycoproteins / Nussieba Ahmed Osman Elhag." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2015. http://d-nb.info/1073724107/34.

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Halecker, Sabrina [Verfasser], and Gerd [Akademischer Betreuer] Sutter. "Peste des Petits Ruminants Virus (PPRV): optimization of diagnostic procedures and pathogenesis studies / Sabrina Halecker ; Betreuer: Gerd Sutter." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2021. http://d-nb.info/1238017223/34.

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22

Portilla, Jarufe Katherine Vanessa. "Determinación de la persistencia de los niveles de anticuerpos pasivos contra el virus de la peste porcina clásica en lechones nacidos de marranas con distinto programa de vacunación." Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2006. https://hdl.handle.net/20.500.12672/658.

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El objetivo del estudio fue evaluar la persistencia de los anticuerpos pasivos contra el virus de la Peste Porcina Clásica (vPPC) en lechones de dos granjas tecnificadas A y B con distintos programas de vacunación contra el vPPC. En la granja A, las marranas son vacunadas a los 90 días de gestación y en B a los 18 a 21 días post-parto. Se colectaron 60 muestras de sangre de lechones por granja, durante la primera (n igual a 15), tercera (n igual a 15), quinta (n igual a 15) y séptima (n igual a 15) semana de edad, así como de las marranas (n igual a 15) de cada granjas para la detección de los anticuerpos mediante la prueba de ELISA indirecta o de cloqueo. En la primera semana de edad el 100% de lechones de ambas granjas presentaron anticuerpos pasivos, persistiendo dichos anticuerpos en la mayoría de lechones por encima de séptima semana de edad. Se detectaron diferencias en los niveles de anticuerpos pasivos en los lechones durante la primera y tercera semana de edad de ambas granjas siendo estadísticamente significativa (p menor a 0.05). Al comparar los coeficientes de variación de los resultados de los lechones de ambas granjas, se observó una mayor variabilidad en los niveles de anticuerpos en lechones de la granja A. Así mismo, hubo variación en los niveles de anticuerpos en las marranas de la granja A en comparación a los resultados de las marranas de la granja B pero, esta variación no tuvo significancia estadística (p mayor a 0.05). Estos resultados sugieren que los niveles y la persistencia de los anticuerpos pasivos dependen del sistema de manejo de cada granja. Palabras Claves: virus de la Peste Porcina Clásica (vPPC), anticuerpos maternales, lechones, marranas, vacunación, granjas porcinas.
--- The persistence of the levels of maternal antibodies against the of the Classical Swine Fever virus (CSFV) in piglets born of vaccinated sows from two farms (A and B) with different vaccination programs against CSFV located in Lima valley, Peru, was evaluated. In the farm A, the sows are vaccinated at 90 days of gestation and in farm B at 18 - 21 days post furrowed. Serum samples were taken from a total of 60 piglets by farm, at first (n is equal to 15), third (n is equal to15), fifth (n is equal to 15) and seventh (n is equal to 15) weeks old and from sows from farm A (n is equal to 15) and B (n is equal to15) for antibodies detection against CSFV by indirect ELISA test. The 100% (30/30) of piglets of both farms had maternal antibodies against CSFV at first week old. In the majority of piglets the maternal antibodies persisted up to seventh week old. The levels of maternal antibodies in the piglets from both farms showed a statistically significant (p menor a 0,05) differences at first and third week old. The comparison of the maternal antibodies titers indicated more variation in piglets from farm A, a high and uniform antibodies titers were observed in piglets from farm B during the study. The sows had high level of antibodies against CSFV indicating a good passage of these antibodies to their piglets. These results suggest that the level and persistence of the maternal antibodies in the piglets depend of the management system of each pig farms. Key Words: Classical Swine Fever Virus (CSFV), maternal antibodies, piglet, sows, vaccination, pig farms.
Tesis

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Holz, Correia Carine Lidiane. "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." Thesis, Montpellier 2, 2011. http://www.theses.fr/2011MON20126.

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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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López, Fernández Elisabet. "Caracterización de la seguridad y eficacia de BA71ΔCD2: una vacuna experimental contra el virus de la peste porcina africana". Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667336.

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El virus de la peste porcina africana (VPPA) representa, hoy en día, una gran amenaza para la industria porcina. La presencia descontrolada de la PPA en África ha favorecido su exportación a otros países, tal y como demuestra la última entrada del VPPA en el Cáucaso, en el año 2007, a través de la República de Georgia. Desde entonces, el VPPA se ha extendido a países vecinos, alcanzando la Unión Europea en 2014, y más recientemente llegando a China en verano de 2018, desde donde ya ha comenzado a extenderse por el resto de Asia. La falta de vacunas disponibles contra la PPA hace su control difícil y, hasta ahora, solamente la utilización de virus atenuados vivos como vacunas experimentales, había demostrado una sólida protección homóloga, contra la infección letal utilizando los virus virulentos parentales. Más recientemente, resultados obtenidos en nuestro laboratorio (Monteagudo et al., 2017) , han permitido describir un nuevo candidato vacunal con capacidades únicas de protección cruzada: BA71ΔCD2. Así, la inoculación de cerdos con BA71ΔCD2 fue capaz de proteger no sólo contra el desafío experimental con BA71, la cepa virulenta parental, sino también contra virus heterólogos, incluyendo Georgia2007/1, la cepa del genotipo II del VPPA que circula actualmente en la Europa continental y en Asia. A pesar de su demostrada eficacia y capacidad cros-protectora, BA71ΔCD2 presenta una serie de limitaciones de bioseguridad para su uso comercial, al menos en zonas libres de la enfermedad. Así pues, dos son los objetivos principales de esta tesis: primero, desarrollar una vacuna basada en BA71ΔCD2 más segura, reduciendo, en lo posible, la presencia de virulencia residual detectable sin disminuir su eficacia. Segundo, caracterizar en la medida de lo posible, su potencial protector pensando en escenarios muy distintos; como el de la Europa que nos rodea, así como en zonas endémicas donde la enfermedad está causando grandes estragos sin poder aplicar ninguna medida de control; como en el caso del África subsahariana y más recientemente de Asia. Para el primer objetivo se abordaron diferentes estrategias de vacunación que implicaban desde la inactivación del virus, cambiar las pautas de administración, buscar rutas de inoculación alternativas, usar adyuvantes de última generación e incluso llegar a manipular el genoma del virus, creando virus dobles mutantes e incluso inducibles. Los datos recopilados durante esta tesis nos demuestran que existe un fino equilibrio entre atenuación, virulencia y apatogenicidad para el caso del VPPA, pudiendo pasar con facilidad del éxito al fracaso en términos de protección. Si bien no podemos garantizar una formulación óptima para la administración de BA71ΔCD2 que conjugue su máxima eficacia y bioseguridad, estamos hoy más cerca de ello, exigiendo en el futuro más esfuerzos en investigación. Como sucede con casi todas las vacunas vivas en el mercado, incluyendo las que se administran en niños, su éxito depende precisamente de su capacidad de replicar in vivo. La dificultad añadida en el caso de una vacuna frente a la PPA en cerdo es que, como sucedería con el caso del Ébola para humanos, su administración en Europa, zona libre de ambas enfermedades, exige tolerancia cero en lo que se refiere al riesgo. El segundo objetivo de nuestra tesis se realizó pensando en zonas más desfavorecidas, endémicas para el VPPA, como podría ser el África subsahariana. Zonas en las que, en ocasiones circulan concomitantemente diversos genotipos del virus y multitud de subvariantes del mismo, y en los que habría que generar vacunas individuales para cada uno de ellos. De forma muy resumida, a lo largo de esta tesis hemos podido demostrar, por un lado, la capacidad de BA71ΔCD2 de proteger no sólo frente a una infección con Georgia2007/1, administrada intramuscularmente, sino además frente a la inoculación intranasal o por contacto con el mismo virus, extendiendo la protección incluso a la infección con garrapatas infectadas naturalmente con el genotipo XIX del VPPA; virus que se encuentra circulando actualmente en África. Finalmente, hemos podido demostrar que aquel animal vacunado frente a BA71ΔCD2 que sobrevive a una infección con un virus virulento, queda a su vez protegido frente a posteriores infecciones incluso con aislados muy alejados filogenéticamente de BA71ΔCD2. Virus contra los que por sí solo su eficacia había demostrado ser muy baja. Así pues, la vacunación con BA71ΔCD2 podrá no sólo tener efectos beneficiosos directos, sino también, indirectos, sobre todo para aquellos animales que han de vivir en zonas con una presión epidemiológica muy fuerte. De nuevo, futuras investigaciones habrán de desentrañar los mecanismos intrínsecos implicados en esta protección dual. La demostración, en esta tesis, de que BA71ΔCD2 podía inducir una respuesta de anticuerpos diferenciales respecto a la cepa circulante, basadas en un ensayo de inhibición de la hemadsorción, junto con el hecho de que BA71ΔCD2 sea el único prototipo vacunal existente, hoy en día, capaz de crecer en una línea celular estable añade muchas posibilidades a su utilización como vacuna de emergencia en zonas endémicas y muy castigadas por el virus. Mientras esperamos la vacuna ideal, estamos orgullosos de poder estar en el punto de mira, de aquellos que necesitan herramientas para controlar una más que inminente epidemia global.
African swine fever virus (ASFV) represents today a major threat to the swine industry. The uncontrolled presence of ASF in Africa has favoured its exportation to other countries, as evidenced by the last entry of ASFV in the Caucasus in 2007 through the Republic of Georgia. Since then, ASFV has spread to neighbouring countries, reaching the European Union in 2014, and more recently China in the summer of 2018, where it has already begun to spread throughout the rest of Asia. The lack of available vaccines against ASF makes its control even more difficult, and to date, only the use of live attenuated viruses as experimental vaccines, had demonstrated a strong but homologous protection against lethal infection with parental virulent viruses only. Recent results obtained in our laboratory (Monteagudo et al., 2017), allowed us to describe a new candidate with cross-protective capabilities: BA71ΔCD2. Thus, the inoculation of pigs with BA71ΔCD2 was able to protect not only against the experimental challenge with BA71, its virulent counterpart strain, but also against heterologous viruses, including Georgia2007/1, the strain of genotype II, currently circulating in Europe and Asia. Despite its demonstrated efficacy and cross-protective capacity, BA71ΔCD2 presents a series of biosecurity limitations for commercial use, at least in disease-free areas. So, two are the main objectives of this thesis: first, the development of a safer vaccine based on BA71ΔCD2, reducing as much as possible its residual virulence without decreasing its effectiveness. Second, the characterization of its protective potential in different scenarios, as it could be the Continental Europe, as well as endemic areas where the disease is causing great damage, without being able to apply any control measure, like Sub-Saharan African countries, or, more recently, Asia. For the first objective, different vaccination strategies were approached, involving the inactivation of the virus, changing the administration guidelines, searching for alternative inoculation routes, using next-generation adjuvants and even manipulating the genome of the virus for the generation of double mutants and inducible viruses. Although we cannot guarantee an optimal formulation for the administration of BA71ΔCD2 that combines its maximum efficacy and biosecurity, we can state that today we are closer than ever to reach such goal. Nevertheless, more efforts in research are needed to obtain the aforementioned objective. As it happens for almost all live vaccines in the market, including those that are currently administered in children, its success depends on its ability to replicate in vivo. A vaccine against ASF in pigs presents an additional difficulty. Similarly, to the case of Ebola for humans, its administration in Europe, a free area of both pathogens, requires zero tolerance in terms of risk. The second objective of our thesis was carried out thinking in the most disadvantaged areas such as Sub-Saharan Africa, which is endemic for ASFV. In these regions, diverse genotypes of the virus and a multitude virus subvariants circulate concomitantly at the same time, and individual vaccines would have to be generated for each one. Briefly, throughout this thesis we have been able to demonstrate the capacity of BA71ΔCD2 to protect not only against an infection with Georgia2007/1, administered intramuscularly, but also against intranasal, contact inoculation with the same virus, even expanding its protection to infection with ticks naturally infected with a genotype XIX of ASFV; currently circulating in Africa. Finally, we have been able to demonstrate that animals vaccinated with BA71ΔCD2 that survive an infection with its virulent counterpart, are protected against subsequent ASFV-infections even with phylogenetically very distant isolates from the BA71ΔCD2, against which its effectiveness is limited. These experiments demonstrate that vaccination with BA71ΔCD2 has direct and indirect effects that might be extremely beneficial, especially for those animals living in areas with a very high epidemiological pressure. Again, future investigations will have to unravel the intrinsic mechanisms involved in this dual protection. This thesis demonstrates that BA71ΔCD2 could induce a differential antibody response to the circulating strain, based on a hemadsorption inhibition assay. Moreover, BA71ΔCD2 is the only prototype vaccine capable of growing in a stable cell line, which represents a high benefit for its use as an emergency vaccine in endemic areas. While waiting for the ideal vaccine, we are proud to be in the spotlight of those who need tools to control a more than imminent global epidemic

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Salami, Habib. "Diffusion d'un virus et évolution de son génome dans les populations de ruminants domestiques : application à l'épidémiosurveillance de la "Peste des petits ruminants"." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS155/document.

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La peste des petits ruminants (PPR), causée par un Morbillivirus, est l'infection virale la plus grave des caprins et ovins. Elle est largement répandue en Asie, au Moyen Orient et en Afrique. En Afrique elle est en émergence au nord et au sud du continent et représente un facteur majeur d'insécurité alimentaire pour la population agricole (70% des populations pauvres des régions considérées). La PPR est un modèle d'étude des maladies transfrontalières ; sa diffusion est très liée aux mouvements régionaux d'animaux vivants. La compréhension de cette diffusion est une condition essentielle à la mise en place de mesures de contrôle efficaces (vaccination, quarantaine, contrôle aux frontières,…). A notre connaissance aucune étude n'a été entreprise pour connaître l'ampleur de la diversité génétique du PPRv au cours d'infections naturelles de petits ruminants et l'accumulation des mutations virales dans un circuit de diffusion. Or dans les pays d'élevages extensifs tropicaux l'identification et la traçabilité animale sont inexistantes, ce qui rend difficile reconstruction des circuits de diffusion des animaux et du virus. Dans ces conditions, la diversité génétique du virus peut être utilisée comme marqueur de diffusion épidémiologique. L'objectif de cette thèse est d'utiliser la variabilité génétique du PPRV pour caractériser les lignées virales circulantes et retracer les processus de transmission du virus à travers un large territoire centré sur le Sénégal. En analysant 2 gènes de PPR nous avons estimé la vitesse d'évolution du virus sur une période de 4 années comprise en 2010 et 2014.Les résultats montrent que les premières souches de la lignée 2 de PPRv ont été introduites en 2005 au Sénégal et dans les pays voisins. L'horloge moléculaire et l'arbre phylogéographique rapportés ici indiquent clairement que la lignée II maintenant enzootique en Afrique de l'ouest prend son origine au Nigeria. Les mouvements trans-africains à l'origine du déplacement est-ouest de la lignée II trouvent leur origine dans le commerce de bétail à la croisée des frontières, une évidence économique et culturelle en Afrique de l'Ouest.Mots clés : peste des petits ruminants ; gène viral ; mutation virale ; circuit de transmission ; phylogénie ; phylogéographie ; surveillance épidémiologique, Sénégal
Peste des petits ruminants (PPR), caused by a Morbillivirus is one of the most important viral infections in sheep and goats. It is widely spread in Asia, Middle East and Africa. In Africa, it is an emerging disease in the north and the south of the continent. It is a major factor of food insecurity for the farming population (70% of the poor population in the tropical regions). PPR is a study model of transboundary diseases; its spread is highly related to regional movements of livestock. Understanding the spread of PPR is an essential condition for the implementation of efficient control measures (vaccination, quarantine, border controls etc.). Up to our knowledge, no studies have investigated the range of genetic diversity of PPR virus (PPRv) during natural infections in small ruminants and the accumulation of virus mutations during its spread. Further on, in tropical countries with extensive farming, animal identification and traceability are a current problem. In such conditions, the genetic diversity of the PPRv can be used as a marker of animal movement and spread of the virus. The objective of this study was to investigate the genetic diversity of the PPRv in order to characterise the actual viral lineages and to retrace the transmission of the virus in Senegal and its surrounding countries. Analyzing two complete viral genes of the PPR, we have estimated the rate of evolution of this virus, in a four year period, between 2010 and 2014. The results of the study show that the first strains of lineage II of PPRv have been introduced in 2005 in Senegal and its surrounding countries. Molecular clock analysis and phylogeographical reconstitution of the PPRv indicate that the lineage II, actually enzootique in western Africa, has its origins in Nigeria. This viral introduction from the direction east towards west, corresponds to the transboundary movement and commerce of livestock in the countries of western Africa, which represents the economic and cultural tradition of the people of this region.Key words: Peste des petits ruminants, viral gene, virus mutation, transmission, phylogeny, phylogéographie, epidemiosurveillance, Senegal, West Africa

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Nizamani, Zaheer Ahmed. "Délivrance in vivo de siRNA et évaluation de leur effet antivirale contre le virus de la peste des petits ruminants (PPRV)." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20100/document.

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L'interférence ARN est un processus biologique permettant la dégradation d'un ARN messager par un ARN double brin de courte taille spécifique de cet ARNm. Elle a un potentiel d'application en thérapie antivirale pour peu que les ARN interférents (ARNi) soient délivrés efficacement in vivo. Dans le genre Morbillivirus, on trouve des pathogènes importants en santé publique et vétérinaire tels que le virus de la rougeole et les virus de la peste des petits ruminants (PPR) et de la peste bovine. Il n'existe aucun traitement contre les infections à morbillivirus. L'objectif de ce travail était d'évaluer la possibilité d'administrer in vivo un ARNi actif contre le virus PPR in vitro. Une formulation basée sur des liposomes complexés avec des ARNi ou un adénovirus non réplicatif exprimant des ARN courts en tête d'épingle (shARN) ont été testés chez des chèvres dans un modèle d'épreuve infectieuse avec une souche virulente de PPR. Les différences observées n'étaient cependant pas significatives au plan statistique. Pour améliorer la délivrance par vecteur viral, nous avons comparé un autre vecteur de type baculovirus qui s'est avéré plus efficace in vitro que l'adénovirus précédent. Par ailleurs, nous avons testés in vitro également deux peptides capables de pénétrer dans les cellules. L'un d'entre eux, le Perfect 6 (PF6) a presque complètement inhibé l'expression du gène de la nucléoprotéine par le virus PPR. En revanche, l'autre (PF14) a été moins efficace mais a relativement mieux résisté à l'inhibition de son activité par la présence de fortes concentrations de sérum dans le milieu. Dans le but d'évaluer in vivo ces nouveaux systèmes de délivrance en s'affranchissant du modèle chèvre lourd et couteux à mettre en œuvre, nous avons initié une stratégie de mise au point d'un modèle non infectieux de suivi dynamique de l'interférence ARN chez la souris par imagerie in vivo. Dans ce travail, nous montrons qu'il est possible de mesurer et de standardiser l'expression d'un gène rapporteur comprenant une séquence du virus PPRV et ensuite de quantifier le niveau de dérégulation de l'expression induit par un ARNi dirigé contre le virus PPR. Après calibration, ce modèle est désormais pour tester différents systèmes de délivrance de siRNA chez la souris
RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner. RNAi has a potential of developing into an effective and specific antiviral therapy if small interfering RNAs (siRNAs) can be efficiently delivered in vivo. Morbillivirus genus includes important pathogens of humans and animals, which include measles virus, peste des petits ruminants virus (PPRV) and rinderpest virus. No treatment exists for morbillivirus diseases. The aim of this work was the in vivo delivery of siRNA against PPRV infection. The delivery of siRNA by a liposome and short hairpin RNA (shRNA) by means of a replication deficient adenovirus was tested in goats which were later challenged with PPRV. However, significant therapeutic effects were not obtained. To find more efficient vectors, the PPRV inhibition efficiency of recombinant replication deficient adenovirus and a baculovirus expressing shRNA against nucleoprotein of PPRV were compared in vitro. The baculoviral vector was found to be more efficient. Similarly, two cell penetrating peptides (CPPs) were also compared and PepFect6 (PF6) could deliver siRNA NPPRV1 effectively in vitro resulting in an almost complete inhibition of N gene expression by PPRV. Another CPP, the PF14 though with lower transfection efficiency in vitro, was found to be relatively serum resistant compared to PF6. A small animal model for PPRV infection does not exist. Due to economic, ethical, and biosecurity issues involved with use of small ruminants, a strategy based on the use of a non-infectious mouse model and a dynamic follow up of siRNA treatment by live imaging was developed. We show in this work that it is possible to measure and standardize the expression of a bioluminescent reporter gene containing a PPRV sequence and thus, to quantify a down-regulation of such gene by siRNA against PPRV. After some calibration, siRNA delivery can now be tested in this mouse model for comparing various delivery vectors in vivo

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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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Davila, Sylvie. "Etude de l'influence de la virulence des souches de peste procine classique, sur les risques de propagation de la maladie. Etude de l'immunopathogénicité d'une souche moyennement virulente." Rennes 1, 2004. http://www.theses.fr/2004REN10096.

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La Peste Porcine Classique est une maladie infectieuse virale ayant un grand impact économique sur l’industrie porcine. Les isolats récents sont suspectés être des souches de virulence modérée rendant le diagnostic difficile augmentant ainsi le risque de propagation de la maladie. L’étude de l’influence de la virulence sur la propagation de la maladie en calculant le taux de reproduction de base (R0) ont confirmé : l’influence de la virulence sur le R0 et le haut niveau de contagion en intra-parc. Les porcs infectés présentent une leucopénie et une atrophie des organes lymphoi͏̈des qui semble être causé par l’apoptose indirecte des lymphocytes. L’apoptose induite par une souche moyennement virulente dans le sang et la mise en place d’un modèle in vitro de co-culture ont révélé: 1) une leucopénie dès les premières 48 heures accompagnée d’une apoptose des cellules de la périphérie sanguine, 2) la nécessité d’un contact cellulaire pour induire l’apoptose.

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Minet, Cécile. "Contribution au développement d'un vaccin marqué contre la Peste des Petits Ruminants (PPR) par génétique inverse d'un virus à ARN négatif (Morbillivirus)." Montpellier 2, 2009. http://www.theses.fr/2009MON20160.

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La peste des petits ruminants (PPR) est une maladie virale infectieuse, contagieuse et souvent fatale, qui affecte les animaux domestiques et la faune sauvage de l'Afrique subsaharienne, du Moyen-Orient et de l'Asie du sud-ouest. Cette maladie est due à un virus à ARN négatif monocaténaire non segmenté, de la famille des Paramyxoviridae, genre Morbillivirus et sa réplication est placée sous la dépendance de trois protéines : N, P et L. Le vaccin actuel est une souche virulente atténuée par passages successifs sur cellules VERO. Ce vaccin confère une bonne immunité. N'étant pas un vaccin DIVA, il ne permet de faire, par diagnostic sérologique, la distinction entre les animaux vaccinés-animaux infectés. Le développement, par génétique inverse, d'un clone vaccinal infectieux marqué (vaccin DIVA) est l'objectif principal de ce travail de recherche, associé la mise au point d'outils de diagnostic différentiel. La mise en place de la génétique inverse adaptée au virus de la peste des petits ruminants a été notre premier travail. Elle a été réalisée à l'aide d'un minigénome eGFP, dont le gène ne peut être fonctionnel que dans le contexte d'une expression de type virus PPR. La seconde partie du travail a consisté à assembler, cloner et vérifier l'ADNc du génome complet de la souche virale vaccinale PPRV 75/1. La génération, par génétique inverse, du premier clone infectieux PPR a ensuite été mise en œuvre et est toujours en cours. Les résultats obtenus à ce jour ne permettent pas de conclure à la présence d'un clone infectieux. En parallèle, plusieurs stratégies de marquage du vaccin ont été évaluées : délétion d'un segment de la séquence du génome de la souche vaccinale PPRV 75/1, insertion d'une cassette d'expression d'un gène étranger ou substitution d'une partie d'un gène par une séquence homologue dérivée d'un autre morbillivirus ou d'un peptide commercial. Les marques les plus prometteuses ont été ensuite utilisées pour mettre au point des tests diagnostics ELISA
Peste des Petits Ruminants (PPR) is an infectious, contagious and fatal viral disease of goats, sheep and wildlife in sub-Saharan African countries, Middle-East and South-West of Asia. It is caused by a single strand negative RNA virus belonging to the Morbillivirus genus among the Paramyxoviridae family. Current vaccines consist of viral strains attenuated by several passages on cell cultures. These vaccines protect animals against PPR but they are not DIVA vaccines and thus do not permit the distinction between vaccinated and infected animals. However, the manipulation of negative RNA strand by reverse genetics allows the generation of an infectious and marked clone of PPR vaccine strain. Therefore, the aim of this work was to develop both a PPR marked vaccine using reverse genetics technology and the associated diagnosis tools. The first task was to adapt the reverse genetics to the PPR virus using the eGFP minigenome model. Then the full genome of PPR vaccine strain 75/1 was assembled in a plasmid after translation of genomic negative RNA in cDNA. Attempts to generate the first recombinant PPRV are ongoing but up to now, we cannot conclude on the presence of an infectious rescued virus. In parallel, different strategies for vaccine marks were also evaluated: deletion of a region of PPRV genome, insertion of foreign genes or substitution by homologous sequence derived from another morbillivirus or a commercial peptide. In the same time, ELISA assays corresponding to the most promising markers were developed

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Jove, Martel Ana Luisa. "Evaluación de las cepas H120 y M48 en programas de vacunación contra bronquitis infecciosa aviar en pollos de carne." Bachelor's thesis, Universidad Nacional Mayor de San Marcos, 2004. https://hdl.handle.net/20.500.12672/2266.

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El objetivo de este trabajo fue evaluar el grado de protección conferido por 2 vacunas comerciales de Bronquitis Infecciosa Aviar, (cepas vivas M48 y H120) frente al desafío experimental con la cepa M41, en cuatro grupos de aves (Línea Ross 308) con diferentes programas de vacunación G1 (M48), G2 (H120), G3 (M48/H120), G4 (H120/M48) y un grupo control G5 (sin vacuna). La protección fue medida a través de signos clínicos, títulos de anticuerpos, motilidad ciliar, histopatología y parámetros productivos después del desafío experimental. Los grupos que recibieron 2 vacunas fueron mejor protegidos de los signos clínicos, aunque mostraron una disminución de la actividad ciliar (desde el 1er al 3er día post desafío) y lesiones microscópicas de leves a moderados. De estos grupos, el grupo G4 obtuvo una mayor protección después del desafío, se observó una menor presentación de signos respiratorios, una pronta recuperación de la actividad ciliar ( 6to – 10mo día post desafío), leves a moderados daños microscópicos traqueales, un mejor promedio de peso corporal, logrando 286 g. más que el grupo control y 75 puntos más de eficiencia productiva que el grupo control. En los grupos que recibieron una vacuna se observó menor protección, mayor presentación de signos y lesiones respiratorias, y menores parámetros productivos, existiendo diferencias estadísticamente significativa (p<0.05), frente a los grupos con doble vacunación. Además el grupo control sin vacuna, mostró una severa reacción después del desafío afectando negativamente sus parámetros productivos. En cuanto a los títulos de anticuerpos no se observa una correlación con la ausencia o presencia de signos clínicos y lesiones en los pollos desafiados con BIA. Se sugiere que en condiciones de campo la utilización del programa de vacunación del grupo G4 vacunado con H120 y M48, al día y 14 días de edad respectivamente contra BIA, garantizaría una mayor protección ante un posible reto de BIA.
The objective of this study was to determinate the protection level given by two commercial live of Infectious Bronchitis vaccines (strains M48 and H120) against experimental challenge with M41 strain, in four groups of chicks (Ross 308) with different vaccination programs G1 (M48), G2 (H120), G3 (M48/H120), G4 (H120/M48) and a control group G5 (without vaccination). Protection was measure through clinical signs, antibody levels, ciliary activity, histopathology and productive parameters after experimental challenge. Revaccinated groups were better protected against the onset of clinical signs, although they showed a diminished ciliary activity (first-thirth day after challenge) and microscopic damage (histologicaly change from minimal to moderate). We found that group G4 had a very effective protection level. fewer amount of respiratory signs (snores) and minimal to moderate microscopic tracheal damage They also recovered ciliary activity. This group had the best average body weight at the end of the experiment, they obtened 286 g body weight and 15 point of Index of Productive Efficiency more than G5. In the groups that were vaccinated only once, we saw Less protection levels, also more respiratory signs and microscopic lesions, less average weight and productive parameters statistically significant, (P<0,05). Furthermore, G5 showed a severe reaction after challenge, diminishing its productive parameters. The absence or presence of clinical signs or respiratory lesions after challenge against to BIA was not correlated to antibody titers in chicks. We think that under field conditions, vaccination with H120/M48 program would give a wide protection against IBV infections.
Tesis

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Jamin, Agnès. "Caractérisation de cellules dendritiques dans les organes lymphoïdes secondaires et le sang du porc sain et étude de leur activation après infection par le virus de la peste porcine classique." Rennes 1, 2006. http://www.theses.fr/2006REN1S122.

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Des cellules dendritiques conventionnelles (cDC) et plasmacytoïdes (pDC) immatures et semi-matures ont été identifiées dans les zones T des organes lymphoïdes secondaires et le sang de porcs sains. Après infection des porcs par le virus de la peste porcine classique, les DC deviennent matures et actives en 24 h en prenant en charge le virus pour initier la réponse immunitaire innée dans les zones T de l’amygdale. Les lymphocytes B activés co-localisées avec l’antigène viral E2 migrent des centres germinatifs vers les zones T de l’amygdale après 48 h. Les pDC sont recrutées dans le sang, mais le nombre de cDC et de lymphocytes y diminuent. Les pDC sont transitoirement activées les zones T de la rate, mais les cDC y expriment l’interleukine 10, induisant une réponse humorale et l’inhibition de l’activité des DC. Les grandes productions d’interféron alpha et de TNF alpha par les DC, pourraient induire l’apoptose des cellules non infectées et un dysfonctionnement des lymphocytes T
Conventional and plasmacytoid dendritic cells (cDC and pDC) were identified in secondary lymphoid organs and blood of healthy pigs. These DC were immature and semi-mature in T areas of these tissues. After in vivo classical swine fever virus infection, DC subsets loaded viruses, then became mature and active as early as 24 h in T areas, starting innate immune response in tonsil. B cells processed E2 viral antigen from germinal centres to T areas at 48 h post-infection, probably initiating humoral immune response. PDC were recruited in blood, where cDC and lymphocyte numbers decreased. PDC were transiently activated in T areas of spleen. However cDC expressing interleukin 10 induced humoral immune response and inhibited DC functionalities in spleen. High alpha interferon and tumor necrosis factor alpha secretions by DC subsets might be at the origin of uninfected cell apoptosis and T cell disruption

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Pope, Robert Alan. "A study of the pathogenesis of peste-des-petits ruminants virus : emphasising changes in tissue tropism with time and varying virulence." Thesis, Royal Veterinary College (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518043.

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Bakkali, Kassimi Labib. "Organiation des genes codant pour les proteines non structurales du virus de la peste porcine classique et diagnostic differentiel des pestivirus." Paris 7, 1994. http://www.theses.fr/1994PA077118.

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L'agent responsable de la peste porcine classique (ppc) est un virus enveloppe appartenant au genre pestivirus de la famille des flaviviridae. Son genome est constitue d'une molecule d'arn monocatenaire de polarite positive d'environ 12,3kb, ne possedant pas de sequence polyadenilique a son extremite 3' et probablement non coiffee. L'adnc de l'arn genomique de la souche alfort/lcrv a ete clone et la sequence nucleotidique de sept regions du genome couvrant 8167 nucleotides a ete determinee puis comparee a celle des souches alfort/frc et brescia du virus de la ppc et nadl, osloss et sd-1 du virus de la diarrhee virale bovine (bvd). La souche alfort/lcrv est plus proche de la souche brescia (94,3% en nucleotides et 96,7% en acides amines). L'homologie entre les souches du virus de la ppc et celles du virus de la bvd est, en moyenne, de 67% en nucleotides et de 69% en acides amines. Afin d'etudier l'organisation genomique du virus de la ppc la region 3' terminale du genome a ete exprimee dans e. Coli sous forme de proteines de fusion avec la glutathione-s-transferase. Des anticorps monoclonaux diriges contre les produits d'expression ont ete obtenus. Une methode de criblage des surnageants d'hybridomes faisant appel a la detection par la chimiluminescence a ete mise au point. A l'aide de ces anticorps monoclonaux trois proteines virales ayant une masse moleculaire apparente de 76kda, 107kda et 145kda ont ete mises en evidence par immunoblotting dans les extraits de cellules infectees par la souche alfort/lcrv ainsi que par huit autres souches du virus de la ppc. Un schema de maturation de ces proteines est propose. Par comparaison avec le virus de la bvd une difference dans le nombre, la taille et la stabilite de ces proteines a ete observee. Des sondes moleculaires synthetisees a partir des fragments d'adnc et les anticorps monoclonaux obtenus a partir du produit d'expression ont ete utilises pour la distinction des pestivirus entre eux

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Michaud, Vincent. "Détection et caractérisation moléculaires rapides du virus de la peste porcine africaine (ADNdb) et utilisation des reconstructions phylogénétiques pour reconstituer son histoire évolutive." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20268/document.

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La Peste porcine africaine (PPA) est une maladie contagieuse spécifique du porc domestique due au seul arbovirus à ADN identifié à ce jour. Décrite pour la première fois en 1921 au Kenya, la maladie a ensuite diffusé dans de nombreuses régions du monde. Malgré l'isolement de nombreuses souches virales au cours du temps, peu d'études phylogénétiques ont été menées jusqu'ici pour comprendre les relations unissant ces isolats entre eux. Or, la caractérisation est essentielle à la traçabilité des souches et donc à la compréhension de l'épidémiologie de la maladie. De plus, les conditions climatiques et environnementales des principaux pays atteints rendent difficile l'accès, le transport et la conservation de nouvelles souches. Dans cette thèse, un protocole de prélèvement et de conservation du sang a été développé, pour la détection et la caractérisation rapides des souches. Une étude phylogénétique approfondie a été réalisée en utilisant des données de séquences publiques et inédites de virus isolés depuis 1950. Les analyses ont porté sur les gènes B646L, CP204L et E183L. Les analyses phylogénétiques ont utilisé les méthodes de maximum de vraisemblance et d'inférence bayésienne, qui ont permis de proposer une nouvelle nomenclature virale en 35 clusters différents. De plus, une datation des origines du virus a été menée, après avoir éliminé les biais d'analyse dus à une pression de sélection positive et/ou aux recombinaisons. L'horloge moléculaire a permis de déterminer que l'ancêtre commun le plus proche des souches contemporaines (TMRCA) se situait au début du 18ème siècle
African swine fever (ASF) is a highly lethal disease of domestic pigs caused by the only known DNA arbovirus. It was first described in Kenya in 1921 and since then a substantial number of isolates have been collected worldwide. However, only few phylogenetic studies have been carried out to better understand the relationships between isolates, which is essential for virus traceability and epidemiological understanding of the disease. Access, transport and virus conservation are also complicated by climatic and environmental conditions in affected developing countries. In this thesis, a simple method of blood sampling was developed allowing rapid virus detection and characterization. Comprehensive phylogenetic reconstructions were made using publicly and newly generated sequences of hundreds ASFV isolates of the last 60 years. Analyses focused on B646L, CP204L and E183L genes. Phylogenetic analyses were achieved using maximum likelihood and Bayesian coalescence methods and a new lineage based nomenclature is proposed to designate 35 different clusters. In addition, dating of ASFV origin was carried out from the molecular data sets. To avoid biased diversity, positive selection or recombination events were neutralized. The molecular clock analyses revealed that ASFV strains currently circulating have evolved over 300 years, with a time to the most recent common ancestor (TMRCA) going back to the early 18th century

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Almeida, Henrique Meiroz de Souza [UNESP]. "Epidemiologia e prevalência de infecções pelo Vírus da Diarreia Viral Bovina (BVDV) em suínos de criações não tecnificadas." Universidade Estadual Paulista (UNESP), 2015. http://hdl.handle.net/11449/136047.

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Made available in DSpace on 2016-03-07T19:21:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-10-02. Added 1 bitstream(s) on 2016-03-07T19:24:55Z : No. of bitstreams: 1 000857857.pdf: 713336 bytes, checksum: ab84264d137dfc3385c359eec0183281 (MD5)
Os membros do gênero Pestivirus apresentam grande semelhança antigênica entre si. A reação sorológica cruzada entre o Vírus da Peste Suína Clássica (CSFV) e o Vírus da Diarreia Viral Bovina (BVDV) é de grande importância, e interfere em programas de erradicação da Peste Suína Clássica (PSC). Este trabalho teve como objetivos determinar a prevalência de anticorpos anti-BVDV em suínos de criações não tecnificadas, associar fatores de risco e buscar vínculos epidemiológicos entre rebanhos através do geoprocessaemento. Para tal, 360 amostras de soro de suínos provenientes de 56 propriedades rurais da região nordeste do Estado de São Paulo foram submetidas ao teste de virusneutralização (VN) utilizando os genótipos BVDV-1 estirpe Singer e BVDV-2 estirpe VS253. Foram detectados 4,72% (17) de amostras reagentes na VN, e 26,79% (15) rebanhos apresentaram pelo menos um animal reagente. Não foi possível associar fatores de risco. O estimador de intensidade de Kernel indicou áreas de alto risco de ocorrência da enfermidade e, juntamente com a análise de correspondência múltipla apontou associação com a presença de rebanhos de bovinos, quantidade mediana de leitões e rebanho suíno mediano nas propriedades da região. Ressalta-se a importância dos bovinos na ocorrência de infecções pelo BVDV em suínos, assim como a problemática no diagnóstico e levantamento epidemiológico para vigilância de PSC devido a presença de anticorpos anti-BVDV nos animais
The members of the Pestivirus gender have great antigenic similarity. The serological cross-reaction between the Classical Swine Fever Virus (CSFV) and the Bovine Viral Diarrhea Virus (BVDV) usually interferes with Classical Swine Fever (CSF) eradications programs. This study focused on establishing the prevalence of antibodies for the BVDV in pigs of non-technified rearing farms, associate risk factors and use geospatial analysis tools to correlate positive herds. A set of 360 serum samples from 56 herds located in the northeastern region of the state of São Paulo were analyzed by the virusneutralization test (VN), using the genotypes BVDV-1 strain Singer and BVDV-2 strain VS253. During the sample collection, a questionnaire was applied to the farmers in order to obtain epidemiological information of the herd. 4,72% (17) of the samples were positive in the VN and 26,79% (15) of the herds had at least one positive animal. It was not possible to associate risk factors; however, the variable use of milk serum in the feed showed a tendency to be associated with the occurence of the disease. The Kernel intensity estimator showed three high risk of occurence areas, which were associated with the presence of bovine herds, medium quantity of piglets in the herd and medium size of total swine herd. The results highlight the importance of bovines in the prevalence of swine BVDV infections and the difficulties in diagnostic tests and CSF surveillance actions due to the serological cross-reaction between BVDV and CSFV antibodies

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Almeida, Henrique Meiroz de Souza. "Epidemiologia e prevalência de infecções pelo Vírus da Diarreia Viral Bovina (BVDV) em suínos de criações não tecnificadas /." Jaboticabal, 2015. http://hdl.handle.net/11449/136047.

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Orientador: Luís Guilherme de Oliveira
Coorientador: Samir Issa Samara
Banca: Hélio José Montassier
Banca: José Carlos Figueiredo Pantoja
Resumo: Os membros do gênero Pestivirus apresentam grande semelhança antigênica entre si. A reação sorológica cruzada entre o Vírus da Peste Suína Clássica (CSFV) e o Vírus da Diarreia Viral Bovina (BVDV) é de grande importância, e interfere em programas de erradicação da Peste Suína Clássica (PSC). Este trabalho teve como objetivos determinar a prevalência de anticorpos anti-BVDV em suínos de criações não tecnificadas, associar fatores de risco e buscar vínculos epidemiológicos entre rebanhos através do geoprocessaemento. Para tal, 360 amostras de soro de suínos provenientes de 56 propriedades rurais da região nordeste do Estado de São Paulo foram submetidas ao teste de virusneutralização (VN) utilizando os genótipos BVDV-1 estirpe Singer e BVDV-2 estirpe VS253. Foram detectados 4,72% (17) de amostras reagentes na VN, e 26,79% (15) rebanhos apresentaram pelo menos um animal reagente. Não foi possível associar fatores de risco. O estimador de intensidade de Kernel indicou áreas de alto risco de ocorrência da enfermidade e, juntamente com a análise de correspondência múltipla apontou associação com a presença de rebanhos de bovinos, quantidade mediana de leitões e rebanho suíno mediano nas propriedades da região. Ressalta-se a importância dos bovinos na ocorrência de infecções pelo BVDV em suínos, assim como a problemática no diagnóstico e levantamento epidemiológico para vigilância de PSC devido a presença de anticorpos anti-BVDV nos animais
Abstract: The members of the Pestivirus gender have great antigenic similarity. The serological cross-reaction between the Classical Swine Fever Virus (CSFV) and the Bovine Viral Diarrhea Virus (BVDV) usually interferes with Classical Swine Fever (CSF) eradications programs. This study focused on establishing the prevalence of antibodies for the BVDV in pigs of non-technified rearing farms, associate risk factors and use geospatial analysis tools to correlate positive herds. A set of 360 serum samples from 56 herds located in the northeastern region of the state of São Paulo were analyzed by the virusneutralization test (VN), using the genotypes BVDV-1 strain Singer and BVDV-2 strain VS253. During the sample collection, a questionnaire was applied to the farmers in order to obtain epidemiological information of the herd. 4,72% (17) of the samples were positive in the VN and 26,79% (15) of the herds had at least one positive animal. It was not possible to associate risk factors; however, the variable "use of milk serum in the feed" showed a tendency to be associated with the occurence of the disease. The Kernel intensity estimator showed three high risk of occurence areas, which were associated with the presence of bovine herds, medium quantity of piglets in the herd and medium size of total swine herd. The results highlight the importance of bovines in the prevalence of swine BVDV infections and the difficulties in diagnostic tests and CSF surveillance actions due to the serological cross-reaction between BVDV and CSFV antibodies
Mestre

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Haffar, A. "Etude de la proteine de matrice m du virus de la peste des petits ruminants : clonage, sequencage et expression dans le systeme baculovirus." Paris 6, 1999. http://www.theses.fr/1999PA066231.

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Le virus de la peste des petits ruminants (pprv) est un virus a arn monocatenaire, enveloppe et de polarite negative. Il est responsable d'une grave maladie, souvent mortelle, sur la chevre et le mouton. Son genome, organise comme ceux de tous les morbillivirus, groupe auquel il appartient, code pour huit proteines identifiees: six proteines structurales (n-p-m-f-h-l), et deux proteines non structurales (c et v). Les sequences de la grande majorite de ces proteines sont actuellement connues. La proteine de matrice m a ete clonee et sequencee par des methodes classiques de la biologie moleculaire. Le gene de la proteine m du pprv, long de 1466 nucleotides, code pour une proteine de 335 acides amines, et de poids moleculaire de 38 kda. La proteine m est tres conservee, avec une homologie assez elevee avec celles des autres virus du groupe. La proteine m du pprv a ete exprimee dans le systeme baculovirus/cellules d'insecte, afin d'etudier son comportement, et ses interactions avec d'autres proteines structurales. Ce systeme d'expression a permis de montrer que la proteine m-pprv est capable de former, a elle seule, des pseudo vesicules a la surface de la cellule infectee. Cette interaction n'existe pas avec la proteine m deletee de sa region centrale, la moins conservee. Nous avons pu montrer egalement que la proteine m du pprv interagit avec d'autres proteines, notamment avec la nucleoproteine (n) des virus pestiques (pprv et virus de la peste bovine). Aucune interaction similaire n'a ete observee avec la proteine n d'un autre virus a arn negatif (virus de la rage). La formation par le systeme baculovirus de pseudo vesicules, exprimant la proteine m du pprv seule ou avec la proteine n, pourrait servir de base a la reflexion sur un vaccin recombinant.

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Doubli-Bounoua, Nadia. "Epidémiologie moléculaire des virus dans les voies respiratoires et association avec les signes cliniques d’asthme équin modéré." Caen, 2016. http://www.theses.fr/2016CAEN2056.

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Les virus (Influenza virus équin (EIV), α-herpèsvirus (EHV-1 & EHV-4), Virus de la rhinite équine A et B (ERAV & ERBV), Adénovirus équin 1 et 2 (EAdV1 & EAdV2), γ-herpèsvirus (EHV-2 & EHV-5) et Coronavirus équin (ECoV)) n’ont actuellement pas tous été recherchés par qPCR dans le cadre de l’asthme équin modéré (AEM) chez les chevaux à l’entrainement. Les objectifs de ce projet sont :1) déterminer la prévalence et l'incidence de la détection et/ou quantification des génomes viraux dans les voies respiratoires des chevaux de course à l’entrainement ; 2) étudier la concordance entre deux compartiments des voies respiratoires; 3) préciser les relations entre la détection et/ou quantification virale et a) les signes cliniques d’AEM et b) les performances. Une étude prospective longitudinale a été conduite de Novembre 2012 à Janvier 2015, sur 52 Trotteurs Français différents ont été prélevés mensuellement : des écouvillons naso-pharyngés (ENP) et du liquide de lavage trachéal (LT). Les dix virus d’intérêt ont été systématiquement recherchés par qPCR dans les ENP et les LT. Les signes cliniques d’AEM (jetage nasal, toux) et le score de mucus trachéal ont été notés lors de chaque examen. Les génomes viraux les plus fréquemment détectés dans les ENP et LT sont EHV-5, EHV-2 et ERBV. Aucune association significative n'a été trouvée entre la détection/quantification virale dans les ENP et les signes cliniques. La détection d’EHV-2 dans le LT a été significativement associée à la toux (OR 3,1; P=0,01) et à l’excès de mucus trachéal (OR 2,1; P=0,02). La détection (OR 5,3; P < 0,001) et la quantification d’ERBV (OR 15,0; P < 0,001) dans le LT ont été significativement associés à la toux
Equine influenza virus (EIV), α-herpesvirus (EHV-1 & EHV-4), Equine rhinitis virus A and B (ERAV & ERBV), Equine adenovirus 1 and 2 (EAdV1 & EAdV2) Herpesvirus (EHV-2 & EHV-5) and Equine coronavirus (ECoV)) are not currently being investigated by qPCR for mild equine asthma (MEA) in training horses. The objectives of this project are to: 1) determine the prevalence and incidence of viral genome detection and / or quantification in the respiratory tract of racehorses during training; 2) to study the concordance between two compartments of the respiratory tract; 3) specify the relationship between detection and / or viral quantification and a) the clinical signs of MEA and b) performance. A longitudinal prospective study was conducted from November 2012 to January 2015, both nasopharyngeal swabs (NS) and tracheal washes (TW) were collected monthly on 52 Strandardbred racehorses at training. The ten viruses of interest were systematically investigated by qPCR in NS and TW. Clinical signs of MEA (nasal discharge, cough) and tracheal mucus score were noted during each examination. The viral genomes most frequently detected in NS and TW are EHV-5, EHV-2 and ERBV. No significant association was found between viral detection / quantification in NS and clinical signs. Detection of EHV-2 in TW was significantly associated with cough (OR 3. 1, P = 0. 01) and excess tracheal mucus (OR 2. 1, P = 0. 02). Detection (OR 5. 3; P <0. 001) and quantification of ERBV (OR 15. 0; P <0. 001) in LT were significantly associated with cough

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Bohórquez, Garzón José Alejandro. "Immunopathogenesis of persistent and subclinical infections generated by Classical swine fever virus." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/673869.

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La pesta porcina clàssica (PPC) continua sent una de les malalties més importants en sanitat animal, inclosa a la llista de malalties de declaració obligatòria de l’Organització Mundial de Sanitat Animal (OIE). És causat pel virus de la PPC (VPPC), membre del gènere Pestivirus de la família Flaviviridae. A causa de les greus conseqüències de la PPC, en termes socioeconòmics, el control de la malaltia als països endèmics es considera una tasca de gran importància. Un dels reptes per al control de la malaltia és la presència de formes persistents de PPC al camp, que poden ser congènites o postnatals. Els animals que desenvolupen formes persistents de PPC són clínicament sans, mentre que excreten càrregues virals elevades. A més, no desenvoluparan resposta d’anticossos, cosa que els farà indetectables mitjançant els mètodes serològics per diagnòstic recomanats actualment. Addicionalment, els animals infectats persistentment seran refractaris a la vacunació. En el cas de la persistència congènita de la PPC, aquesta s’ha relacionat amb la immunotolerància, a causa de la incapacitat del fetus de reconèixer patògens després de la infecció pel VPPC. La comprensió dels mecanismes subjacents a aquestes formes de PPC serà crucial per al disseny de noves eines de diagnòstic o estratègies antivirals per aconseguir el control de la PPC. La present tesi es va centrar en l’elucidació dels mecanismes virològics i immunològics implicats en l’establiment i el manteniment de la infecció persistent congènita o postnatal pel VPPC. Amb aquesta finalitat, truges embarassades es van infectar amb soques del VPPC amb diversos graus de virulència en un moment en què s’ha establert que és possible induir una infecció congènita persistent. Es va detectar que la virulència de la soca infectant té un paper important en la transmissió transplacentària, ja que les soques de virulència alta i moderada van mostrar una transmissió més eficient per aquesta ruta que el VPPC de baixa virulència. A més, la soca de virulència moderada va ser capaç de generar persistència viral congènita en garrins. Addicionalment, es va trobar evidència de reconeixement del patogen i d’inducció de resposta immunitària innata, en termes d’interferó alfa (IFN-α) per part dels fetus i els garrins. Això contrastava amb la definició generalment reconeguda del fenomen d’immunotolerància i de la infecció congènita persistent pel VPPC. El paper de l’edat en l’establiment d’una infecció persistent postnatal pel VPPC també es va avaluar mitjançant la infecció de garrins a l’edat de deslletament amb una soca del VPPC moderadament virulenta. En alguns d’aquests animals es va generar infecció persistent postnatal. Tanmateix, es va trobar que la proporció de porcs infectats persistentment era inferior a la dels experiments anteriors en què es va dur a terme la infecció en les primeres hores després del naixement. Aquest resultat suggereix un paper molt important de l’edat en la inducció de la infecció persistent postnatal pel VPPC. En els animals infectats persistentment es van detectar marcadors d’estats immunitaris alterats. Finalment, es va determinar que les poblacions de cèl·lules precursores augmentades en animals persistentment infectats amb VPPC eren similars, en fenotip i funcionalitat, a poblacions de cèl·lules immunosupressores descobertes anteriorment en humans. Els resultats de la present tesi proporcionen informació sobre les bases virològiques i immunològiques de la persistència viral en VPPC. És possible que els porcs amb infecció postnatal persistent pel VPPC es puguin utilitzar com a model per a l’estudi de les vies d’evasió de la resposta immunitària implicades en malalties humanes i animals.
La peste porcina clásica (PPC) sigue siendo una de las enfermedades más importantes en sanidad animal, siendo incluida en la lista de enfermedades de declaración obligatoria de la Organización Mundial para la Sanidad Animal (OIE). La enfermedad es causada por el virus de la PPC (VPPC), un miembro del género Pestivirus en la familia Flaviviridae. Debido a las tremendas consecuencias de la PPC, en términos socioeconómicos, el control de la enfermedad en países endémicos es considerado como una labor de gran importancia. Uno de los desafíos para el control de la enfermedad es la presencia de formas persistentes de PPC en el campo, las cuales pueden ser congénitas o postnatales. A pesar de estar excretando altas cargas virales, los animales que padecen formas persistentes de PPC son aparentemente sanos. Estos animales tampoco desarrollan respuesta de anticuerpos, siendo indetectables por los métodos serológicos recomendados para diagnóstico. Los animales persistentemente infectados con VPPC también son refractarios a la vacunación. En la forma persistente congénita de PPC, esto ha sido asociado a inmunotolerancia, debido al fallo del sistema inmune fetal en el reconocimiento del patógeno tras la infección. El conocimiento de los mecanismos que subyacen estas formas de PPC será crucial para el diseño de nuevas herramientas diagnosticas o estrategias antivirales para lograr el control de la PPC. La presente tesis se enfocó en elucidar los mecanismos virológicos e inmunológicos involucrados en el establecimiento y mantenimiento de infección persistente congénita y postnatal por el VPPC. Con este fin, cerdas preñadas fueron infectadas con cepas de VPPC de diversos grados de virulencia, en un momento de la gestación en el que se ha reportado la inducción de persistencia congénita. Se determinó que la virulencia de la cepa infectante juega un papel importante para la transmisión trans-placentaria, ya que las cepas de alta y moderada virulencia fueron transmitidas más eficientemente a través de esta ruta que la cepa de baja virulencia de VPPC. Además, la cepa de moderada virulencia fue capaz de generar persistencia congénita con VPPC en lechones. Adicionalmente, se encontró evidencia de reconocimiento del patógeno e inducción de respuesta inmune innata, en términos de interferón alfa (IFN- α) por los fetos y lechones. Este resultado contrastó con la definición generalmente reconocida del fenómeno de inmunotolerancia y la infección persistente con VPPC. El papel de la edad en el establecimiento de infección persistente postnatal con VPPC fue estudiado infectando cerdos a edad de destete con una cepa de moderada virulencia. Infección persistente postnatal fue inducida en algunos de estos animales, sin embargo, la proporción de cerdos persistentemente infectados fue más baja que estudios anteriores en los cuales la infección fue llevada a cabo durante las primeras horas después del nacimiento. Esto sugiere fuertemente que la edad a la que se infecta el animal juega un papel en la inducción de la forma persistente postnatal de PPC. Marcadores de estados inmunes alterados fueron detectados en los animales persistentemente infectados. Finalmente, se encontró que poblaciones celulares precursoras, las cuales se encuentran aumentadas en animales persistentemente infectados con VPPC, son similares en fenotipo y funcionalidad a poblaciones celulares inmunosupresoras reportadas anteriormente en humanos. Los resultados de la presente tesis ayudan a entender las bases virológicas e inmunológicas de la persistencia viral causada por VPPC. Es posible que los animales que padecen las formas postnatales persistentes puedan ser usados como un modelo para ayudar en el estudio de los mecanismos de evasión de la respuesta inmune implicados en enfermedades de sanidad animal y humana.
Classical swine fever (CSF) remains one of the most important diseases in animal health, being included into the list of notifiable diseases for the World Organisation for Animal Health (OIE). It is caused by the CSF virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family. Due to the major consequences of CSF, in socio-economical terms, the control of the disease in endemic countries is considered a task of great importance. One of the challenges for disease control is the presence of persistent forms of CSF in the field, which can be congenital or postnatal. Animals that develop persistent CSF forms will be clinically healthy, while excreting high viral loads. Furthermore, they will not develop antibody response, which will make them undetectable by the currently recommended serological methods for diagnosis. Moreover, CSFV persistently infected animals will be refractory to vaccination. In the case of congenital CSF persistence, this has been linked to immunotolerance, due to a failure in pathogen recognition by the foetus after CSFV infection. The understanding of the mechanisms underlying these forms of CSF will be crucial for the design of new diagnostic tools or antiviral strategies to achieve CSF control. The present thesis focused in the elucidation of the virological and immunological mechanisms involved in the establishment and maintenance of congenital and postnatal persistent CSFV infection. To this end, pregnant sows were infected with CSFV strains of varying degrees of virulence at a time point in which congenital persistent infection has been reported to be induced. The virulence of the infecting strain was found to play a major role for trans-placental transmission, as high and moderate virulence strains showed more efficient trans-placental transmission than low virulence CSFV. Moreover, the moderate virulence strain was able to generate congenital viral persistence in piglets. In addition, evidence of pathogen recognition and induction of innate immune response, in terms of Interferon alpha (IFN-α) by the foetuses and piglets was found. This was in contrast with the generally acknowledged definition of the immunotolerance phenomenon and CSFV congenital persistent infection. The role of age in the establishment of postnatal persistent infection with CSFV was also assessed by infecting piglets at weaning age with a moderately virulent CSFV strain. Postnatal persistent infection was generated in some of these animals, however, the proportion or persistently infected pigs was found to be lower than previous experiments in which infection was carried out in the first hours after birth. This strongly suggests the role of age for the induction of CSF postnatal persistent form. Markers of altered immune states were detected in the persistently infected animals. Finally, precursor cell populations found to be increased in CSFV persistently infected animals were determined to be similar, in phenotype and functionality, to immunosuppressive cell populations previously reported in humans. The results of the present thesis provide insight into the virological and immunological basis of viral persistence in CSFV. It is possible that CSFV postnatal persistently infected pigs could be used as a model which can aid in the study of immune evasion pathways implicated in human and animal diseases.
Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animals

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Piriou-Guzylack, Laurence. "Contribution à l'étude des réponses immunitaires cellulaires du porc, par de nouveaux outils méthologiques, dans un modèle d'infection par le virus de la peste porcine classique." Rennes 1, 2002. http://www.theses.fr/2002REN10028.

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Freitas, Ferdinando. "Functional characterization of unassigned african swine fever virus proteins putatively involved in transcription and replication towards an efficient vaccine design." Doctoral thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2019. http://hdl.handle.net/10400.5/18129.

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Tese de Doutoramento em Ciências Veterinárias na especialidade de Ciências Biológicas e Biomédicas
African swine fever (ASF) is an infectious disease of domestic pigs and wild boars with mortality rates reaching up to 100% and is endemic in most of the Sub-Saharan countries. In 2007 it was introduced in Georgia and spread to neighbouring countries, reaching the Russian Federation, several European countries and, more recently, China and Vietnam (February 2019). Currently, there is neither a vaccine nor a treatment against ASF and the control of the disease depends strictly on sanitary measures, including stamping out and trade bans of animals and pork products leading to devastating socio-economic losses to affected countries. The etiologic agent of the disease is African swine fever virus (ASFV), a large (approx. 190 nm) double-stranded DNA (170 to 193 kbp) enveloped virus. ASFV genome encloses more than 150 open reading frames (ORFs) and to this date most of them lack any known or predictable function. ASFV is quite independent from cellular machinery encoding enzymes required for replication, transcription and virion assembly, including the putative I215L E2 Ubiquitin-conjugating enzyme, QP509L, Q706L RNA Helicases and the P1192R type II topoisomerase. The E2 ubiquitin-conjugating enzymes are part of the essential cellular post-transcriptional regulation ubiquitin-proteasome pathway. In this study, the pI215L binding activity was characterized as being mono and poly-ubiquitinated in the Cys85 at different temperatures and pH values. Moreover, I215L gene is transcribed from 2 hours post infection (hpi), and immunoblot analysis confirmed that pI215L is expressed from 4 hpi being detected all over the cell specially in the viral factories from 8 hpi. Downregulation assays by siRNA suggested that pI215L plays a critical role in the transcription of late viral genes and in viral DNA replication. RNA helicases are described as essential for infections, modulating RNA-RNA and RNA-protein interactions, gene expression, viral egress and host antiviral responses. In the present work, we found that QP509L, Q706L are conserved between ASFV virulent and non-virulent isolates. Furthermore, ASFV-QP509L and Q706L are actively transcribed from 2 hours post infection, and both proteins are localized in the viral factories at 12 hours post infection. However, QP509L was also detected in the cell nucleus. Transcript downregulation uncovered the essential role of these proteins during viral cycle progression, in particular for the late transcription. Type II topoisomerases are involved in resolving DNA tangles and supercoils by cutting the duplex and allowing the DNA replication to proceed. In this study, we report that P1192R is actively transcribed throughout infection, being detected from 2 hpi and reaching a maximum concentration around 16 hpi. P1192R knockdown experiments revealed its critical role for viral infection, given by a reduction in viral transcripts, cytopathic effect, the number of viral factories per cell, and virus yields. We also demonstrated that enrofloxacin exposure during the late phase of infection induces viral genomes fragmentation, whereas, when added at early phase of infection completely abolishes replication. The data obtained from I215L, QP509L. Q706L and P1192R characterization studies opens new venues to the rational design of a mutant virus lacking these genes, and also points new pathways to be targeted by antiviral drugs.
RESUMO - Caracterização funcional de proteínas do vírus da peste suína Africana putativamente envolvidas na transcrição e replicação com o intuito de desenvolvimento de uma vacina. - A peste suína africana é uma doença viral infeciosa que afeta os suínos domésticos e os selvagens, com taxas de mortalidade perto dos 100%, originando perdas económicas elevadas nos países afetados. A doença é endémica na maioria dos países subsaarianos, e desde 2007, assistiu-se uma expansão nos países Europeus, incluindo membros da União Europeia, e mais recentemente, na China e Vietname. Atualmente não existe vacina ou tratamento para esta infeção e o controlo da doença baseia-se no diagnóstico rápido, na eliminação compulsiva dos suínos e no bloqueio ao comércio de suínos e produtos derivados. O agente etiológico é o vírus da peste suína africana (VPSA), um vírus composto por uma molécula de ADN de cadeia dupla (170 to 193 kbp) contendo mais de 150 grelhas de leitura. Algumas destas estão devidamente caracterizadas codificando para proteínas estruturais ou regulatórias, contudo, a grande maioria foi identificada por homologia de sequência com outros vírus não se conhecendo, até à data, qual a sua função durante a infeção. Apesar dos inúmeros esforços ao longo dos anos, a complexidade viral, a falta de conhecimento sobre muitos dos aspetos da biologia do vírus e das suas interações com o hospedeiro invalidaram a obtenção de uma vacina segura e eficaz. Por um lado, as abordagens clássicas embora promissoras não garantem proteção contra estirpes heterólogas, enquanto a produção de vacinas de ADN ou proteína, mesmo com adjuvantes, não induzem imunidade contra uma segunda infeção. No entanto, a identificação de suínos previamente infetados e que resistem a novas infeções reforça a ideia da possibilidade de se obter uma imunidade protetora. Dadas as circunstâncias atuais de expansão da doença, estudos recentes apontam a necessidade de se aprofundar o conhecimento sobre os aspetos da biologia do VPSA com vista a identificação de novas estratégias para o desenvolvimento racional de vacinas ou de identificação de novos alvos para o uso de fármacos com vista a controlar a infeção. Neste contexto, os estudos apresentados neste trabalho caracterizam a I215L, QP509L, Q706L e P1192R, identificadas inicialmente, por homologia de sequência com outras proteínas tipicamente envolvidas na replicação e transcrição de outros vírus. A I215L foi identificada por partilhar identidade com as enzimas E2 de conjugação da ubiquitina. Estas enzimas pertencem a uma cadeia de sinalização do sistema de regulação pós-transcricional ubiquitina-proteossoma. Os estudos realizados revelaram que a pI215L tem a capacidade de receber uma ou duas ubiquitinas (mono e di-ubiquitinada) no resíduo Cisteína-85, a diferentes temperaturas e valores de pH, evidenciando a sua plasticidade em participar em diferentes fases da infeção quer no hospedeiro quer no vetor. Além disto, o gene é transcrito precocemente (2 horas após infecção, hpi) e a proteína expressa desde as 4h, sugerindo que esta deverá ser necessária desde o início da infecção. Paralelamente, os nossos estudos por imunofluorescência revelaram uma distribuição da pI215L por toda a célula, e em especial, nas fábricas virais, sugerindo um papel ativo na regulação de vários processos, incluindo replicação de ADN e da transcrição. Os ensaios de ARN de interferência (siRNA) contra o I215L demonstraram um papel essencial desta proteína durante a infeção, originando uma redução dos transcritos tardios, do número de genomas (-63 a -68%) e na libertação de partículas infeciosas (até -94%). [...]
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Martin, Lydie. "Développement et caractérisation d'un modèle d'infection non lytique de cellules de Leydig par le virus de l'Artérite Virale Equine." Thesis, Normandie, 2018. http://www.theses.fr/2018NORMC201.

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Le virus de l’Artérite Virale Équine (EAV) est un virus à ARN simple brin positif, appartenant à la famille des Arteriviridae, dans l’ordre des Nidovirales. C’est un virus spécifique des équidés qui peut être transmis par voies respiratoire et vénérienne. Lors de la primo-infection, l’EAV peut entraîner des signes cliniques grippaux, mais de façon plus grave, il peut aussi provoquer l’avortement des juments gestantes ainsi que la mort des nouveau-nés. L’EAV représente donc un enjeu économique majeur pour la filière équine. Suite à la primo-infection, ce virus peut persister dans de l’appareil reproducteur de certains étalons. Les mécanismes de cette persistance ne sont pas connus.Au cours de cette thèse, le premier modèle in vitro d’infection non lytique d’une lignée issue de l’appareil reproducteur mâle par l’EAV a été développé. L’infection de ces cellules de Leydig a montré une induction de l’expression de nombreux gènes de l’immunité innée dont ceux codant pour des cytokines pro-inflammatoires et des chimiokines qui permettraient le recrutement de cellules de l’immunité innée au niveau des testicules, et qui pourraient expliquer l’orchite observée chez certains étalons lors de la phase aiguë de l’infection. Pour les étalons infectés de façon persistante, la castration et les traitements anti-GnRH peuvent permettre la suppression de la persistance du virus, suggérant ainsi une implication de la testostérone dans la persistance du virus. Les cellules TM3 exprimant le récepteur aux androgènes, des essais de traitements ont été réalisés. Les premiers résultats préliminaires semblent indiquer que les cellules TM3 ne répondent pas ou peu au stimulus hormonal. Cependant, des tests de prétraitement par la testostérone seraient à envisager afin d’en étudier les conséquences sur le cycle viral. Ce modèle d’infection non lytique reste cependant un modèle intéressant pouvant être utilisé afin d’étudier les relations hôte-pathogène et pouvant aider à comprendre les mécanismes impliqués dans la persistance de l’EAV
Equine Arteritis Virus (EAV) is a positive-strand RNA virus, which belongs to the Arteriviridae familly, in the Nidovirales order. It is an equid specific virus that can be transmitted by respiratory and venereal routes. During primary infection, EAV can induce flu-like clinical signs, but worse, it may also cause the abortion of pregnant mares and newborn foal death. EAV is therefore a main economic challenge for the horse industry. Following primary infection, this virus is able to persist in the reproductive tract of some stallions. The mechanisms of this persistence remain unknown.During this thesis, the first in vitro model of an EAV non-lytic infection of a male reproductive tract cell line has been developed. EAV infection of these Leydig cells induced the expression of numerous innate immune genes including those coding for pro-inflammatory cytokines and chemokines, which could recruit innate immune cells to testicles and which could explain the orchitis observed in some stallions during primary infection.For persistently infected stallions, castration and anti-GnRH treatments can suppress EAV persistence, suggesting an involvement of testosterone in the virus persistence. Since TM3 cells express the androgen receptor, treatment trials have been performed. The first preliminary results suggest TM3 cells do not respond to the hormonal stimulus, or only a little. However, pretreatment trials should be realized to study the consequences on the viral cycle.Nevertheless, this non-lytic infection model is still an interesting model that can be used to study the host-pathogen relationship and that could help understanding the mechanisms involved in EAV persistence

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Roger, François. "Recherches épidémiologiques et microbiologiques sur une maladie émergente du dromadaire (Camelus dromedarius) dans la Corne de l'Afrique : rôles possibles du virus de la peste des petits ruminants (Paramyxoviridae, Morbillivirus) et de "Streptococcus equi subsp. equi"." Montpellier 2, 2000. http://www.theses.fr/2000MON20216.

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En ethiopie, en 1995 et 1996, une maladie epizootique a touche la majeure partie de la population cameline. C'etait une pathologie aigue tres contagieuse, caracterisee par un syndrome febrile et respiratoire. La morbidite etait tres elevee et la mortalite variable, surtout apres un traitement antibiotique. En moins de deux ans, la maladie a ete enregistree dans toutes les zones d'elevage camelin de la corne de l'afrique. La similitude des symptomes avec ceux de la peste des petits ruminants (ppr), affectant a la meme periode des caprins et ovins des memes zones, et la propagation remarquablement rapide de cette maladie, ont oriente les recherches etiologiques vers une maladie virale et plus precisement vers les morbillivirus des ruminants (virus de peste bovine et de la ppr). Une souche du virus ppr (pprv) a ete detectee par un test d'immunocapture d'antigenes et par amplification genique (rt-pcr). Le sequencage de fragments amplifies a montre une proximite genetique avec des souches connues du virus ppr. Un test elisa pour la detection des anticorps anti-pprv a ete effectue sur des echantillons de serums de regions epidemiologiquement differentes. Une augmentation des taux de sero-prevalence a ete observee. Par ailleurs, des travaux bacteriologiques ont ete conduits en utilisant plusieurs milieux de culture. Une souche de streptococcus equi subsp. Equi a ete isolee. C'etait apparemment le premier isolement de l'agent de la gourme des equides chez des camelides. Il est plausible que cette maladie ait ete initiee par une souche du virus ppr. Ce virus pouvait avoir un role immunosuppresseur favorisant l'apparition d'infections bacteriennes secondaires. Toutefois, considerant la specificite de la gourme des equides, maladie tres contagieuse, le role exact de streptococcus equi subsp. Equi reste a etre evaluer. Ces hypotheses sont discutees, notamment dans le contexte des maladies emergentes et du transfert interespeces d'agents infectieux.

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Bernard, Jennifer. "Caractérisation de la compétence vectorielle des tiques Ornithodores pour le virus de la peste porcine africaine et étude de deux déterminants : la relation souche virale – vecteur et l’influence de la salive de tiques sur l’infection chez le porc domestique." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS099/document.

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La peste porcine africaine (PPA) est une maladie hémorragique contagieuse dévastatrice pour l’élevage porcin pour laquelle aucun traitement ni vaccin ne sont disponibles. Cette infection est due à un virus à ADN, unique membre de la famille des Asfarviridae, qui se transmet directement entre suidés ou via un vecteur, la tique du genre Ornithodoros. Le rôle de ces tiques dans le cycle épidémiologique de la PPA consiste principalement à maintenir le virus dans les populations de suidés sauvages en Afrique. Elles ont aussi été identifiées à l’origine de certains cas de résurgence de la maladie dans des bâtiments d’élevage porcin, notamment dans la péninsule ibérique, lors des années 1970-80. La PPA, éradiquée fin des années 1980 en Europe (à l’exception de la Sardaigne), a été de nouveau introduite en 2007 d’abord en Géorgie pour progresser jusqu’à atteindre l’Est de l’Union Européenne. La question de la compétence vectorielle des tiques Ornithodores pour le virus de la PPA et des déterminants qui influencent cette compétence est posée dans l’évaluation des risques d’endémisation et/ou de dispersion de la maladie en Europe et ailleurs. Le premier chapitre de cette thèse vise à caractériser la compétence vectorielle des tiques Ornithodores pour le virus de la PPA et faire ressortir les patrons généraux qui la qualifient. Pour cela, a été réalisée une revue systématique des études ayant testé la compétence vectorielle d’une ou plusieurs espèces de tiques pour une ou plusieurs souches virales de PPA durant ces 50 dernières années. Au final, il en ressort une forte variabilité de résultats selon les couples tique-virus. En outre, il semble difficile de comparer ces résultats et d’établir des « profils types » du fait de l’évaluation partielle de la compétence vectorielle pour de nombreux couples tique-virus et de la diversité des méthodologies utilisées pour tester et mesurer la compétence. Pour autant chaque modalité d’étude révèle une partie des mécanismes et des adaptations auxquels sont soumis les couples tique–virus et suggère l’effet de certains déterminants dont deux sont traités dans les deux autres chapitres de la thèse. Le second chapitre de la thèse traite de l’adaptation tique-virus, par l’étude expérimentale de l’infection des tiques O. erraticus, O. porcinus et O. moubata à l’aide de deux souches de génotype II du virus de la PPA, et par un essai de transmission du virus des tiques aux porcs. Des souches du génotype II ont été choisies car ce génotype circule actuellement en Europe et risque d’infecter les tiques européennes O. erraticus s’il se propage jusqu’en péninsule ibérique. Alors qu’O. erraticus est capable de s’infecter et de transmettre différentes souches virales du génotype I, sa compétence à transmettre la souche Georgia2007/1 (génotype II) n’a pour l’instant pas été démontrée. Toutefois, nos études suggèrent que les résultats de compétence dépendent aussi des conditions d’expérimentation telles que la nature des colonies de tiques utilisées ou encore le titre viral utilisé pour infecter les tiques. Le dernier chapitre de la thèse porte sur l’effet de la salive de tique sur l’étape de transmission du virus de la PPA par la tique au porc. Durant le repas de sang, la salive est un élément essentiel qui va permettre l’accroche et le gorgement durable de la tique sur son hôte, avec des propriétés immuno-modulatrices importantes agissant directement sur l’hôte. Ainsi a été réalisée une étude expérimentale in vivo faisant intervenir la tique O. porcinus et le virus Ambat02 (génotype II) et testant l’effet local et systémique chez le porc d’un extrait de glandes salivaires de tique co-inoculé ou non avec le virus de la PPA, versus la piqûre naturelle de tiques non infectées. Les résultats de cette expérience nous montrent que la salive de tique est capable de moduler au niveau local le recrutement de cellules immunitaires dans la peau du porc et potentiellement influencer l’infection locale chez le porc
African swine fever (ASF) is a contagious hemorrhagic disease with disastrous financial consequences for pig industry, as no vaccine or treatment exists. This infection is caused by a DNA virus, only member of the Asfarviridae family that can be directly transmitted between swine or by a non-compulsory vector, the Ornithodoros tick. Ornithodoros ticks play a role in the persistence of the disease within wild and domestic suids in Africa. They were also involved in resurgences of outbreaks in some pig farms in the Iberian Peninsula in 1970-1980. ASF, eradicated in Europe at the end of the 1980’s except in Sardinia, was reintroduced in Georgia in 2007 then spread towards the Eastern European Union. The question of the tick vector competence for ASF virus (ASFV) and its related determinants is of importance in the risk assessment of endemisation/spread of the disease in Europe or elsewhere.The first chapter of this thesis aims to characterize Ornithodoros tick vector competence for ASFV and to highlight a common pattern to qualify it. For this purpose, a systematic review of the studies carried out on the vector competence of one or more tick species for one or more ASFV, was performed on the last 50 years publications. A high variability of the results obtained for different couples “tick-virus” was highlighted. As most of the papers describe partial evaluation of the vector competence and because of the high number of methods used to perform these assessments, it was definitively very difficult to compare these results, and to propose common patterns. However, each of these studies revealed a part of the mechanisms that participated to the adaptation in the couple “tick-virus”, and suggested the importance of different determinants, out of them, two were experimentally assessed as described in the two other chapters.The second chapter of this thesis describes the adaptation “tick-virus” through the experimental infection of three different ticks, O. erraticus, O. porcinus and O. moubata by two ASFV strains belonging to the genotype II. O erraticus’s competence is known for ASFV strains belonging to the genotype I but has never demonstrated the ASFV Georgia 2007/1 strain (genotype II) and currently circulating in Europe. However, the experiments we performed, suggest that many experimental conditions could influence the results obtained on vector competence as the tick colony or the virus dose used for the tick infection.The third chapter describes the effect of the tick saliva on the ASFV transmission from the tick to the pig. Tick saliva contains important immunomodulatory molecules that interfere with the pig immune system permitting complete engorgement of the tick on its host. The host-vector and pathogen interactions were studied through an in vivo experimentation involving pig, O porcinus tick and ASFV Ambat02 strain (genotype II). The local and systemic effects on the pig immune responses were assessed with the ASFV alone or combined with tick gland extract, versus a healthy tick bite. Data analysis highlighted the tick saliva role on skin immune cell recruitment and its potential effect on local infection

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Bosch, Camós Laia. "Unmasking African swine fever virus antigens inducing CD8+ T-cell responses with protective potential." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667371.

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La contínua propagació de la pesta porcina africana (PPA) a l’Europa continental després de la seva introducció a Georgia l’any 2007, així com l’expansió a Àsia a partir del 2018, posa en evidència la gran amenaça que aquesta malaltia representa per la indústria porcina arreu del món. La PPA és una malaltia hemorràgica porcina de declaració obligatòria a l’Organització Mundial de Sanitat Animal (OIE) que provoca enormes pèrdues econòmiques. L’agent causant, el virus de la pesta porcina africana (VPPA), és un virus embolcallat i amb simetria icosaèdrica, amb un genoma d’ADN de doble cadena d’unes 180 kpb. Actualment no es disposa de vacuna contra el VPPA. El sistema de control recomanat per la OIE es basa en un diagnòstic eficient, seguit pel sacrifici dels animals infectats o potencialment en contacte amb el virus, mesures poc assequibles en regions menys afavorides. El desenvolupament de vacunes contra la PPA es veu dificultat per la manca de coneixement sobre aspectes crítics de la infecció i la immunitat contra el VPPA. En aquest sentit, tot i que s’ha demostrat que els limfòcits T CD8+ juguen un paper clau en la resposta protectora contra el VPPA, els antígens del virus capaços d’induir respostes T CD8+ segueixen majoritàriament sense haver estat descrits. La identificació d’aquests antígens podria facilitar el disseny racional de vacunes, així com l’enteniment dels mecanismes subjacents a la immunitat contra el VPPA. Així doncs, l’objectiu principal de la present tesi era la caracterització de proteïnes del VPPA que continguessin epítops T CD8+ amb potencial protector contra la soca Georgia2007/1 del VPPA, actualment circulant per Europa i Àsia. S’han explorat diferents metodologies i estratègies amb l’objectiu d’identificar tant epítops T CD8+ com proteïnes enteres del VPPA codificant epítops T CD8+ promíscuament reconeguts i amb potencial protector. Per identificar epítops T CD8+ específics contra el VPPA, es va utilitzar una estratègia doble: i) una anàlisi bioinformàtica multiparamètrica emprant el proteoma de la soca Georgia2007/1 com a patró; i ii) un estudi immunopeptidòmic basat en l’anàlisi de pèptids units a les molècules de SLA I en macròfags alveolars porcins infectats in vitro amb VPPA. Els resultats obtinguts a partir d’assajos in vitro realitzats amb PBMCs de porcs recuperats de la infecció amb VPPA han permès definir els estudis immunopeptidòmics com a una estratègia més fiable que les prediccions in silico per a la identificació d’epítops T CD8+ del VPPA. Tal com s’esperava, els pèptids no van ser promíscuament reconeguts per PBMCs de tots els animals, confirmant la seva marcada restricció per al·lels específics de SLA I. Anàlisis posteriors utilitzant proteïnes completes van permetre la caracterització d’una sèrie d’antígens del VPPA capaços de ser reconeguts promíscuament. Així, mitjançant fibroblasts autòlegs transitòriament transfectats amb plasmidis codificant ORFs completes del VPPA fusionades a ubiquitina, per millorar la presentació antigènica per classe I, va permetre la identificació d’antígens del VPPA immunodominants i promíscuament reconeguts per cèl·lules T CD8+ específiques del VPPA. Finalment, experiments d’immunització amb ADN van permetre demostrar el potencial protector dels antígens identificats contra la infecció experimental amb una dosi letal de la soca Georgia2007/1 del VPPA. La protecció total contra el VPPA molt probablement requereixi un ampli repertori d’especificitats de cèl·lules B i T, i per tant caldrà seguir aprofundint en l’estudi d’antígens del VPPA amb potencial protector. Serà també necessari explorar plataformes d’expressió alternatives que permetin obtenir respostes immunitàries més robustes que les conferides per les vacunes d’ADN, una eina ideal per a la investigació bàsica però que resta lluny de ser òptima com a formulació vacunal final d’ús veterinari.
The continuous spread of African swine fever (ASF) through Continental Europe after its introduction in Georgia in 2007, and its subsequent expansion in Asia from 2018, evidence this disease as a major threat to swine industry worldwide. ASF is a pig hemorrhagic disease of obligatory declaration to the World Organization for Animal Health (OIE) and causes enormous economic losses to the affected countries. The causative agent, African swine fever virus (ASFV), is a large, enveloped, icosahedral virus with a dsDNA genome of about 180 kbp in length. There is currently no commercial vaccine against ASFV. Early and efficient diagnosis followed by slaughtering of infected and in contact animals are the only control methods today recommended by the OIE, measures unfortunately not affordable by less favored regions. ASF vaccine development is largely hindered by lack of knowledge about critical aspects of ASFV infection and protective immunity. In this regard, CD8+ T lymphocytes have been widely shown to play a critical role in protective response against ASFV. However, the identity of the ASFV antigens capable of inducing protective CD8+ T-cell responses remains largely unknown. Identification of such protective antigens could lead to rationale vaccine design as well as better understanding the mechanisms underlying ASFV immunity. Therefore, the present thesis aimed to determine ASFV proteins containing CD8+ T-cell epitopes with potential to elicit protective responses against the Georgia2007/1 ASFV, the isolate currently circulating in Continental Europe and Asia. Different methodologies and strategies have been explored aiming to identify both ASFV-specific CD8+ T-cell epitopes and full-length proteins encoding promiscuously recognized CD8+ T-cell epitopes with protective potential. In order to identify ASFV-specific CD8+ T-cell epitopes, a double strategy was employed: i) a multiparametric bioinformatics analysis using the Georgia2007/1 proteome as template; and ii) an immunopeptidomic approach based on the analysis of SLA I-bound peptides found in porcine alveolar macrophages in vitro infected with ASFV. The results observed when evaluating the in vitro recognition of the peptides by ASFV-specific PBMCs obtained from ASF-recovered pigs, suggested immunopeptidomics analysis as a more reliable strategy than in silico predictions for the identification of ASFV CD8+ T-cell epitopes. As expected, peptides were not promiscuously recognized by PBMCs from all animals, confirming their marked restriction for specific SLA I alleles. Further analysis using full-length proteins allowed determining few ASFV antigens promiscuously recognized by ASFV-specific PBMCs. Thus, stimulation of ASFV-specific PBMCs with autologous fibroblasts transiently transfected with plasmids encoding full-length ASFV ORFs fused to ubiquitin to improve SLA I antigen presentation, led to the identification of four ASFV proteins as immunodominant and promiscuously recognized antigens by ASFV-specific CD8+ T cells. Finally, DNA immunization experiments allowed demonstrating the protective potential of the ASFV antigens here identified against the Georgia2007/1 ASFV challenge. Sterilizing protection against ASFV most likely will require a broad repertoire of B and T-cell specificities and thus, further investigations will be needed to determine other ASFV antigens eliciting protective responses. Likewise, it will most likely be indispensable the use of alternative expression platforms to encode the potential vaccine antigens, aiming to induce more solid immune response than that afforded by DNA vaccines, an ideal tool for antigen discovery but far from optimal for final veterinary vaccine formulations.

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Frouco, Gonçalo Daniel dos Santos. "Modulating chromatin structure and gene expression during African swine fever virus infection : new strategies for an efficient vaccine rational design." Doctoral thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2018. http://hdl.handle.net/10400.5/14927.

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Tese de Doutoramento em Ciências Veterinárias, na especialidade de Sanidade Animal.
African swine fever virus (ASFV) is a nucleo-cytoplasmic large DNA virus which infects all members of the family Suidae, causing a fatal disease of domestic swine and wild boar. Since no effective vaccine or treatment is available, ASF is considered a global threat for pig husbandry. The ASFV genome encodes among others, enzymes required for virion assembly, genome transcription and replication, including a putative histone-like protein, pA104R. In bacteria, these proteins perform topological modification of the chromosome (twisting, bending and folding), playing important structural and regulatory functions. Since ASFV has a large genome, a viral histone-like protein may be important for packaging its genome within the virion particle and/or for viral replication and transcriptional events. In this study, the ASFV-pA104R activity was characterized and its DNA-binding activities were evaluated. pA104R binds both to ssDNA and dsDNA, although having higher affinity to ds-DNA, over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. The arginine residue located in pA104R’s DNA-binding domain, at position 69, also revealed to be important for an efficient DNA-binding. Additionally, since pA104R together with the viral type II topoisomerase, pP1192R, displayed DNA-supercoiling activity, a synergistic effect between these viral is proposed. The expression of pA104R was observed in the late phase of infection in infected cells with the Vero-adapted ASFV isolate Ba71V, co-localizing with cell nucleus and viral factories. siRNA experiments showed that the knockdown of A104R induce a reduction of viral progeny, copy numbers of viral genomes and transcription of a late viral gene, revealing that pA104R plays a critical role in viral DNA replication and gene expression. Results obtained on these studies prompted us to pursue the objective to generate a defective infectious single cycle (DISC) ASFV lacking the A104R gene. Recombinant virus was successfully obtained, however the complementary cell line previously developed did not support its replication. The antiviral activity of four HDACi against ASFV was also evaluated in this study. The results showing the abrogation of viral replication by NaPB open new insights on its use as an antiviral strategy to control ASFV spreading. Overall our data strongly support that pA104R plays an important role on ASFV replication opening a new window for the design of ASF control measures through the development of efficient and safe vaccines and antivirals.
RESUMO - Modulação da estrutura cromatínica e da expressão génica durante a infeção do Vírus da Peste Suína Africana – novas estratégias para o desenvolvimento de uma vacina eficaz - O vírus da peste suína africana (VPSA) é um vírus de DNA nucleo-citoplasmático que infeta todos os membros da família Suidae, causando uma doença com elevada mortalidade em suínos domésticos e nos javalis. Atualmente não existe uma vacina ou tratamento eficaz, tornando a peste suína africana (PSA) uma ameaça para a suinicultura mundial. O genoma do VPSA codifica aproximadamente 150 proteínas, algumas delas bem caracterizadas, estando envolvidas na transcrição, replicação ou na montagem do virião. No entanto, e apesar de todos os esforços realizados nas últimas décadas, a função biológica de numerosas proteínas virais não é ainda conhecida. Esta lacuna aliada à necessidade de um melhor entendimento sobre a biologia do VPSA e as suas interações com o hospedeiro têm contribuído em grande parto para a dificuldade no desenvolvimento de uma vacina eficaz contra PSA. Por homologia de sequências proteicas, o genoma do VPSA codifica para uma proteína tipo histona (pA104R). Nas bactérias, estas proteínas são responsáveis por modular a topologia do DNA (torção, flexão e dobramento), desempenhando assim importantes funções estruturais e controlando a expressão de diferentes genes. O facto do genoma do VPSA codificar entre outras uma proteína viral semelhante a histonas bacterianas, reveste-se assim de enorme relevância pelo papel que que estas proteínas possam desempenhar na compactação do genoma na partícula viral e/ou para a sua replicação e transcrição. Neste contexto, este estudo pretendeu caracterizar o papel da VPSA-pA104R na replicação viral, tendo como objetivo contribuir para o conhecimento da biologia deste vírus e para averiguar se o gene A104R será um bom candidato para desenvolver uma vacina DISC (do inglês “defective infectious single cycle). Além disso, diferentes inibidores das histonas deacetilases (HDACs) foram testados como potenciais antivirais, eventualmente úteis no controlo da PSA Os principais objetivos deste trabalho foram assim os seguintes: (1) estudar VPSA-pA104R, através da clonagem, expressão, purificação e caracterização de sua atividade in vitro; (2) Compreender a relevância funcional de dois resíduos conservados de pA104R; (3) Avaliar os níveis de mRNA e proteína, bem como a localização intracelular de pA104R em células infetadas com VPSA, em diferentes tempos de infeção; (4) Desenvolver uma estratégia que permita a deleção da ORF A104R do genoma do VPSA e a obtenção de uma vacina DISC; (5) avaliar os níveis de acetilação das histonas das células infetadas para melhor compreensão do mecanismo de modulação dos mecanismos epigenéticos do hospedeiro pelo VPSA; (6) Avaliar o efeito dos inibidores das HDACs na infeção pelo VPSA. Neste estudo, a VPSA-pA104R foi expressa num sistema procariota baseado em Escherichia coli. Após a sua purificação, a sua atividade foi caracterizada através de ensaios EMSA (do inglês “electrophoretic mobility shift assay”) e concluiu-se que esta proteína viral se liga tanto a DNA de cadeia simples como dupla, embora tenha maior afinidade para o de cadeia dupla, e estimou-se que o local de ligação seja cerca de 14 a 16 nucleótidos. Esta ligação ao DNA continua presente em variadas condições de temperaturas, pH e concentrações de sal e é independente de ATP. A perda de atividade da proteína mutada pontualmente no resíduo de arginina localizado na posição 69, revelou que este resíduo é importante para uma ligação eficaz ao DNA. Além disto, foi possível concluir que a carga positiva deste aminoácido é determinante para a capacidade de ligação do pA104R ao DNA. Adicionalmente, uma vez que se observou atividade de superenrolamento de DNA quando a pA104R e uma topoisomerase tipo II viral, pP1192R, foram adicionados a DNA plasmídeo relaxado, este trabalho suporta que o VPSA codifica de facto para proteínas necessárias a compactação do seu genoma e ainda é proposto a existência de um efeito sinérgico entre as duas proteínas virais acima descritas. Como o objetivo de melhor compreender a importância da pA104R na infeção do VPSA, avaliou-se a dinâmica da sua expressão e a sua localização intracelular. A expressão de pA104R foi observada na fase tardia da infeção em células Vero infetadas com o isolado viral Ba71V, co-localizando com núcleo da célula e fábricas virais citoplasmáticas. Em relação à dinâmica de transcrição do gene A104R, apesar de ser típica de um gene tardio, foi possível detetar transcritos a partir das 2 horas pós-infeção (hpi). As experiências usando siRNA (do inglês “small interference RNA”) contra os transcritos do gene A104R, mostraram que a redução dos níveis de RNA deste gene induzem uma redução da progenia viral, do número de cópias de genomas virais e da transcrição de um gene viral tardio (B646L), revelando que o pA104R desempenha um papel crítico na replicação do DNA viral e na expressão de genes virais. Atualmente, as únicas medidas de controlo do VPSA são baseadas na deteção precoce da doença e na rápida aplicação de medidas biossanitárias como o abate de animais infetados, controlo do movimento de animais e a vigilância. As tentativas falhadas até agora em obter uma vacina inativada ou atenuada permitem que novas estratégias, como as vacinas DISC ganhem espaço na investigação do VPSA como uma revigorante estratégia para controlar o VPSA. Os resultados obtidos neste estudo, anteriormente descritos, suportam a ideia que um mutante de deleção no gene A104R replicará o seu genoma nas células hospedeiras, mas não poderá compacta-lo dentro do virião, resultando num virião não-infecioso "vazio" que será incapaz de iniciar um segundo ciclo de infeção. Assim a infeção com este vírus mutante será capaz de estimular o sistema imunitário do hospedeiro, mas ao mesmo tempo será seguro, não produzindo progenia infeciosa. Assim outro objetivo deste trabalho foi então obter um vírus DISC deletado no gene A104R. Para isto, o vírus recombinante foi obtido por recombinação homóloga e uma linha Vero complementar, que expressa a pA104R, foi desenvolvida. Embora, o vírus recombinante tenha sido obtido com sucesso, a linha celular complementar desenvolvida não suporta a sua replicação e, como tal, a seleção e propagação do vírus recombinante não foi possível. Os baixos níveis de expressão de pA104R destas células quando comparados com os de células infetadas poderão explicar esta não complementação. Com base em estudos anteriores que mostram que VPSA regula o estado epigenético da célula hospedeira, o grau de acetilação das histonas de células infetadas foi avaliado e a atividade antiviral de quatro inibidores das HDACs (NaPB, VPA, TSA e SAHA) contra a infeção pelo VPSA também foi testada neste estudo. O VPSA induz uma hipoacetilação dos resíduos de lisina 9 e 14 da histona H3 (H3K9K14). Esta modificação epigenética corrobora outras reportadas noutros estudos e todas elas estão classicamente correlacionada com o silenciamento de genes em células eucarióticas e pode indicar que o VPSA subverte diferentes mecanismos celulares, controlando o acesso da maquinaria de transcrição aos genes hospedeiros. Adicionalmente, um dos inibidores das HDACs testados, o NaPB, reverte este estado de hipoacetilação da histona e inibe a replicação do VPSA, interferindo com a expressão de gene virais tardios. Os resultados obtidos neste estudo sugerem fortemente que a pA104R participa da modulação da topologia do DNA viral, estando envolvida na replicação, transcrição e/ ou compactação do DNA viral. Com o objetivo de desenvolver uma vacina DISC, o gene A104R poderá assim constituir um bom alvo a deletar. Esta nova estratégia poderá ser uma alternativa às tentativas até agora falhadas de obter uma vacina contra a PSA. Contudo, antes de uma vacina DISC ser realidade, um esforço científico no desenvolvimento de uma linha celular complementar será imperativo. Os resultados obtidos sugerem ainda que as HDACs celulares estão envolvidas no estabelecimento de infeção pelo VPSA e revelaram que o NaPB pode ser usado como uma estratégia antiviral adicional para controlar a propagação de vírus nas áreas de surto.
N/A

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Basto, Afonso Silva Pinto. "Um novo sistema de clonagem baseado na lipoproteína OprI para obtenção de formulações imunogénicas derivadas da parede celular bacteriana." Doctoral thesis, Universidade Técnica de Lisboa. Faculdade de Medicina Veterinária, 2011. http://hdl.handle.net/10400.5/3620.

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Анотація:

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Sanidade Animal
A modulação de imunidade específica por conjugação de antigénios com ligandos de receptores de reconhecimento de padrão (PRR) constitui uma estratégia emergente para o desenvolvimento de vacinas subunitárias. Neste trabalho, desenvolve-se um novo sistema de clonagem em Escherichia coli para expressão de antigénios em fusão com a lipoproteína OprI, um ligando TLR da membrana externa de Pseudomonas aeruginosa. O sistema permite um controlo apertado da expressão proteica e a purificação por cromatografia de afinidade com iões metálicos, ultrapassando as principais limitações de versões anteriores. Confirmou-se o processamento, translocação e triacilação da lipoproteína e desenvolveram-se protocolos para a produção de outras formulações recombinantes derivadas da parede bacteriana (fragmentos e vesículas de membrana externa) com potencial distinto para activação PRR. Como modelo, clonaram-se as sequências dos antigénios A104R do vírus da peste suína africana (VPSA), ovalbumina e EGFP. Demonstrou-se a capacidade adjuvante das três formulações, avaliando a resposta humoral e a indução de linfócitos T CD8+ in vivo e o perfil de citocinas e quimiocinas induzidas em células dendríticas estimuladas in vitro. Os resultados observados validam o sistema para a obtenção de formulações imunogénicas com aplicação no desenvolvimento de vacinas subunitárias experimentais e em estudos de modulação de resposta adaptativa.
ABSTRACT - A new cloning system based on the OprI lipoprotein for the production of bacterial cell wall-derived immunogenic formulations - The modulation of specific immunity through the conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the development of subunit vaccines. Here, a new Escherichia coli cloning system for the expression of antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane, is described. The system offers tight regulation of expression and allows for purification by metal affinity chromatography, circumventing the major drawbacks of former versions. Lipoprotein processing, translocation and triacylation were confirmed and protocols for the productions of other recombinant bacterial cell wall-derived formulations (outer membrane fragments and vesicles) with distinct potential for PRR activation were developed. As models, the sequences coding for the antigens A104R from African swine fever virus (ASFV), ovalbumin and EGFP were cloned. The adjuvant capacity of the three formulations was demonstrated evaluating the induction of humoral and CD8+ T cells responses in vivo and the cytokine and chemokine profile induced in dendritic cells stimulated in vitro. The results observed validate the system for the production of immunogenic formulations suitable for the development of experimental subunit vaccines and for studies on the modulation of adaptive immunity.
FCT-Fundação para a Ciência e Tecnologia. CIISA-Centro de Investigação Interdisciplinar em Sanidade Animal da Faculdade de Medicina Veterinária-UTL.

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48

Comerlato, Juliana. "Contributions au développement de modèles petit animal et cellulaire pour les études de pathogenèse des morbillivirus." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT070.

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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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49

Martins, Maria Solange Gonçalves Barbas Baptista 1991. "A comparison of genomic and viral expressed sequences of a virus gene manipulation TLR responses." Master's thesis, 2014. http://hdl.handle.net/10451/15758.

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Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014
The I329L ORF of the African swine fever virus is a host evasion gene coding for a transmembrane protein inhibiting the induction of interferon through activation of Toll Like Receptor 3 (TLR3). Additionally, by sharing sequence similarities with TLR3, and also inhibiting TRIF mediated NF-kb activation, the protein has been characterized as a viral TLR3 antagonist. The key observation formed backing the project was that the I329L protein expressed in transfected cells not only yielded the predicted 50 kDa component, but also another one of 35 kDa. Thus the objective of this work was to explain the origin of the 35 kDa component. Two hypotheses were proposed to explain the origin of the 35 kDa molecule: (a) Translation of the I329L from a second AUG, predicted to yield a molecule of the expected size. (b) Proteolytic degradation of the 50 kDa full length protein, as indeed has been observed for other TLRs. Clear cut evidence for the first hypothesis was obtained as an I329L mutated at the second AUG only yielded a 50 kDa component in transfected cells. Importantly, both full length and second AUG 35 kDa constructs induced a similar inhibition of TLR3 signalling in luciferase reporter assays. Moreover, the full length and 35 kDa components were found to similarly localize to the endosome after activation of the TLR3 with double stranded RNA.
A peste suína africana (PSA) é uma doença hemorrágica letal, muito contagiosa que afecta os porcos domésticos, com implicações socio-ecónomicas, tendo em conta que sem uma vacina o único controlo da doença implica o sacrifício dos porcos afectados e quarentena dos animais da mesma exploração. O vírus causa uma infecção persistente e assintomática nos seus hospedeiros naturais, o porco-bravo, javalis e carraças. Já existe sinais de contaminação na Rússia e o risco da doença se espalhar pela europa depende da população animal selvagem e das quintas com pouca segurança biológica, mas também do comércio entre os países africanos e a Europa. Quando uma célula é infectada por um vírus, ela é capaz de reconhecer o microorganismo invasor ao reconhecer os padrões moleculares associados ao patogéneo (PMAP) graças aos seus receptores de reconhecimento de patogéneos (RRP), e por vários caminhos diferentes é capaz de induzir a expressão do interferão (IFN), uma citocina, de tipo I. A célula desenvolve um estado anti-viral e pelo impacto do IFN secretado, estabelece um estado anti-viral semelhante nas células vizinhas. Os receptores TLR fazem parte dos RRP e têm um papel importante na imunidade inata contra infecções, ao se ligarem a moléculas microbianas. As células infectadas por vírus dependem principalmente dos TLR3, TLR7/TLR8 e TLR9 para induzir a expressão do IFN do tipo I e assim detectar os ácidos nucleicos. Foi relatado que estes TLR, localizados em endossomas, são clivados por catepsinas sendo essencial para uma sinalização mais eficiente e maior estabilidade. Os vírus de ADN possuem vários mecanismos estratégicos/genes que modulam de forma positiva ou negativa a biologia celular do hospedeiro, bem como a resposta imunitária. Existem várias estratégias contra o sistema do IFN, que incluem a inibição da sua produção, a inibição das suas vias de sinalização, e bloqueio de enzimas induzidas pelo IFN com actividade antiviral. Neste caso, o Vírus da Peste suína africana (VPSA), desenvolveu vários genes de evasão às respostas imunes do hospedeiro, entre os quais a ORF I329L, que codifica uma proteína transmembranar do tipo I, contendo quatro zonas de repetição de leucinas no seu domínio extracelular e com homologia no domínio intracelular com as Box1 e Box2 do domínio TIR intracelular do TLR3. Recentemente, análises à proteína revelaram que inibe um componente da resposta inata, a indução do IFN de tipo I pela activação da via do TLR, isto é, a indução e secreção do IFN-β, bem como a activação do NF-kB. Essa inibição é adquirida por dois mecanismos distintos, nomeadamente pelo domínio extracelular que inibe a interacção do TLR com o ligando de RNA de dupla cadeia (dsRNA), e pelo domínio intracelular que causa um impacto na transdução do sinal pela associação com o intermediário TRIF. O I329L foi então caracterizado como um antagonista viral do TLR3, afectando de forma negativa a resposta antiviral do hospedeiro pela via do IFN tipo I. Por este motivo, seria uma abordagem racional eliminar o I329L do vírus para a construção de uma vacina viral atenuada e por isso é essencial compreender os mecanismos de acção deste gene. Como o TLR3, assim como o TLR7 e TLR9, precisam de ser proteoliticamente processados para uma melhor capacidade de sinalização, existe a possibilidade de que o I329L pode ser igualmente processado por um mecanismo semelhante. Ao estudar o I329L em células transfectadas, foi possível observar dois componentes proteicos, um com a massa molecular prevista de ~50 kDa, e outro de ~35 kDa. A origem do componente de menor massa molecular pode ter origem em um processamento da proteína de 50 kDa ou, alternativamente, ser o produto de uma tradução a partir do segundo codão AUG que codifica uma metionina, no dominio extracellular do I329L, o que levaria à produção de uma proteína do tamanho esperadode 35 kDa. Deste modo, os principais objectivos da tese foram determinar se o I329L sintetizado a partir do segundo AUG inibe a activação do TLR3, e se existe processamento proteolítico após activação com RNA de dupla cadeia, isto é poly (I:C). Para responder a estas questões, foi primeiro necessário clonar o I329L com uma “immunotag” Myc no N terminal e uma “immunotag” HA no C terminal. A seguir, de modo a responder à questão colocada pelo primeiro objectivo, o I329L foi mutado no segundo codão ATG, usando mutagénese dirigida. Células transfectadas com o I329L mutante e TLR3 foram estimuladas com poly (I:C) durante 30 minutos. Após as amostras das células previamente lisadas após o estímulo terem sido transferidas para uma membrana por Western blot e reveladas com anticorpo anti-HA, foi possível ver que a banda de 35 kDa tornou-se ausente no mutante. Tal indicou que o I329L pode ser traduzido por ambos os codões AUG, originando dois produtos diferentes. De modo a verificar que o produto originado a partir do segundo AUG é funcional, foram feitos ensaios de luciferase, medindo a activação do IFN-β após a activação de células a expressar o TLR3, estimuladas com poly (I:C) e sobre expressão do TRIF. Os resultados demonstraram que ambos os constructs (o I329L de tamanho completo e o “curto”) eram funcionais, podendo inibir a activação do TLR3 pela via do IFN-β após estimulação com dsRNA. Foi igualmente demonstrado que a inibição deriva do domínio extracelular ao interferir com a estimulação por poly (I:C) e deriva do domínio intracelular ao interagir com a sinalização mediada por TRIF. Como o TLR3 se localiza nos endossomas, foi igualmente importante verificar a localização intracelular do I329L após estimulação com poly (I:C). Para tal, células foram cultivadas em lamelas e transfectadas com plasmídeos contendo a sequência completa ou “curta” do I329L marcado com as “tags” Myc e HA. Foi possível visualizar que ambas as formas se encontravam no endossoma, provando que seguem vias semelhantes de localização intracelular. Infelizmente as experiências realizadas com o anticorpo para a “tag” do Myc não deu um bom sinal para poder analisar a localização com mais afinco. Por último, de forma à responder à questão do segundo objectivo, foi necessário transfectar células com o plasmídeo contendo o I329L completo, e após estimulo com poly (I:C), revelar as amostras transferidas por Western blot com um anticorpo para Myc. De modo desapontante os resultados foram inconclusivos devido ao alto ruído de fundo, que impediu verificar correctamente as bandas apresentadas. Em todo o caso, foi possível observar que após 30 minutos de estímulo com poly (I:C), a banda de 50 kDa desapareceu, o que não descartou a hipótese de um processamento proteolítico do I329L completo, e é necessário mais investigação sobre o assunto. Resumindo e concluindo, I329L é uma proteína antagonista de TLR3 com duas formas funcionais sintetizadas a partir do primeiro e do segundo codão AUG, capazes de inibir a activação de IFN-β extracelularmente por dsRNA e intracelularmente pelo TRIF; ambas co-localizam nos endossomas, e existe ainda a possibilidade do I329L ser processado como um TLR3. Para trabalho futuro é necessário confirmar as conclusões obtidas pelos ensaios funcionais de luciferase, fazendo análises de expressão por microarrays, de modo a comparar os domínios extracelulares dos I329L completo e “curto”, o que pode ser importante para futura exploração da sua capacidade de manipulação celular tal como da resposta imune. Adicionalmente, também é importante determinar se o I329L traduzido a partir do segundo AUG é igualmente produzido em células infectadas com o VPSA de modo a perceber melhor o mecanismo de acção do I329L após infecção viral.

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50

Costa, Emanuel Nery de Oliveira Quartin 1986. "Viral modulation of interferon (IFN) responses to african swine fever virus (ASFV)." Master's thesis, 2011. http://hdl.handle.net/10451/5033.

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Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2011
A imunidade inata constitui a primeira resposta dada por um hospedeiro quando atacado por agentes patogénicos. A resposta imune baseia-se em genes codificados na linha germinativa, chamados receptores de reconhecimento de padrões (PRRs). Estes conseguem distinguir o “Eu” do “não-Eu”, reconhecendo padrões moleculares conservados ao longo da evolução dos vários agentes patogénicos, chamados padrões moleculares associados a agentes patogénicos (PAMPs). No caso dos vírus, um parasita intracelular obrigatório, os PAMPs mais importantes e mais estudados são o seu material genético, tal como o DNA genómico viral, RNA de cadeia dupla (ds) ou simples (ss) ou a estrutura RNA viral, 5’- trisfosfato-RNA. Existem vários PRRs, que podem ser agrupados em classes: os receptores transmembranares do tipo Toll (TLRs), os receptores citoplasmáticos do tipo RIG-I (RLRs), os receptores do tipo Nod (NLRs) e os receptores do tipo AIM2 (ALRs). Os PRRs iniciam uma sinalização em cascata que culmina com a activação de factores de transcrição, que entre outros, vão induzir a produção e excreção duma citoquina, o interferão (IFN). Este grupo de citoquinas é composto por três classes, IFN tipo I (p.e IFN-α/β) , tipo II (p.e IFN-γ) ou do tipo III (p.e. IFN-λ). O IFN pode despoletar variados efeitos anti-virais. A cascata de sinalização estimulada pelo IFN inicia-se com a ligação do IFN ao seu respectivo receptor extra celular que ,através de fosforilações, permite a activação de receptores intracelulares. Já no interior da célula, sinalizadores de transdução e ativadores da transcrição (STATs) são recrutados e fosforilados, o que permite a formação de homo ou heterodímeros que migram para o núcleo. No núcleo, as STATs ligam-se a zonas promotoras de genes estimulados pelo IFN (ISGs), para promover a transcrição de mais de 300 ISGs com propriedades anti-virais. No caso do estímulo causado por IFN do tipo I, os complexos formados pelas STATs vão ligar-se ao elemento de resposta estimulado pelo IFN (ISRE). No caso do IFNs do tipo II, os complexos ligam-se à sequência activada pelo IFN-λ (GAS). Os ISGs facultam ao hospedeiro diversas estratégias para combater a infeção viral. Apesar de os mamíferos possuírem um sistema imune bastante evoluído, os vírus também têm evoluído estratégias para evitar e/ou manipular as defesas do hospedeiro, dedicando uma parte substancial do seu genoma a estas estratégias. Estas podem ir desde uma interferência global na expressão e/ou síntese de proteínas das células do hospedeiro, ou serem mais específicas, diminuindo o impacto dos IFNs. O estudo destas interações, pode não só ser útil para conhecer os mecanismos de infecção do vírus, mas também para perceber melhor os mecanismos de defesa do hospedeiro. Estes conhecimentos podem permitir o desenvolvimento de terapias e tratamentos anti-virais ou mesmo anticancerígenos. A peste suína africana (ASF) é uma doença que nos porcos domésticos (Sus sacrofa) é tipicamente hemorrágica e leva normalmente à morte do hospedeiro. Contudo, as infecções são assintomáticas nos hospedeiros naturais, o javali, o porco selvagem e a carraça, sendo esta última, um dos principais vectores de transmissão do vírus da peste suína africana (ASFV), tornando o seu controlo difícil sem uma vacina. Nos últimos anos, devido ao grande desenvolvimento urbano e consumo de carne de porco, têm havido surtos de ASF em África, causando perdas devastadoras. O ASFV é um virus de DNA de cadeia dupla, o único arbovírus de DNA e o único membro da familia Asfaviridae, infectando principalmente macrófagos e monócitos. Tal como todos os vírus, o ASFV contém genes que manipulam a biologia da célula do hospedeiro, como por exemplo, genes que inibem a apoptose e respostas imunes controladas pelo factor nuclear kappa B (NFκB), entre outros. Contudo, ainda não foi demonstrado que algum gene do ASFV consiga inibir a resposta do IFN. Isto é surpreendente, pois o ASFV infecta macrófagos, um tipo de célula sensível ao IFN e porque a sua infecção persistente, é incompatível com uma resposta efectiva mediada por IFN. O K205R é um gene do ASFV sem função definida, mas ensaios preliminares de luciferase mostraram que este gene consegue inibir a resposta do IFN. Contudo, os mecanismos utilizados pelo K205R nesta inibição são desconhecidos. O objectivo desta dissertação de mestrado é tentar perceber melhor estes mecanismos e determinar a sequência mínima necessária para que o K205R tenha o efeito observado. O K205R foi isolado através de PCR, utilizando como molde o DNA genómico da estirpe do AFSV, BA71. Subsequentemente, foiclonado no plasmídeo pcDNA3, que contém um marcador molecular, a hemaglutinina (HA), a montante da zona de inserção do gene. Para determinar a extensão da ação do K205R, foram feitos ensaios de luciferase utilizando células transfectadas com repórteres de luciferase sobre o controlo dos promotores de IFN- β, ISRE e GAS. O K205R mostrou inibição para todos os reporteres. Para tentar definir a zona do K205R responsável pelo efeito observado, fez-se uma previsão da estrutura secundária da proteína do K205R, recorrendo à bioinformática, que permitiu identificar uma sequência “coiled-coil” putativa, uma estrutura secundária associada a interações entre proteínas. Também é sugerida uma sequência putativa para um sinal de exportação nuclear (NES). Com base nesta análise foram construídos quatro fragmentos do K205R e posteriormente clonados no pcDNA3. Depois de se verificar a sequencia correcta de DNA de cada um dos clones e expressão das suas proteinas em células vero transfectadas , o passo seguinte foi verificar a localização celular destes fragmentos através de imunofluorescência nestas mesmas células. Esta experiência permitiu verificar que de facto, os fragmentos que não tinham a sequência putativa NES, em comparação com células transfectadas com o K205R inteiro, tinham uma maior acumulação nuclear. Para estudar o mecanismo, e a que nivel o K205R actua para inibir a via de sinalização do ISRE, foi feito um “western blot” utilizando extractos proteicos de células VERO transfectadas com os diferentes fragmentos do K205R e posteriormente estimuladas com IFN-β durante 15 minutos e durante 45 minutos. Esta experiência permitiu verificar que a fosforilação da STAT1 diminui na presença do K205R, contudo, apenas um fragmento reproduziu este efeito. Este fragmento de 75 aminoácidos não contém a sequência, nem para a sequência “coiled-coil”, nem para NES. Esta dissertação de mestrado apresenta resultados consistentes com a existência de um NES funcional na sequência do K205R, uma inibição da fosforilação da STAT1 mediada pelo K205R, mas também apresenta uma abordagem para determinar os mecanismos utilizados pelo K205R para inibir a indução e o impacto do IFN-β. Contudo, mais experiências têm de ser feitas para realmente se comprovar a existência de um NES, como por exemplo, ensaios de imunofluorescência de células transfectadas com K205R na presença de Leptomicina B, um inibidor da exportação nuclear. Também será necessário estudar as vias de sinalização inibidas pelo K205R que não foram abordadas neste trabalho, tal como a via de indução de IFN-β e a via do GAS.
A key part of the innate response to virus infections is the interferon (IFN) response. This can limit virus replication and dissemination and is a critical factor in controlling virus infections, particularly persistent viruses. Many viruses encode proteins which interfere with induction of IFN and IFN-activated pathways and these can have important roles in virus pathogenesis and persistence. African Swine Fever (ASF) causes major economic losses in many African countries and is a threat to pig farming worldwide. There is no vaccine and therefore options for disease control are limited. In Europe, there is always the danger of accidental introduction of the virus, as indeed occurred in Portugal in 1957, causing severe financial losses. Thus, defining the mechanism of proteins involved in evasion of the host’s defense response and in virus virulence is of extreme interest, so we can understand the virus and try to develop strategies to reduce ASF impact. ASFV is a large cytoplasmic DNA virus which encodes between 160 to 175 open reading frames. Many of its genes are not essential for replication in vitro, but are host evasion strategies facilitating virus replication and transmission in vivo. These include proteins which inhibit host defence systems. Surprisingly, since ASFV can survive as a persistent virus, no ASFV proteins have been described which inhibit the IFN response. However, the early gene K205R, might have an impact on IFN response. Luciferase assays, shown inhibitions of IFN induction (IFN-β) and IFN-signalling (ISRE, GAS) pathways. Using a bioinformatics tool (Jpred), we got a predication of K205R protein secondary structure. Based on this prediction, deletion mutant fragments of K205R were constructed and used in immunofluorescence and western blot assays. The immunofluorescence results suggest the presence of a functional nuclear export signal (NES) motif in the K205R protein sequence. Western blot experiments suggested that K205R is affecting the phosphorylation status of STAT1, in cells stimulated with IFN-β (ISRE pathway). Although it was not possible to clearly determine the minimum sequence needed for all the functions of K205R, the results suggest that K205R inhibition of the impact of IFN type I, depends on a sequence within amino acids 130 and 205, which affects STAT1 phosphorylation. Further experiments should be done to investigate the mechanism of K205R inhibition in the pathways not covered on this thesis (IFN-β induction pathway and GAS pathway). The existence of functional NES also needs confirmation.

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Дисертації з теми "Virus de la peste équine"

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