Dissertations / Theses on the topic 'Orbiviruses'

Polymerase chain reaction (PCR) sequencing of specific viral gene segments was used to investigate the phylogenetic relationships among the orbiviruses. Sequence comparisons of the bluetongue virus (BTV) RNA3 from different regions of the world (North America, South Africa, India, Indonesian, Malaysia, Australia and the Caribbean region) showed that geographic separation had resulted in significant divergence, consistent with the evolution of distinct viral populations. There were at least 3 topotypes (Gould, 1987); the Australasian, African - American and another topotype represented by BTV 15 isolated in Australia in 1986. The topotypes of BTV had RNA3 nucleotide sequences that differed by approximately 20 per cent. Analysis of BTV-specific gene segments from animal and insect specimens showed that bluetongue viruses had entered northern Australia from South East Asia, possibly by wind-borne vectors. Nucleotide sequence comparisons were used to show the close genetic relationship between BTV 2 (Ona-A strain) from Florida and BTV 12 from Jamaica, and to investigate the reassortment of BTV genome segments in nature. The mutation rates of the BTV RNA2 and RNA3 segments were estimated to be of the order of 10(-4) nucleotide changes/site/year, similar in magnitude to that reported for other RNA viruses.

African horse sickness (AHS) is a life threatening disease of equids caused by African horse sickness virus (AHSV), a member of the genus Orbivirus in the family Reoviridae. The virus is transmitted to horses by midges (Culicoides spp.) and the disease is most prevalent during the time of year, and in areas where the Culicoides spp. are most abundant, namely in late summer in the summer rainfall areas of the country. Whilst the clinical signs and presentation of the disease were well documented by Sir Arnold Theiler (1921), very little is known or documented about AHSV dynamics or the clinical pathological and serological responses of horses to natural infection with AHSV. This dissertation describes the history and current knowledge on AHS, and the methods and results of a prospective study on natural AHSV infection of horses, undertaken between 2009 and 2010 by the Equine Research Centre (ERC) at the University of Pretoria, Faculty of Veterinary Science, Onderstepoort. This study is the first documented study of its nature and included animals of various ages and therefore variable vaccination status. The objectives of the study were to describe the viral dynamics of AHSV infection in horses, to gain a better understanding of the clinical pathological and serological responses to natural AHS infection and to demonstrate early detection of AHS infection in horses under field conditions.
Dissertation (MSc)--University of Pretoria, 2010.
Veterinary Tropical Diseases
unrestricted

The aim of this study was to initiate a structural analysis of the capsid polypeptides from several serotypes of bluetongue virus in order to provide insight into the relatedness and possible origins of the different serotypes. Tryptic peptide mapping of ¹²⁵I-labelled group antigen by ion exchange chromatography was used to assess the structural relatedness of seven BTV serotypes from Southern Africa, North America and Australia. Each serotype had several tyrosine containing tryptic peptides which were unique, but approximately 35% of the peptides analyzed were found to be highly conserved between all 7 serotypes. BTV-20 appeared to be closely related to BTV-B and these two serotypes with BTV-4 and BTV-17 appeared to form a closely knit central cluster.
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Les invasions biologiques constituent une source de préoccupation majeure du fait des conséquences écologiques, économiques et sanitaires dont elles sont responsables. Déterminer et comprendre les facteurs sous-jacents au succès invasif des espèces envahissantes permet de prédire de nouvelles invasions et de mettre en place des stratégies de contrôle. Culicoides imicola est un vecteur majeur d’Orbivirus d’intérêt vétérinaire incluant le virus de la fièvre catarrhale ovine (FCO). Suite à l’émergence de la FCO dans le bassin méditerranéen, les populations de C. imicola ont été découvertes dans des territoires où elles étaient considérées comme absentes, caractérisant alors cette présence comme la résultante d’une expansion récente de l’espèce. Cette thèse décrit un ensemble de travaux visant à comprendre l’histoire de la colonisation du bassin méditerranéen par C. imicola. L’utilisation d’une approche multi-marqueurs combinant des analyses de génétique de populations, des inférences basées sur la méthode Approximate Bayesian Computation (ABC) et la simulation mathématique de la dispersion atmosphérique de l’espèce, a permis (i) de déterminer l’origine des populations installées au Maghreb et au Moyen Orient et de décrire les routes de colonisation et la chronologie de ces évènements, (ii) de définir les caractéristiques démographiques, évolutives et temporelles de la colonisation du sud de l’Europe et (iii) de caractériser les principaux facteurs expliquant le succès d’expansion géographique des populations installées. Les principaux résultats de cette thèse permettent de proposer des hypothèses pour expliquer le succès de l’installation des populations de C. imicola dans le bassin méditerranéen
Biological invasions are of major concern because of their environmental, economic and health consequences. Determining and understanding the factors underlying the invasion success of species allow predicting potential other biological invasions, and developing vector control strategies. Culicoides imicola is a major vector species of Orbivirus, including the bluetongue virus (BTV) which affects domestic ruminants. Following BT emergence in the Mediterranean basin, C. imicola populations were recorded in territories where the species was considered to be absent, and consequently was described as expanding its range expansion on a short period. This Phd work describes a set of studies aiming at understanding the colonization history of the Mediterranean basin by C. imicola. The use of a multi-loci approach combining population genetics analyses, Approximate Bayesian Computation (ABC) methods and mathematical simulations of the atmospheric dispersion of the species enabled to (i) determine the origin of the established populations in the Maghreb and the Middle-East and describe the routes of colonization and the chronology of such events, (ii) define the demographic, evolutionary and temporal characteristics of south-western Europe colonization and (iii) characterize the main factors explaining the successful range expansion of the established populations. The main results of this thesis allow suggesting hypotheses to explain the successful establishment of C. imicola populations in the Mediterranean basin

African horse sickness (AHS) is a non-contagious viral disease transmitted by arthropod vectors namely Culicoides (Avaritia) imicola Kieffer and Culicoides (Avaritia) bolitinos Meiswinkel endemic to sub-Saharan Africa. The disease affects all equine species, where its severity increases in horses foreign to Africa. Currently, vaccination is the only means of controlling the disease. African horse sickness poses a great risk to South African equines, not only due to the high mortality rate, but also due to the large scale restrictions implemented on the movement of horses for breeding or competition and on the international exportation of horses by the Department of Agriculture, Forestry and Fisheries (DAFF) and the World Organisation for Animal Health (OIE). A prospective study was undertaken between 2013 and 2014 by the Department of Veterinary Tropical Diseases and the Equine Research Centre (ERC), Faculty of Veterinary Science (FVS), University of Pretoria to determine the presence of Culicoides midges, the vector of the African horse sickness virus (AHSV) and the prevalence of disease at two equine establishments on the East Rand, Gauteng Province, South Africa. The two establishments differed extremely, when looking at infrastructure, management and vaccination protocols, this being the primary reason for their inclusion into the study. In the study, which started in December 2013, EDTA blood samples were collected and rectal temperatures recorded every 14 days over six months, from 28 Friesian / Lusitano and Appaloosa horses both resident in stables and open camps at the two establishments. The horses ranged in age from yearlings to four years. The EDTA samples were tested for the presence of AHSV and equine encephalosis virus (EEV) dsRNA by RT-qPCR (Quan et al. 2010). The clinical picture of the horses was recorded and rectal temperatures monitored for presentation of clinical cases caused by both viruses. It was shown that a total of nine (32%) cases of AHSV and five (18%) cases of EEV were identified in the 28 horses included in this study, where 89% of the horses had been vaccinated against AHS. As part of the risk assessment at each establishment it was essential to monitor the presence of the known vectors of AHSV. Therefore the conventional down-draught Onderstepoort black-light trap was operated overnight at various intervals throughout the study. The infection rate using RT-qPCR of the collected Culicoides midges was lower than the previous assumptions made by the owner and consulting veterinarians based on the mortality rate during the previous AHS season. Both AHSV and EEV were detected in separate single pools of collected midges. The low number of positive midges found in this study during 2014 could be explained by the occurrence of both diseases followed by the very active midge season of 2013. It is hypothesized that the prevalence of these diseases is dependent on seasonal patterns where a build-up of virus must reach a critical level after which spilling over will occur into associated equine populations (Venter et al. 2014). The present study also investigated the relationship between prevention strategies; primarily vaccination with a registered vaccine and the incidence of both diseases, where it shows that the prevalence of disease is dependent on the various prevention strategies implemented at each establishment. The presence of subclinical infection as seen in this study requires further investigation as it has a major impact on the movement of equines and the possible introduction of disease into naïve populations. The analysis of EE in the study, which is more prevalent than AHS, however does not cause severe disease, assists in the evaluation of wild-type virus transmission, as there is no commercial vaccine is available for EE. The presence of the virus assists in the study of the virus/host dynamics, natural maintenance cycles and the transmission of orbiviruses amongst South African horses. (Venter et al. 1999).
Dissertation (MSc)--University of Pretoria, 2015.
tm2016
Veterinary Tropical Diseases
MSc

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The objective of this work was to determine the epidemiological situation of Bluetongue virus (VLA) infection in sheep herds and to characterize the technological and sanitary aspects in the states of Alagoas (AL), Ceara (CE), Maranhao (MA) , Paraiba (PB), Piaui (PI), Rio Grande do Norte (RN) and Sergipe (SE). For this purpose, 226 farms visited and applied a questionnaire where blood of 2.692 apparently healthy sheep collected. In the present study, a mean prevalence in the northeast of 60,62% (137/226) of positive animal properties and 26,52% (714/2.692) of seropositive sheep. A mean VLA seroprevalence of 33,06% (162/490) in sheep and 82,93% (34/41) in the herds, with at least one positive animal observed in the state of CE. In AL, a seroprevalence of 2,55% was observed (7/275) from the animals and 21.74% (5/23) on farms. In the MA State, 64,13% (177/276) of the animals and 100% (23/23) of the herds were positive. Regarding the RN State, of the 33 properties surveyed, 12 (36,36%) had seroreagents and 16 of the animals studied (4,04%) were positive. In PB State, 2,82% (8/284) of the sheep were seroreagent and of the 24 farms analyzed, 2 (8,33%) presented a positive animal. In the PI, 76,98% (291/378) of the animals and all the properties (32/32) were seroreagent. In SE State, 894% (53/593) of the sheep were positive and 58,0% (29/50) in the sampled herds showed positivity. There was a significant association (p <0,05) regarding the sex, age and degree of kinship of the animals. The acquisition of animals for replacement of the herds (p <0,05) (odds ratio = 5,87, 95% CI = 2,06-16,76, p = 0,001) was identified as a risk factor for BTV in the evaluated states. In this study, the technological and sanitary aspects verified that the breeding system most adopted in the Northeast was the extensive (84,07%), with the activity directed mainly to meat (84,07%). It was verified that the animals were handled in sheepfolds on 68,14% of the properties. The predominance of joint rearing with goats and cattle was too observed. It was verified that 81,42% of the properties had some kind of technical assistance and that only 31,86% of the owners invest in their professional qualification of the employees. The most adopted practices on farms were cleaning the facilities (67,70%) and disposal of animals (80,09%). It was observed that 60,18% of the farms apply some type of vaccine. It was also observed that worm was the biggest problem faced in sheep farms (97,80%), and 96,02% of the producers used vermifugation as the control method. Other health problems frequently reported by the interviewees were Myiasis (93,83%), Caseous Lymphadenitis (89,87%), Pododermatitis (87,67%) and Bronchopneumonia (81,94%). The results obtained in this work indicate that the BTV is present in the sheep herds of the states analyzed and that the exploitation of these in the Northeast has low technological level. It was also verified that the control of the diseases of these states is deficient, which explains, in part, the low productivity of the herds
O escopo deste trabalho foi determinar a situação epidemiológica da infecção pelo vírus da Língua Azul (VLA) e caracterizar os aspectos tecnológicos e sanitários nos rebanhos ovinos dos estados de Alagoas (AL), Ceará (CE), Maranhão (MA), Paraíba (PB), Piauí (PI), Rio Grande do Norte (RN) e Sergipe (SE). Para tanto, foram visitadas e aplicados questionários em 226 propriedades, onde coletou-se o soro de 2.692 ovinos, aparentemente saudáveis. Observou-se uma prevalência média no nordeste de 60,62% (137/226) de propriedades com animais positivos e 26,52% (714/2692) de ovinos soropositivos. No CE houve uma soroprevalência média do VLA de 33,06% (162/490) nos ovinos, e de 82,93% (34/41) nas propriedades com pelo menos um animal positivo. Em AL, foi verificada uma soroprevalência de 2,55% (7/275) nos animais, e de 21,74% (5/23) nos criatórios. Já no MA, 64,13% (177/276) dos animais e 100% (23/23) dos rebanhos foram positivos. Em relação ao RN, das 33 propriedades pesquisadas, 12 (36,36%) tiveram sororreagentes e dos 396 animais estudados, 16 (4,04%) foram positivos. Na PB, 2,82% (8/284) dos ovinos foram sororreagentes e dos 24 rebanhos analisados, 2 (8,33%) apresentaram animal positivo. No PI, 76,98% (291/378) dos animais e todas as propriedades (32/32) foram sororeagentes. Em SE, 8,94% (53/593) dos ovinos foram positivos e nos rebanhos amostrados, 58% (29/50) apresentaram positividade. Houve associação significativa (p<0,05) quanto ao sexo, idade e grau de sangue dos animais. A aquisição (compra) de animais para reposição do plantel (p<0,05) (odds ratio = 5,87; IC 95% = 2,06-16,76; p=0,001) foi identificada como fator de risco para Língua Azul nos estados avaliados. No estudo dos aspectos tecnológicos e sanitários verificou-se que o sistema de criação mais adotado no Nordeste foi o extensivo (84,07%), com a atividade voltada majoritariamente para corte (84,07%), sendo verificado que os animais eram manejados em apriscos em 68,14% das propriedades. Foi observada a predominância de criação conjunta com caprinos e com bovinos. Verificou-se que 81,42% das propriedades possuíam algum tipo de assistência técnica e que apenas 31,86% dos proprietários investiam na qualificação profissional de seus funcionários. As práticas mais adotadas nas fazendas foram a limpeza das instalações (67,70%) e o descarte de animais (80,09%). Quanto à vacinação dos rebanhos, foi observado que 60,18% dos criatórios aplica algum tipo de vacina. Observou-se, também, que a verminose foi o maior problema enfrentado nos criatórios de ovinos (97,80%), sendo que 96,02% dos produtores utilizam como método de controle a vermifugação. Outros problemas sanitários frequentemente relatados pelos entrevistados foram a Miíase (93,83%), Linfadenite Caseosa (89,87%), Pododermatite (87,67%) e Broncopneumonia (81,94%). Os resultados obtidos neste trabalho indicam que o VLA encontra-se presente nos ovinos dos estados analisados e que a exploração destes no Nordeste possui baixo nível tecnológico. Verificou-se, também, que o controle das enfermidades destes estados é deficiente, o que explica, em parte, a baixa produtividade dos rebanhos
2018-03-19

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.

La maladie épizootique hémorragique (EHD) est une arbovirose inscrite sur la liste de l'OIE (organisation mondiale de la santé), qui affecte aussi bien les ruminants sauvages (essentiellement les cervidés) que les ruminants domestiques (les bovins) par la morsure de petits moucherons hématophages, les Culicoïdes (Diptera: Ceratopogonidae). Au début du 21ème siècle, cette maladie a émergé au Maghreb et au Moyen Orient (Turquie, Israël et Jordanie), générant de lourdes pertes économiques pour les éleveurs.L'émergence inattendue de l'EHDV en Tunisie en 2006, simultanément avec l’observation de foyers d'EHDV-6 au Maroc et en Algérie, a suscité une vive inquiétude dans le monde agricole tunisien, qui avait déjà eu à faire face à la survenue d’épizooties d'autres orbivirus : la peste équine africaine en 1960 et de la fièvre catarrhale ovine en 1999 donnant l’exemple qu'une maladie, réputée exotique, puisse émerger et devenir endémique (cas pour la fièvre catarrhale ovine).Ce projet a donc été réalisé pour améliorer les connaissances sur le virus de l’EHDV responsable de l'infection en Tunisie, en 2006, qui entrainait l’apparition de signes cliniques semblables à ceux du BTV.Dans un premier temps, nous avons disposé d'un échantillonnage de différents prélèvements de bovins, Culicoïdes de type imicola collectés en 2006 et conservés à la sérothéque de l'IRVT. Nous y avons donc recherché le génome de l’EHDV par RT-PCR afin de le caractériser et d'isoler le virus. Les résultats ont démontré que l'EHDV-6 était bien le sérotype qui circulait en Tunisie en 2006. Cette partie du travail a fait l'objet d'une publication dans Acta Veterinaria Hungarica.Le deuxième travail exposé s'est intéressé à la recherche d'une présence éventuelle de l’EHDV-6 en Tunisie avant et après l'épidémie de 2006 dans deux espèces animales : les bovins et les dromadaires. Pour cela, nous avons recherché les anticorps ou le génome viral à partir de prélèvements de terrain réalisés de 2000 à 2014 sur des bovins et des dromadaires. A l'issue de cette étude, nous avons détecté une possible circulation à très bas bruit de l'EHDV-6 chez les bovins. Les résultats obtenus sont discutés et ont fait l'objet d'une publication en cours de publication dans le journal Véterinaria Italiana.Concernant l’étude réalisée chez le dromadaire, espèce sensible au BTV, nous voulions évaluer son rôle de réservoir potentiel pour le virus de l’EHD. L’ensemble des résultats sérologiques et virologiques de notre étude nous indique que cette espèce ne semble pas jouer un rôle dans l’épidémiologie de l’EHD.Enfin, parallèlement à ces recherches sur le virus de l’EHD, nous avons mené une enquête sur la présence du virus de la Bluetongue à partir des échantillons de dromadaires et de bovins tunisiens. Les résultats ont été discutés. L'ensemble de ces études contribue à une meilleure connaissance de l'EHDV-6 présent en Tunisie, et permet de rendre compte des espèces potentiellement réservoirs. Certains travaux présentés pourraient être poursuivis pour évaluer le rôle du dromadaire comme réservoir d’Orbivirus et mieux déterminer les espèces de l'inventaire faunique des Culicoïdes impliquées dans la transmission des Orbivirus
The epizootic hemorrhagic disease (EHD) is an arthropod-borne virus that is on the OIE’s list (World Animal Health Organisation, formerly Office international des épizooties), this disease is mainly transmitted to wild (mainly deer) as well as domestic (primarily cattle) ruminants, by the bites of minute size midges, the culicoides (Diptera: Ceratopogonidae) also known as biting midges. In the beginning of the 21st century, EHD was emerged in Maghreb (North Africa) and in the Middle East (Turkey, Israel, Jordan), causing severe losses for the farmers and ranchers.The unexpected emergency of EHDV in Tunisia in 2006, simultaneously with the observation of EHDV-6 cases in Morocco and Algeria, has aroused great concern in the Tunisian agricultural sector, which had already to face the occurrence of other animal diseases orbivirus: African horse sickness in 1966 and bluetongue in 1999 giving examples of the possibility that deemed exotic disease could emerge and become endemic (case bluetongue).This project was carried out to raise the knowledge on the EHDV virus causing the infection in Tunisia in 2006, which led to the appearance of clinical signs similar to those of BTV.First, we prepared a sampling of various samples of cattle, Culicoides type imicola collected in 2006 and stored at the serum bank of IRVT. So, we searched therefore the genome of the EHDV by RT-PCR in order to characterize and isolate the virus. Results showed that EHDV-6 was actually the serotype circulating in Tunisia in 2006. This part of the job was published in Acta Veterinaria Hungarica.The second working paper concerned the potential presence of the EHDV-6 in Tunisia before and after the epidemic of 2006 in two animal species: cattle and camels. For this we looked for antibodies or viral genome from field samples collected from 2000 to 2014 cattle and camels. Following this study, we detected a possible circulation of EHDV-6 at a very low level of intensity among the cattle. The found results were discussed and made the subject of a publication to be in the newspaper Veterinaria iItaliana.Regarding the study on the dromedary species sensitive to BTV, we wanted to examine its potential role as a reservoir species for EHD virus. All serological and virological results of our study indicate that this species does not seem to play a role in the epidemiology of EHD.Finally, alongside these researches on EHD virus, we have investigated the presence of Bluetongue Virus in Tunisian samples from camels and cattle. The results were discussed.All these studies contribute to a better knowledge of EHDV-6 present in Tunisia and allows taking into account some species that are potentially reservoir. Some presented researches could be pursued to assess the role of the camel as a reservoir for Orbivirus and better identify species of the faunal inventory of Culicoides involved in transmission of orbivirus

Les zoonoses constituent plus des deux tiers des pathologies virales qui concernent l’homme. Le développement et la démocratisation des outils de métagénomique en font de bons outils d’inventaire et de surveillance de virus potentiellement émergents.Dans un premier temps j’ai développé et validé un protocole expérimental de purification des viromes à ARN qui permettait le maintien de l’infectivité des particules virales. Ce protocole a ensuite été appliqué pour caractériser les communautés virales d’arthropodes hématophages et de prélèvements de faune sauvage. J’ai par la suite réalisé l’inventaire des communautés virales de viande de singe fumée illégalement importée en France et confisquée par les douanes, qui a révélé la présence de nombreux bactériophages, dont certains pourraient infecter des bactéries potentiellement pathogènes pour l’homme.Enfin j’ai caractérisé les communautés virales de culicoïdes collectés au Sénégal, ce qui a permis de mettre en évidence la présence de nombreux virus géants à ADN infectant les amibes. Le séquençage des viromes à ARN a quant à lui révélé la présence d'un certain nombre d'arbovirus qui pourraient constituer un risque d’émergence pour la santé humaine. Du fait de nombreux facteurs intrinsèques et extérieurs à l’agent infectieux, la prédiction des futures émergences de virus zoonotiques est très compliquée voire utopique, mais elle reste un challenge crucial et d’actualité. La stratégie de réalisation d’inventaires des communautés virales présentes dans les différents acteurs des cycles de transmission zoonotique est un premier pas indispensable dans la connaissance des risques potentiels d’émergence en population humaine
Zoonoses are responsible of more than two thirds of human viral infections. The development of high-throughput sequencing tools and their application in metagenomics allow inventorying the viral communities of various reservoirs in order to detect the emergence of viruses before their infection to humans. In this context, I characterized the viral communities of simian bushmeat illegally imported into France and of Culicoides biting midges, recognized vectors of several viruses of human and veterinary medicine importance. I have first developed a protocol for the purification of RNA viromes which allowed maintaining the infectivity of viral particles. This protocol was subsequently applied to characterize viral communities of bloodsucking arthropods and wildlife samples. In a second part I realized the inventory of viral communities of smoked simian bushmeat illegally imported into France and confiscated by the French customs. This study revealed the presence of a wide diversity of bacteriophages, in which some of them could infect bacteria potentially pathogenic for humans.Finally I characterized the viral communities of Culicoides biting midges collected in Senegal, which revealed the presence of sequences related to several giant DNA viruses infecting amoeba. Sequencing of the RNA virome revealed the presence of several arboviruses that could constitute a risk of emergence of zoonoses for humans.The prediction of future emerging zoonotic viruses is very difficult, if not impossible. However the characterization of viral communities present in the different actors of zoonotic transmission cycle is a first step to evaluate potential risks of transmission to humans

Les élevages bovins de La Réunion connaissent régulièrement des foyers de « bavites », appellation locale des maladies causées par les virus de la fièvre catarrhale ovine (bluetongue virus, BTV) et de la maladie hémorragique épizootique (epizootic hemorragic disease virus, EHDV), deux orbivirus transmis par des moucherons hématophages du genre Culicoides (Diptera : Ceratopogonidae). Plusieurs sérotypes de ces deux orbivirus circulent sur l’île et sont responsables de pertes économiques et de dégradations de l’état sanitaire du cheptel bovin. Parmi les 5 espèces de Culicoides décrites à La Réunion, Culicoides imicola, Culicoides bolitinos, Culicoides enderleini, Culicoides grahamii, et Culicoides kibatiensis, au moins les deux premières sont vectrices du BTV et de l’EHDV aux ruminants. Dans ce contexte, il paraît important de caractériser l’écologie et de préciser le rôle vectoriel des Culicoides à La Réunion. Ce travail vise à i) déterminer les facteurs environnementaux et climatiques qui gouvernent la présence et l’abondance de chaque espèce de Culicoides dans l’île ; ii) modéliser leurs dynamiques temporelles et spatio-temporelles ; iii) identifier les espèces potentiellement impliquées dans la transmission des deux orbivirus et caractériser la circulation de ces deux virus dans les populations locales de Culicoides. Un suivi longitudinal reposant sur des collectes bimensuelles de moucherons réalisées dans 11 sites durant 26 mois a permis de caractériser puis d’analyser avec des modèles statistiques de haie les dynamiques temporelles des différentes espèces de Culicoides afin d’identifier les paramètres environnementaux et climatiques qui les gouvernent. Ces modèles de dynamique ont ensuite été spatialisés en utilisant le logiciel Ocelet et validés en s’appuyant sur les données issues d’une large campagne de captures dans 101 sites répartis sur toute l’île. Enfin, la recherche de BTV et d’EHDV, effectuée par PCR (réaction en chaîne par polymérase) dans 1500 pools monospécifiques de Culicoides collectés lors du suivi longitudinal, a permis de détecter chacun des deux orbivirus chez 4 des 5 espèces de Culicoides : le BTV a été détecté chez toutes les espèces sauf C. grahamii et l’EHDV chez toutes les espèces sauf C. enderleini. Les travaux de cette thèse ont donc permis d’approfondir les connaissances sur la composante vectorielle de la fièvre catarrhale ovine et de la maladie hémorragique épizootique à La Réunion, et, en l’absence de méthodes de prévention comme la vaccination et de stratégies de contrôle des vecteurs, de développer des approches permettant de préciser le risque de transmission de ces deux virus
Cattle farms in Reunion Island regularly experience outbreaks of "bavites", a local term designing diseases due to epizootic haemorrhagic disease virus (EHDV) and bluetongue virus (BTV), two orbiviruses transmitted by hematophagous midges of the genus Culicoides (Diptera: Ceratopogonidae). Several serotypes of these two orbiviruses circulate in Reunion Island and are responsible for economic losses and deterioration in the health status of bovine livestock. Among the 5 species of Culicoides recorded in Reunion Island, Culicoides imicola, Culicoides bolitinos, Culicoides enderleini, Culicoides grahamii, and Culicoides kibatiensis, at least the first two are vectors of BTV and EHDV to ruminants. In this context, it seems important to characterize the ecology and to specify the vector role of Culicoides in Reunion Island. The aims of this work are to i) determine the environmental and climatic factors that govern the presence and abundance of each Culicoides species on the island; ii) model their temporal and spatio-temporal dynamics; iii) identify the species potentially involved in the transmission of the two orbiviruses and characterize the circulation of these two viruses in local Culicoides populations. A longitudinal survey consisting in bimonthly collections of midges in 11 sites during 26 months enabled characterizing and modelling the temporal dynamics of the different Culicoides species using hurdle statistical models in order to identify environmental and climatic drivers of their dynamics. These dynamic models were then spatialized using Ocelet spatial dynamics software and validated using data originating from a large trapping campaign carried out in 101 sites throughout the island. Finally, the screening of 1500 monospecific pools of Culicoides for EHDV and BTV by polymerase chain reaction enabled detecting each virus in 4 of the 5 species of Culicoides: BTV was detected in all species except C. grahamii and EHDV in all species except C. enderleini. The work carried out during this thesis therefore contributed to improve knowledge of the vector ecology in relation to EHDV and BTV transmission in Reunion Island and, in the absence of prevention methods such as vaccination and vector control strategies, to develop approaches to specify the risk of transmission of these two viruses

African horsesickness (AHS), caused by the African horsesickness virus (AHSV) (Coetzer&Erasmus, 1994), has 10 segments of double stranded RNA (Verwoerd et al., 1970). These encode seven structural proteins, namely VP1, VP2, VP3, VP4, VP5, VP6 and VP7 (Verwoerd et al., 1972; Huismans&Van Dijk, 1990) as well as four nonstructural proteins (NS1, NS2, NS3 and NS3A) (Van Dijk&Huismans, 1988). NS3 is cytotoxic when expressed in insect cells causing membrane permeabilisation and cell death (Van Staden et al., 1995). This characteristic was postulated to be associated with the two hydrophobic domains (Van Staden et al., 1998; Van Niekerk et al., 2001(a)). Although NS3 has the same basic structure and function in all orbiviruses it differs greatly on nucleic acid sequence level as well as in the degree of variation that is found within the protein (Sailleau et al., 1997; Huismans et al., 2004). Therefore the main aim of this study was to identify which regions or amino acids remain conserved and might therefore play a role in the structure or function of NS3. The focus was mainly on the two hydrophobic domains as well as the spacer region between the hydrophobic domains. The sequence variation analyses between AHSV, BTV and EHDV NS3 revealed that there is a conserved threonine-serine motif in HD1, a conserved aspartic acid at the beginning of the spacer region as well as a conserved asparagine in HD2. The conservation of these residues between the different serogroups indicates that they might have some functional or structural role in the NS3 protein. NS3 protein sequences in the AHSV ã phylogenetic cluster has three extra amino acids in the spacer region at aa 149-151. There is also a significant difference in the spacer region length between EEV NS3 and AHSV NS3, which might have an effect on the topology of the respective proteins. In the spacer region of AHSV NS3, three regions were targeted for mutation analyses: substitution of the conserved isoleucine (aa 141) with a neutral amino acid, deletion of three amino acids (aa 149-151), and insertion of 15 additional amino acids. Three mutants were generated and assayed: the IÄS mutant, the KGDdel mutant and the 15 aa insert mutant. The cytotoxicity of the IÄS and KGD deletion mutants was exactly the same as the native NS3 protein, while the 15 aa insertion resulted in a slightly decreased cytotoxicity. This was the same as the EEV NS3 cytotoxicity level in insect cells. The fluorescent localisation and subcellular fractionation studies showed that both the IÄS mutation and 15 aa insertion affected the localisation of NS3 within the cell and also increased the solubility of the protein. This indicated that even though the membrane localisation of NS3 was disrupted it still had a cytotoxic role in the cell. It could be that wild type NS3 has both a membrane associated and a soluble fraction within cells, and that the soluble fraction is indeed responsible for the observed cytotoxicity and not the membrane fraction as assumed previously. It is therefore postulated that NS3 might have different domains that have various roles within the cell.
Dissertation (MSc)--University of Pretoria, 2009.
Genetics
unrestricted

African horse sickness virus (AHSV) affects equine populations around the world. It is the cause of a high rate of morbidity and associated large economic losses in affected regions. The virus is a segmented double stranded RNA virus and a member of Orbivirus genus in the Reoviridae family. The prototype member of the orbiviruses is bluetongue virus (STY) and other members include Chuzan virus and St. Croix River virus. These viruses are all characterized by a genome of ten dsRNA segments that encode at least ten different proteins. Three of the minor core proteins are found within the core of BTV. These are all associated with the RNA transcription complex and the enzymatic activities with which they are associated include an RNA polymerase (VP1), an RNA capping enzyme (VP4) and an RNA helicase (VP6). Genes homologous to the BTV genes that encode these proteins are found in all members of the Orbivirus genus. The aim of this thesis is to characterize VP4 of AHSV, the capping enzyme candidate, and to compare it to other orbivirus capping enzymes. Possible functional motifs and regions of importance within the orbivirus capping enzymes will be identified. The gene will also be expressed and used to perform assays to characterize the different enzymatic activities of VP4. The VP4 cDNA of AHSV serotype 3 was cloned and sequenced. From the full-length verified nucleotide sequence an open reading frame was identified and used to predict the amino acid sequence. These were compared to other orbivirus species including STY, Chuzan virus and St. Croix River virus. These alignments identified a number of highly conserved regions, consisting of four or more amino acids conserved between all the sequences analyzed. A fibronectin type 3-like motif, containing 12 conserved amino acids, was identified which could be responsible for protein binding. This motif contains 12 conserved amino acids making it a good candidate for a functional motif. Conservation does not, however, always predict regions of importance. In BTV a lysine-containing motif was identified to be responsible for GMP binding. This region is not conserved between the different viruses. AHSV has a motif containing a lysine residue similar to the motif identified in rotavirus and reovirus. Two other motifs described in BTV were also not conserved in the other viruses. One of them, a leucine zipper, was shown to dimerize BTV VP4. Phylogenetically, AHSV and Chuzan virus are the most closely related while BTV is more distant and St. Croix River virus forms a distinct out-group when the different VP4 sequences are compared. AHSV-3 VP4 was expressed as a histidine-tagged protein in the baculovirus expression system. Not unexpectedly, the protein was found to be insoluble, similar to BTV VP4 produced by means of the same system. However, whereas BTV VP4 could be solubilized by the addition of salt the AHSV VP4 remained insoluble at high salt concentrations. Several adjustments were made. Cells were lysed in a high salt buffer, the pH of the buffers was adjusted and sucrose cushions were used but none of the methods was found to improve the yield of soluble VP4 significantly. However, the pellet containing VP4 was relatively empty of contaminating protein and, therefore, a number of enzymatic assays were performed with the pellet. Assays for inorganic phosphatase and nucleotide phosphatase were performed. Strikingly, both assays indicated the presence of active phosphatases in the WT and VP4 pellets. Also, an assay was performed for guanylyltransferase activity but no activity was observed for this assay. The sequence data therefore points to VP4 as the probable capping enzyme although it may have a different structural complex. The failure to produce a reliable source of soluble purified AHSV VP4 made it impossible to provide evidence to confirm the associated enzymatic activities.
Dissertation (MSc(Genetics))--University of Pretoria, 2005.
Genetics
unrestricted