Academic literature on the topic 'Bluetongue virus; Ruminants'

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.

 Bluetongue is a non-contagious disease of domestic and wild ruminants caused by a virus within the Orbivirus genus of the family Reoviridae and transmitted by Culicoides biting midges. It is a reportable disease of considerable socioeconomic concern and of major importance for the international trade of animals and animal products. In the past, bluetongue endemic areas were found between latitudes 40°N and 35°S; however, bluetongue has recently spread far beyond this traditional range. This is in accordance with the extension of areas in which the biting midge Culicoides imicola, the major vector of the virus in the “Old World”, is active. After 1998 new serotypes of bluetongue virus (BTV) were discovered in Southern European and Mediterranean countries. Since 2006 BTV-serotype 8 has also been reported from the countries in Northern and Western Europe where Culicoides imicola has not been found. In such cases, BTV is transmitted by Palearctic biting midges, such as C. obsoletus or C. dewulfi, and the disease has thus spread much further north than BTV has ever previously been detected. New BTV serotypes have recently been identified also in Israel, Australia and the USA. This review presents comprehensive information on this dangerous disease including its history, spread, routes of transmission and host range, as well as the causative agent and pathogenesis and diagnosis of the disease. It also deals with relevant preventive and control measures to be implemented in areas with bluetongue outbreaks.  

Abstract Bluetongue disease is an infectious non-contagious disease of domestic and wild ruminants, transmitted by hematophagous insects of the genus Culicoides. In endemic areas the disease has a seasonal character, occurs usually in summer when the population of vectors is at its peak. Culicoides are active at temperatures in the range from 13oto 35oC. The replication of the virus stops when the environmental temperature is below 13oC. It has been reported that the temperature and humidity of the environment affect to a great extent the biology of the vector and the survival of the virus in the reservoirs. During the summer, the number of infected cattle and sheep is directly dependent on the density of the population of the vector, the length of vectors’ life-span, the temperature of the environment and by precipitation, the affi nity of the vector to different hosts, and the ability of the vector to locate the host. Bluetongue has been spreading worldwide due to climatic changes and increasing average daily temperatures. The seasonal occurrences of the disease and the climate change have conditioned the need for adopting new strategies. The stochastic SEIRD mathematical model has been developed in order to simulate the transmission of the Bluetongue virus through the susceptible ruminant population on the territory of the Republic of Serbia, as well as to investigate the effect of climatic factors on the vector population and the magnitude of a possible epizootia. Besides the effects of climatic factors, we have analyzed a number of different approaches in the control of the disease based upon the vaccination of ruminants and control of vectors.

ABSTRACTBluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence.IMPORTANCEBluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals byCulicoidesbiting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.

La llengua blava (LB) és una malaltia transmesa per vectors Culicoides de declaració obligatòria causada pel virus de la llengua blava (VLB) i considerada emergent i re-emergent a Europa. Aquesta malaltia afecta principalment a remugants domèstics i salvatges i també camèlids, causant importants pèrdues econòmiques en el sector ramader. La implicació de diferents hostes i vectors en el cicle de transmissió del VLB dificulta el control de la malaltia. El cicle de transmissió del virus està condicionat per factors externs, com són el canvi climàtic i l’alteració dels ecosistemes, els quals han afavorit l’expansió dels vectors en les últimes dècades. Entre les mesures de control, destaquen la vacunació, la restricció de moviment d’animals susceptibles en zones de risc i els programes de vigilància epidemiològica, tant d’animals domèstics com de vectors. Tot i que les vacunes comercials actuals han demostrat ser efectives en remugants domèstics, l’elevat nombre de serotipus del VLB presents (fins a 26 descrits actualment), fa que sigui complicat desenvolupar una vacuna universal que ofereixi protecció creuada. Totes aquestes variables han fet impossible fins al moment l’eradicació d’aquesta malaltia. La majoria d’espècies de remugants salvatges presents a Europa, si no tots, són susceptibles a la infecció pel VLB, que és majoritàriament asimptomàtica. Aquest fet fa que tinguin importància com a possibles reservoris i transmissors del virus, tant entre animals salvatges com de salvatges a domèstics. La informació relativa a la seva implicació en el cicle de transmissió del virus entre animals salvatges i domèstics és encara escassa. Això, juntament amb el fet que els programes de vacunacions massives s’apliquin exclusivament a remugants domèstics, posa de manifest la necessitat de portar a terme estudis addicionals a fi de determinar el paper dels remugants salvatges en l’epidemiologia de la LB. La present tesi s’ha centrat en l’estudi de la LB en els remugants salvatges presents a la Península Ibèrica. El seu contingut segueix l’estructura típica d’un treball científic. Comença amb una Introducció, en la qual es realitza una breu revisió actual sobre la LB i el VLB, seguit d’un apartat amb els Objectius que seran tractats en cada capítol. A continuació es presenten dues Seccions estructurades en cinc Capítols, que corresponen a articles científics en diferent estat de publicació (tres publicats i dos enviats). A l’apartat de Discussió general es pretén donar una breu visió del conjunt de capítols i, finalment, s’ennumeren totes les Conclusions obtingudes d’aquesta tesi doctoral. En la primera secció de la tesi (Epidemiologia) s’han realitzat estudis serològics i virològics retrospectius per tal d’aportar més informació de l’evolució de la LB en les espècies de remugants salvatges presents a la Península Ibèrica. Aquests estudis indiquen que aquestes espècies estan implicades en el manteniment del VLB i que poden actuar com a reservoris del virus a la Península Ibèrica. En la segona secció (Vacunació i infecció experimental) s’ha demostrat la susceptibilitat a la infecció amb els serotipus 1 i 8 del VLB del cérvol (Cervus elaphus) i la cabra salvatge (Capra pyrenaica). A més, s’ha avaluat la protecció induïda per una dosi (en cabra salvatge) o dues dosis (en cérvol) vacunals enfront la inoculació experimental amb soques homòlogues del virus. Finalment, s’ha realitzat un estudi longitudinal del desenvolupament d’anticossos neutralitzants fins a 18 mesos després de la immunització a la cabra salvatge.
La Lengua azul (LA) es una enfermedad de declaración obligatoria causada por el virus de la lengua azul (VLA) y considerada emergente y reemergente en Europa. Esta enfermedad afecta especialmente a rumiantes domésticos y salvajes y también camélidos, causando importantes pérdidas económicas en el sector ganadero. La implicación de diferentes hospedadores y vectores en el ciclo de transmisión del VLA dificulta el control de la enfermedad. Dicho ciclo está condicionado por factores externos como son el cambio climático y la alteración de ecosistemas, los cuales han favorecido la expansión de los vectores en las últimas décadas. Entre las estrategias de control destacan la vacunación, la restricción del movimiento de animales susceptibles en zonas de riesgo y los programas de vigilancia epidemiológica tanto de animales domésticos como de vectores. Aunque las vacunas comerciales actuales han demostrado ser efectivas en rumiantes domésticos, el elevado número de serotipos del VLA presentes (hasta 26 descritos actualmente), hace que sea complicado desarrollar una vacuna universal que ofrezca protección cruzada. Todas estas variables han hecho imposible hasta el momento la erradicación de la enfermedad. La mayoría de especies de rumiantes salvajes presentes en Europa, si no todos, son susceptibles a la infección por el VLA, que es mayoritariamente asintomática. Esto los hace importantes como posibles reservorios y transmisores del virus, tanto entre animales salvajes como de salvajes a domésticos. La información relativa al estudio de su implicación en el ciclo de transmisión entre salvajes y domésticos es todavía escasa. Ésto, unido al hecho de que los programas de vacunaciones masivas se apliquen exclusivamente a rumiantes domésticos, pone de manifiesto la necesidad de llevar a cabo estudios adicionales con el fin de determinar el papel de los rumiantes salvajes en la epidemiología de la LA. La presente tesis se ha centrado en el estudio de la LA en rumiantes salvajes presentes en la Península Ibérica. Su contenido se organiza siguiendo el orden habitual de un trabajo científico. Comienza con un apartado de Introducción, en el que se realiza una breve revisión actual sobre la LA y el VLA, seguido de un apartado de los Objetivos que se abordarán en cada capítulo. A continuación se presentan dos Secciones estructuradas en cinco Capítulos, que corresponden a artículos científicos en diferente estado de publicación (tres aceptados y dos enviados). En el apartado de Discusión general se pretende dar una breve visión del conjunto de capítulos y, para finalizar, se enumeran todas las Conclusiones obtenidas en la tesis doctoral. En la primera sección de la tesis (Epidemiología) se han realizado dos estudios serológicos y virológicos retrospectivos con el fin de aportar más información sobre la evolución de la LA en las especies de rumiantes salvajes presentes en la Península Ibérica. Estos estudios indican que estas especies están implicadas en el mantenimiento del VLA y que pueden actuar como reservorios del virus en la Península Ibérica. En la segunda sección (Vacunación e infección experimental) se ha demostrado la susceptibilidad a la infección con los serotipos 1 y 8 del VLA en el ciervo (Cervus elaphus) y la cabra montés (Capra pyrenaica). En estos experimentos, se ha evaluado la protección inducida por una dosis (en cabra montés) o dos dosis (en ciervo) vacunales frente a la inoculación experimental con cepas homólogas del virus. Finalmente, se ha realizado un estudio longitudinal del desarrollo de anticuerpos neutralizantes hasta 18 meses después de la inmunización en la cabra montés.
Bluetongue (BT) is a reportable disease caused by bluetongue virus (BTV) considered emerging and reemerging in Europe. BT affects especially domestic and wild ruminants and also camelids, causing important economic losses in the animal industry. The implication of different hosts and vectors in the transmission cycle of BTV makes difficult to control the disease. The transmission cycle is affected by external factors, as climate change and ecosystems’ alteration, which have favored vector expansion in the last decades. Among the control measures, vaccination, restricted movement of susceptible hosts during risk periods and epidemiologic surveillance programs including livestock and vectors are the most implemented. Although available commercial vaccines have proven to be effective in domestic ruminants, the high number of BTV serotypes (up to now, 26 described) makes difficult the development of a universal vaccine able to confer cross-protection. All these factors have made impossible the eradication of this disease. Most, if not all, wild ruminant species present in Europe are susceptible to BTV infection, although it is mainly asymptomatic. This fact makes wild species important as potential reservoirs and transmitters among wildlife or from wild to domestic ruminants. Data related to the implication of wild ruminants in the BTV transmission cycle between domestics and wildlife is still limited. This point, and also the fact that mass vaccination campaigns are applied exclusively to domestic ruminants highlights the need to carry out additional studies with the aim of determining the role of wild ruminants in the epidemiology of BT. The present thesis is focused in the study of BT in wild ruminants present in the Iberian Peninsula. The structure is the typical of a scientific paper. It starts with an Introduction, which contains a brief review of BT and BTV, followed by the Objectives that will be developed in each chapter. Afterwards, there are two Sections structured in five Chapters. All the studies are published or submitted to publish in international peer-reviewed journals. In the General discussion section is given a summary of the main findings and, finally, all the Conclusions obtained are listed at the end of the thesis. In the first section of the thesis (Epidemiology) two retrospective serological and virological studies have been carried out in order to provide new information regarding the evolution of BT in wild ruminant species present in the Iberian Peninsula. These studies indicate that wild ruminants are implicated in maintaining BTV, and they may play a relevant role as BTV reservoirs in the Iberian Peninsula. In the second section (Vaccination and experimental infection), the susceptibility to BTV-1 and BTV-8 infection has been demonstrated in red deer (Cervus elaphus) and Spanish ibex (Capra pyrenaica). Moreover, the efficacy of two commercial vaccines has been evaluated by means of specific neutralising antibodies and absence of viraemia in both species, vaccinated and experimentally inoculated with homologous strains. Finally, it has been carried out a longitudinal study of the development of neutralising antibodies until 18 months postimmunization in Spanish ibex.

Les poxvirus sont considérés comme des vecteurs vaccinaux de choix. Parmi eux le virus myxomateux (MYXV) prototype du genre leporipoxvirus a été positivement évalué chez différentes espèces animales, excepté les ruminants. Dans ce travail, nous avons évalué la souche atténuée SG33 du MYXV en tant que vecteur vaccinal non réplicatif chez les moutons. Dans un premier temps, nous avons étudié les interactions entre le SG33 et les cellules dendritiques (DC). Nous avons montré in vitro que le SG33 est capable d'infecter les DC, et préférentiellement la sous-population de DC de type Langerhans. Bien que le cycle de réplication du SG33 soit abortif, l'expression du transgène dans ces cellules a été démontrée. Après maturation, les DC ovines restent sensibles au SG33 mais entrent en apoptose dans les huit heures qui suivent l'infection. In fine, l'analyse du profil d'expression génique des DC infectées, montre que la majorité des gènes surexprimés est impliquée dans la réponse inflammatoire, la réponse immune et les voies de signalisation des interférons de type I. Par la suite, l'efficacité du SG33 comme vecteur vaccinal chez le mouton a été démontrée en utilisant comme modèle d'épreuve une souche hypervirulente du virus de la bluetongue (BTV). Des moutons ont été immunisés avec du SG33 exprimant la protéine VP2 associée ou non à la protéine VP5 du BTV. Seule l'immunisation avec le SG33 exprimant VP2 a permis d'induire une protection clinique et virologique contre une inoculation d'épreuve avec le BTV homologue. La protection est corrélée à la présence d'anticorps neutralisants chez les moutons avant inoculation d'épreuve. Les niveaux de VP2 produite par les 2 virus recombinants seraient à l'origine de la différence de protection observée. Finalement, la protéine VP7 étant responsable d'une protection croisée partielle entre sérotypes du BTV, nous avons montré que l'inoculation intradermique de SG33 exprimant VP7 à des moutons, induisait l'apparition d'une réponse humorale et cellulaire CD4+ spécifique de VP7. Toutefois, cette réponse immunitaire ne s'est pas révélée protectrice lors d'une inoculation d'épreuve hétérologue. Ces résultats indiquent que le MYXV est capable d'induire une réponse immune adaptative efficace, conduisant à une protection significative contre une inoculation d'épreuve sévère, faisant de ce virus, un vecteur prometteur pour la vaccination des ruminants
Poxviruses are deemed as vaccine vectors of choice. Among them, the myxoma virus (MYXV), the prototype of the Leporipoxvirus genus has proved its efficacy in different animal species but not in ruminants. In this work, we evaluated SG33, an attenuated strain of MYXV as a non-replicative vaccine vector in sheep. In a first study, we examined interactions between SG33 and ovine dendritic cells. We showed in vitro that SG33 infect ovine dendritic cells (DC) mainly the subpopulation of Langerhans-type. Expression of the recombinant antigen was demonstrated although the cycle of SG33 replication was abortive. After maturation, the ovine DC remained susceptible to MYXV, although apoptosis occured within eight hours after infection. Finally, analysis of the gene expression profile of infected DC highlighted that most overexpressed genes are involved in the inflammatory and immune responses, and the signaling pathways of type I interferon. Subsequently, the final demonstration of the SG33 efficacy as a vaccine vector in sheep was carried out using as challenge experiment. We used a highly virulent bluetongue virus challenge model to test protection of sheep previously immunised with SG33 expressing the major VP2 protein antigen of BTV, associated or not with the VP5 protein. We showed that only two immunisations with SG33 expressing VP2 alone elicited a significant clinical and virological protection in sheep against the homologous BTV challenge. Protection was mainly correlated with the presence of neutralising antibodies in sheep before challenge. Differences of VP2 expression between the two SG33 recombinant viruses may explain the differences of protection observed in this study. Finally, since VP7 protein of BTV was suggested to induce cross protective immunity between BTV serotypes, we showed that intradermal inoculation of sheep with SG33 expressing the VP7 protein, can generate an humoral and a CD4+ cellular immune response specific to VP7. However, this immune response did not provide protection against an heterologous BTV challenge. Taken together, these results indicate that MYXV is able to induce an effective adaptive immune response in sheep, leading to significant protection against a severe challenge, making this virus a promising vector for vaccination of ruminants

Les Poxviridae, dont fait partie le virus de la myxomatose (MYXV), sont des outils de choix pour la génération de vaccins recombinés réplicatifs ou non réplicatifs chez différentes espèces animales. Ayant pour but de développer un vaccin contre la Fièvre Catarrhale Ovine, ou Bluetongue (BT), chez les ruminants, nous avons étudié le tropisme cellulaire du vecteur myxomateux chez les bovins et chez les ovins. Les résultats obtenus montrent que le MYXV ne se réplique pas dans les cellules de ruminant et gagent en partie de l'innocuité de tels vaccins. L'inoculation d'ovins par un virus myxomateux recombiné a montré la mise en place d'une réponse humorale contre le produit d'un transgène témoin. Un essai de vaccination-protection contre le BTV avec des MYXV recombinés exprimant des antigènes du BTV montre que nous n'avons pas de protection efficace. Ces résultats nous ont conduit à étudier l'interaction entre les cellules dendritiques (DC) ovines, clés de l'immunité, et le MYXV. Parmi les DC, les cellules de Langerhans sont les cibles préférentielles du MYXV. Bien que les DC infectées deviennent apoptotiques après 16h d'infection, la capacité à induire une réponse immunitaire via un mécanisme de cross-présentation est envisageable. De plus nous avons développé une stratégie originale visant à utiliser la protéine structurale M022L du MYXV comme molécule présentatrice d'antigène pour améliorer ce vecteur. Les virus recombinés sont non pathogènes pour les lapins et capables d'induire une réponse immunitaire aussi bien humorale (inoculation de souris) que cellulaire (inoculation d'ovins) contre le produit du transgène fusionné à M022L chez des espèces non-cibles
Myxoma virus (MYXV) belongs to the Poxviridae family. These viruses are attractive tools for replicative or non replicative recombinant vaccines development in different species. Aiming at developing a vaccine against Bluetongue in ruminants, we first studied bovine and ovine cellular tropism of MYXV. We showed that MYXV can not replicate in ruminant cells attesting of the inocuity of such vaccines. Inoculation of recombinant MYXV in sheep was followed by humoral response against specific antigen. A vaccination/protection assay against Bluetongue using recombinant MYXV expressing BTV-2 antigens showed no efficient protection against a virulent challenge. These results promped us to investigate interactions between MYXV and ovine dendritic cells (DC), since these cells are one of the key of immunity. Results showed that Langerhans DC are the main target of MYXV. Infected DC cells become apoptotic at 16 h post-infection. However, the capacity of immune response induction by a cross-presentation mechanism is conceivable. Moreover, we developed a new strategy of vector design which consists in generating recombinant MYXV expressing a fusion between M022L gene, encoding for a structural protein of the virus membrane, and the transgene. We showed that this virus is dramatically attenuated for rabbits and induces specific humoral and cellular immune responses against the product of the transgene in rabbits and non host animal species

Dissertação (mestrado) - Pontifícia Universidade Católica do Paraná, São José dos Pinhais, 2011
Inclui bibliografias
A Língua Azul (LA) é uma doença viral de ruminantes, não contagiosa, transmitida por artrópodes e economicamente importante, com distribuição mundial. No Brasil, a LA foi primeiramente descrita em bovinos e ovinos no Estado de São Paulo. Desde então, a ci
Bluetongue (BT) is an arthropod-borne, non contagious and economically important viral disease of ruminants with worldwide distribution. In Brazil, BT was first described in cattle and sheep in the State of São Paulo. Since then, viral circulation has bee

La Fièvre Catarrhale Ovine est une arbovirose due à un Orbivirus touchant les ruminants. Récemment, une épizootie majeure, notamment due au sérotype 8 du virus (BTV-8), a touché les ruminants Européens. Ce sérotype a présenté plusieurs particularités telles que le spectre d’hôte original, et la transmission transplacentaire. L’impact de l’infection par le BTV-8 chez le caprin a été étudiée dans ce travail d’un point de vue clinique, virologique, hématologique et sérologique, et notamment pour ce dernier aspect à travers le développement de deux outils sérologiques. L’impact d’une infection maternelle sur le fœtus a également été étudié.Ces travaux ont confirmé que l’impact de l’infection par le BTV-8 chez les caprins est modéré, tant d’un point de vue clinique que d’un point de vue hématologique. Ce travail a également permis de faire ressortir plusieurs connaissances nouvelles: un impact modéré sur les comptages leucocytaires ; une transmission transplacentaire du virus lors d’infection en milieu de gestation ; une détection du virus dans la semence ; une possible transmission « directe », non vectorielle.Ces 3 dernières constatations n’avaient jusqu’alors pas été rapportées dans la littérature chez les caprins, mais avaient été observées chez les ovins et les bovins. Ceci confirme que, même si les caprins sont des animaux sensibles mais que l’impact clinique est limité, la plupart des caractéristiques faisant la spécificité de l’infection par la souche européenne du BTV-8 peuvent également être retrouvées dans cette espèce. Néanmoins, l’absence de description de ces particularités dans des conditions d’infection naturelle ne permet pas de conclure quant à leur impact sur le terrain.Des outils sérologiques ont été également développés afin d’étudier les propriétés antigéniques des protéines virales chez le caprin. La production des protéines recombinantes NS1, NS3, VP7 et VP2 en système baculovirus, et de la protéine NS2 en système E. coli, ont permis l’obtention de protéines recombinantes. Ces 5 protéines recombinantes ont permis le développement de tests sérologiques permettant d’étudier leurs propriétés antigéniques et la cinétique d’apparition des anticorps après vaccination ou/et infection par le BTV-8 chez le caprin.Dans un premier temps, des tests ELISA indirect NS1, NS2, NS3, VP7 et VP2 ont été développés, et la capacité des tests ELISA NS et VP7 à permettre une différenciation entre les animaux infectés et vaccinés a été évaluée. Cependant, des anticorps anti-NS2 et NS3 ont été détectés dans les sérums d’animaux vaccinés et une faible réponse obtenue en ELISA NS1 chez les animaux infectés rend difficile l’utilisation d’un test ELISA DIVA basé sur ces 3 protéines non structurales. Enfin, une réponse en anticorps anti-VP2 est détectée par le test ELISA VP2 après vaccination et après épreuve virulente, suggérant une détection d’anticorps spécifiques de type par ce test.Dans un second temps, un test Luminex multiplex, basé sur l’utilisation des protéines VP7 et VP2 a été développé. Le Luminex VP7 présente une très bonne corrélation avec l’ELISA VP7 lorsque les sérums d’animaux infectés sont testés, avec une aire sous la courbe de 0,987. Les performances de ce test paraissent cependant modérées lorsque des sérums issus d’animaux ayant reçu une unique injection vaccinale sont testés. Le Luminex VP2 présente des performances également excellentes, avec une aire sous la courbe de 0,978. Les VPP et VPN ont été calculées pour des prévalences très faibles (0,5%, correspondant à la prévalence devant être détectée par le screening sérologique d’animaux sentinelles) : la VPP est alors très faible mais la VPN est très élevée (99,99% pour VP7, 99,95% pour VP2). Le test Luminex multiplex développé, caractérisé par une VPN très élevée, permet d’exclure avec confiance la présence du BTV-8 dans une région indemne lors de résultat négatif, correspondant parfaitement aux objectifs assignés
Bluetongue is an infectious non contagious arbovirosis caused by Bluetongue virus (BTV), belonging to the genus Orbivirus. Recently, a major epizooty, due to BTV-8, was encountered in European ruminants. This serotype presented several original features such as an original host spectrum and transplacental transmission. This work consisted in studying the impact of BTV-8 infection in goats from a clinical, virological, haematological and serological (after development of two new serological tests) point of view, because of the lack of knowledge in this specie. The impact on foetuses of infection during gestation was also studied.The different animal studies realised confirmed that the BTV-8 infection has a moderate impact in goats from a clinical and haematological point of view. These studies led to obtain new information about BTV-8 impact: moderate impact on leucocytes counts; transplacental transmission of the virus when infection occurs in mid-pregnancy; detection of BTV-8 in bucks’ semen; direct, non vectorial transmission. The last 3 results had never been described in goats with BTV-8 before but had been encountered in sheep and cattle: it proves that, even if goats are susceptible to the infection but are less affected by the virus, most of feature of BTV-8 North European strain can also be encountered in this specie. However, these features have not been described in natural conditions, making impossible to conclude on their impact in the field.In a second part of this thesis, serological tool have been developed in order to study antigenic properties of viral proteins in goats. Recombinant proteins NS1, NS3, VP7 and VP2 were produced in baculovirus system, while NS2 was produced in E. coli system. These recombinant proteins were used to develop serological test in order to study antigenic properties and the kinetic of antibodies response against this 5 proteins after vaccination against and infection by BTV-8 in goats.In a first part, indirect ELISA NS1, NS2, NS3, VP7 and VP2 were developed, and the opportunity to develop DIVA ELISA test using NS and VP ELISA was evaluated. However, detection of antibodies against NS2 and NS3 in vaccinated animals, and the difficulties to detect antibodies against NS1 in infected animals led us to conclude that a DIVA ELISA test using non-structural proteins was difficult. Finally, it was possible to detect antibodies against VP2 in infected and vaccinated animals using our VP2 ELISA, suggesting a detection of antibodies specific of serotype by this test.In a second part, a multiplex Luminex test, using VP7 and VP2, was developed. This test has, for VP7 detection, a strong correlation with cELISA VP7, with an area under the curve of 0.987. Luminex VP7 performance is moderate when sera from goats having only one vaccine administration were tested. Concerning Luminex VP2, test performance are also excellent with an area under the curve of 0.978. When a prevalence of 0.5% was applied (prevalence that should be detected by serological screening in Europe), de predictive negative value was very high (99.99% for Luminex VP7; 99.95% for Luminex VP2). The Luminex test developed, with a very high PNV, can exclude with a high level of confidence the presence of BTV-8 in a free-area

Orientador: Karin Wether
Banca: Edviges Maristela Pituco
Banca: Liria Hiromi Okuda
Banca: Hélio José Montassier
Banca: Maria da Glória Buzinaro
Resumo: RESUMO - Língua azul (LA) é uma doença viral infecciosa que afeta ruminantes domésticos e selvagens e é transmitida por mosquitos vetores do gênero Culicoides (Diptera: Ceratopogonidae). É de notificação obrigatória segundo lista da OIE (Organização Mundial de Saúde Animal) e do Ministério da Agricultura, Pecuária e Abastecimento do Brasil. Os sinais clínicos e lesões em casos agudos de infecção pelo vírus da língua azul (VLA) são sutis ou inexistentes; e quando presentes são muito semelhantes com outras enfermidades hemorrágicas, havendo a necessidade de técnicas laboratoriais complementares para o diagnóstico definitivo dessa enfermidade. O presente trabalho teve como objetivo realizar um estudo epidemiológico da doença da língua azul nos cervídeos pertencentes ao Núcleo de Pesquisa e Conservação de Cervídeos - NUPECCE e nos ruminantes domésticos mantidos nas proximidades, utilizando técnicas sorológicas (ELISA competitivo em fase sólida e imunodifusão dupla em gel de ágar - IDGA) e moleculares (RT-qPCR), além da investigação da população de insetos hematófagos existentes no local. Para pesquisa do VLA e seus anticorpos foi utilizado sangue total e soro dos cervídeos brasileiros provenientes do criatório e dos outros ruminantes domésticos mantidos nas proximidades do criatório. Dos cervídeos que vieram a óbito também foram colhidos fragmentos de órgãos para diagnóstico molecular. Pela técnica de IDGA apresentaram anticorpos anti-vírus da LA: 47,43% (37/78) dos cervídeos, 9... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Bluetongue (BT) is an infectious viral disease that affects domestic and wild ruminants, transmitted by vectors, mosquitoes of the genus Culicoides (Diptera: Ceratopogonidae). The notification is mandatory according to the list of the OIE (World Organisation of Animal Health) and the Ministry of Agriculture, Livestock and Supply of Brazilian Government. Clinical signs and lesions in acute cases of infection are often subtle or nonexistent; and when present are very similar with others hemorrhagic diseases, it is necessary to use complementary techniques for the definitive diagnosis of the disease. This study aimed to perform an epidemiological study of BT disease in captive cervids using serological (solid phase competitive ELISA and double agar gel imunodifusion - AGID) and molecular techniques (RT-qPCR), in addition to the investigation of the population of hematophagous insects in the area. To investigate the Bluetongue virus (BTV) and their antibodies, whole blood and serum from Brazilian captive cervids and domestic ruminants located nearby were used. From cervids that died, fragments of organs were collected for molecular diagnosis. Using the AGID technique performed, the animals presented anti-BTV antibodies: 47.43% (37/78) of cervids, 90.9% (120/132) of cattle, 55.49% (96/173) of sheep and 19.56% (9/46) of goats. By the solid phase competitive ELISA were reagents: 38.46% (30/78) of cervids, 93.18% (123/132) of cattle, 60.69% (105/173) of sheep and 23.91% (11/46) of goats. Using RT-qPCR from the whole blood the positive results were: 10.14% (7/69) of cervids, 0.75% (1/132) of cattle, 10.65% (18/169) of sheep and 9.09% (4/44) of goats. From the 46 deer that died, RT-qPCR was also performed and 8.69% (4/46) animals were positive for BTV. Between 2015 and 2016, bloodsucking insects of the family Ceratopogonidae, Psychodidae and Simuliidae (Complete abstract click electronic access below)
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