African horsesickness virus (AHSV) belongs to the Orbivirus genus in the family Reoviridae. This non-enveloped virus consists of an outer capsid formed by two structural proteins, VP2 and VP5, and an inner core formed by structural proteins VP7 and VP3. Three additional structural proteins associated with viral replication, as well as ten dsRNA molecules responsible for replication, are found inside the core. VP7 is the smallest of the structural proteins and each monomer consist of two domains, a hydrophilic top and hydrophobic bottom domain. Upon expression of VP7, the protein spontaneously assembles into trimers. Recombinant expression of the core protein VP7 results in large hexagonal structures formed by a double layer of these VP7 trimers with the hydrophobic bottom domains on the inside and hydrophilic top domains on the outside. The use of these crystal structures as a general display system for the display of foreign peptides/epitopes is being investigated in our group. In this regard, sites for the insertion of foreign peptides/epitopes were constructed at amino acid positions 177, 144 and 200 of the top domain of the VP7 protein and the resultant proteins named vectors AHSV-9 VP7-177, AHSV-9 VP7-144 and AHSV-9 VP7-200. Various inserts ranging from the HIV-1 ELDKWA epitope and FMDV VP1 epitopes to the eGFP peptide were inserted and subsequently analysed for immunogenicity. Results showed that a significant immune response was only elicited if the soluble trimer component of a chimeric VP7 protein was used for inoculation purposes. The crystal particles initially investigated as a display system did not result in any immune response. These results emphasized the importance of protein solubility for eliciting a significant immune response. The importance of solubility prompted an investigation into the use of the Bluetongue virus (BTV) VP7 protein as a vaccine display system. This protein is inherently more soluble than AHSV VP7 and does not result in crystal hexagonal structures if recombinantly expressed. An insertion site analogous to that of the AHSV-9 VP7-177 vector, located at amino acid 177 within an RGD loop in the top domain of VP7 was constructed. This new BTV VP7 vector, BTV-10 VP7-169, was characterised with regard to solubility and the ability to form trimers. In order to investigate the effect on solubility and trimerisation, FMDV VP1 epitope and eGFP were inserted into the BTV-10 VP7-169 vector. Results showed that following the construction of the insertion site, the vector was largely insoluble compared to the AHSV VP7 vectors and that insertion of the abovementioned peptides/epitopes did not have a significant effect on solubility. Although trimers were present, the yield was low compared to that of the AHSV chimeric VP7 proteins. Methods of improving the solubility of the chimeric VP7 proteins were investigated by treatment with solubilisation agents, sarkosyl and L-arginine. The results indicated that a strong denaturant such as sarkosyl can solubilise the particulate component of all chimeric VP7 proteins whereas Larginine had limited effect. The effect of these agents on the folding of the proteins were evaluated using fluorescence, since the ability to fluoresce is regarded as an indicator of correct folding. A comparison of the different VP7-eGFP proteins treated with these solubilisation agents showed that the sarkosyl solubilised proteins were not necessarily correctly folded. These results combined with the previously performed solubility assays indicated that a large proportion of correctly folded chimeric VP7 proteins associate with the particulate fraction. Investigation showed that expression of a large amount of correctly folded chimeric proteins results in the aggregation of these proteins within the expressing host cell. Once harvested these proteins remain associated with the insoluble fraction but can be solubilised by arginine treatment, or in some cases mere resuspension in a low-salt buffer, and used for vaccination purposes. In conclusion, the comparative analyses of solubility and trimerisation for the display vectors indicated that AHSV-9 VP7-144 vector may be most suitable for the display of foreign epitopes/peptides as it consistently yielded the largest component of correctly folded proteins. Furthermore, considering that large amounts of correctly folded chimeric VP7 proteins occurred in the insoluble component of each the VP7 display proteins, this study emphasize that use of solubility assays alone does not provide adequate information regarding the potential of a display vector for vaccination purposes. / Dissertation (MSc)--University of Pretoria, 2013. / Genetics / unrestricted

This study was undertaken to investigate the structure-function relationship of VP6 protein of bluetongue virus (BTV) using molecular cloning techniques. VP6 is present in small quantities in BTV and its enzymatic activity and role in the viral replication cycle have not been studied. Since the availability of large amounts of purified VP6 is essential for the analysis of VP6, a BTV -11 S3 gene was cloned into a prokaryotic protein expression system. VP6 protein was expressed in large amounts and purified to near homogeneity. A series of C-terminal and internal deletion mutants of S3 gene was constructed and the truncated VP6 proteins were expressed and purified. The nucleic acid binding activities of the VP6 protein towards dsRNA, dsDNA, and ssRNA were confirmed and a new ssDNA binding activity was also determined. The binding activities of VP6 were concentration-dependent. The sites responsible for the binding activities were mapped using the truncated proteins and synthetic sequence-specific oligopeptides. Two domains of VP6 were responsible for the nucleic acid binding activities and have been mapped within 28 amino acids near the middle and 11 residues near the carboxyl terminus of VP6. The binding affinities of the middle domain of VP6 towards single-stranded and double-stranded nucleic acid were slightly different. Three synthetic oligopeptides corresponding to these domains exhibited concentration-dependent nucleic acid binding activities. Based on these results I suggest that synthetic oligopeptides might be useful to screen nucleic acid binding activities and domains responsible for these activities. Expressed VP6 was used to produce polyclonal and monoclonal antibodies. Oligoclonal antibodies were raised by synthetic oligopeptides. Ten epitopes of VP6 were mapped and characterized. The amino acid sequences and sizes of six linear epitopes identified by oligoclonal antibodies were determined, and their locations were mapped and confirmed by deletion mutant analyses. These linear epitopes were surface-accessible except one. Based on these results I suggest that synthetic sequence-specific oligopeptides could mimic major components of antigenic determinants. Four epitopes recognized by four monoclonal antibodies were mapped and characterized. Three determinants were surface-accessible and three were conformational epitopes. These four determinants were distinct and different from the six linear epitopes determined using oligoclonal antibodies.

Bluetongue virus (BTV) is transmitted by arthropod vectors and causes bluetongue disease with serious economic loss in many regions of the world. The replication mechanism of bluetongue virus is still not clear. To have a better understanding regarding the viral replication, the function of each individual protein has to be identified. This study used molecular biology techniques to investigate the function of the inner core protein VP4. The M1 genes of United States bluetongue virus serotypes-2, -10, -11, -13, and -17 were cloned and sequenced. The length of each of the five M1 genes is 1981 nucleotides. The coding region of the M1 gene, which encodes the VP4 protein, possesses an open reading frame with an initiation codon (ATG) at nucleotides #9-11 and a stop codon (TAA) at nucleotides #1941-1943. This open reading frame encodes a protein of 644 amino acid residues with a predicted molecular weight of about 75 kDa. A potential leucine zipper motif was detected near the carboxyl terminus of the deduced VP4 amino acid sequence. The phylogenetic analysis of bluetongue viruses using the sequences of these five cognate M1 genes is consistent with the results of previous phylogenetic studies. Serotypes-10, -11, -13, and -17 are closely related and serotype-2 is the most distantly related among the five US BTV serotypes. Heterologously expressed bluetongue virus VP4 protein was purified to near homogeneity. Six linear epitopes of VP4 were mapped at both termini and in the middle of the protein. By using enzyme-linked immunosorbent assay and peptide competition assay, six linear epitopes were found to be surface accessible. The VP4 protein was shown to be an oligomer by chemical cross-linking. VP4 protein was identified as a ssRNA-binding protein. The VP4 protein has binding activity towards both capped and non-capped ssRNA. RNA-binding activity was not specific to BTV ssRNA. A leucine-zipper motif of VP4 is not required for RNA-binding activity. One RNA-binding domain was mapped between amino acid residues #112-158 by a Northwestern assay and by deletion mutant analysis. Using sequence-specific synthetic peptides corresponding to VP4 in the arginine-and lysine-rich regions, four potential ssRNA-binding domains of VP4 protein were mapped.

In this study, the electrical properties of [Al/(Bi4Ti3O12+V2O5 ) /Ba(Zr0.1Ti0.9)O3/Silicon¡AMFIS ]structure using annealed-BZT films would be improved for the nonvolatile FeRAM device applications. The radio-frequency magnetron sputtering was used to deposit BTV ferroelectric films on Mo/Ti/SiO2/Si and BZT/Si substrates, respectively, and MFMIS and MFIS structures would also be fabricated. The various sputtering parameter effects of BTV films such as the oxygen concentrations, rf power and deposition time would be discussed. Besides, the electrical and physical properties of as-deposited BZT films for (BTV/BZT/Si¡AMFIS) structure would be improved by different rapidly thermal annealing temperature. The physical characteristics of BTV films and annealed-BZT films for various sputtering parameters were obtained from the XRD pattern and SEM morphology. Besides, the memory window, dielectric constant and leakage current of MFM and MFIS structure using BTV films and annealed-BZT films would be found from the HP4284A, RT66A and HP4156C. From the experimental result obtained, the maximum dielectric constant of BTV films for 40 % oxygen concentration and 130W power were 10.79. The leakage current density was about 10-5A/cm2, as the applied electrical field of 200 kV/cm. In addition, the coercive field (Ec) and remanent polarization (2Pr) were 750 kV/cm and 12 £gC/cm2 from the P-E curves, respectively. Finally, the maximum memory window and lower leakage current density of [Al/(Bi4Ti3O12+V2O5) /Ba(Zr0.1Ti0.9)O3/Silicon¡AMFIS) structure would be found and they were 25V and 10-8A/cm2, respectively.

Die Blauzungenkrankheit (Bluetongue, BT) ist eine weltweit verbreitete Infektionskrankheit der Wiederkäuer. Sie wird von einem Orbivirus, dem Bluetongue virus (BTV) ausgelöst, welches durch belebte Vektoren übertragen wird. Nach ursprünglich limitierter Verbreitung zwischen dem 35. südlichen und dem 40. nördlichen Breitengrad, hat sich das BT-Virus ausgehend vom Mittelmeerraum, auch in Teilen Südeuropas manifestiert. Im Spätsommer 2006 wurde BTV erstmals auch in Zentraleuropa nachgewiesen (Belgien, Niederlande und Deutschland) und breitete sich in den folgenden Jahren in weitere angrenzende europäische Länder aus. Der nachgewiesene Serotyp 8 (BTV-8) war bis dahin nur auf Gebiete südlich der Sahara, sowie Mittel- und Südamerika beschränkt. Die Bedeutung von Wildwiederkäuern für die Epidemiologie der Blauzungenkrankheit in Deutschland und in Mitteleuropa war bis zu diesem Zeitpunkt unbekannt. Sind die einheimischen Wildwiederkäuer (Rothirsch (Cervus elaphus), Damhirsch (Dama dama), Europäischer Mufflon (Ovis aries musimon) und Reh (Capreolus capreolus)) für das BTV empfänglich, so könnten sie eine Rolle als Erregerreservoir spielen und müssen somit auch in einer wirksamen Bekämpfungsstrategie berücksichtigt werden. Von April 2008 bis März 2011 wurde ein Monitoring zum BTV-Nachweis in Thüringen durchgeführt. Es sollte untersucht werden, ob die heimischen Wildwiederkäuerarten während des BTV-8-Seuchenzuges eine Rolle in der Epidemiologie der BT gespielt haben. Im Untersuchungszeitraum wurden 2535 Blut- und 3922 Muskelfleischproben von 4204 gehaltenen und erlegten Wildwiederkäuern (Rot-, Dam-, Muffel-, Reh- und Sikawild) auf BTV untersucht. Für Rot , Dam und Muffelwild lag der Anteil der untersuchten Proben an der Jagdstrecke im Jagdjahr 2008/09 mit 3,7 %, 4,1 % und 24,3 % jeweils am höchsten und nahm in den folgenden Jagdjahren auf 0,4 %, 0,3 % und 5,3 % ab. Beim Rehwild lag der Anteil in allen Jagdjahren zwischen 0,8 % und 1,7 %. Die Seroprävalenzen waren in den Jagdjahren 2008/09 bis 2010/11 rückläufig und lagen beim Muffelwild (n=354) zwischen 4,1 % und 1,3 %, beim Damwild (n=1721) zwischen 0,7 % und 0,0 % und beim Rotwild (n=283) zwischen 3,2 % und 0,0 %. Das bestätigt die Empfänglichkeit und Exposition dieser Spezies für BTV 8 in Thüringen. Bei dem untersuchten Rehwild wurden im gesamten Betrachtungszeitraum keine Antikörper gegen BTV im Blut (n=132) gefunden. In keiner der untersuchten 2535 Blutproben wurde BT-Virusgenom mittels RT-qPCR nachgewiesen. Die rückläufigen Seroprävalenzen bei Rot-, Dam- und Muffelwild und der fehlende Virusgenomnachweis deuten darauf hin, dass es keine Neuinfektionen und keine Persistenz des BTV-8 in der Wildwiederkäuerpopulation gab. Es ist anzunehmen, dass es im Untersuchungszeitraum in Thüringen zu keiner Zirkulation des BTV-8 in der Wildwiederkäuerpopulation, vergleichbar zu dem aus Spanien berichteten „Wildzyklus“, gekommen ist. Rehwild scheint weit weniger empfänglich für eine BTV-8 Infektion zu sein als andere Wildwiederkäuerarten, weshalb die Bedeutung bei der Verbreitung des BTV-8 vernachlässigbar ist. Zur frühestmöglichen Erkennung eines erneuten Auftretens von BTV Infektionen in Deutschland oder Mitteleuropa, zur Ausbruchsverfolgung, zur Abschätzung des Infektionsdruckes im Wildwiederkäuerbestand und zur Einschätzung, ob sich ein eigener Wildwiederkäuer Zyklus etabliert hat, ist neben dem Monitoring im Nutztierbereich auch eine Untersuchung von Wildtieren (Rot-, Dam- und Muffelwild) zu empfehlen. Hierbei sollten ganz gezielt insbesondere Tiere aus Gehegen einbezogen werden. Im Rahmen dieser Studie erwies sich EDTA Blut als geeignete Probenmatrix, welche den Antikörper- und Virusgenomnachweis von BTV in Wildwiederkäuern zulässt und leicht im Rahmen der Schlachttier- und Fleischuntersuchung gewonnen werden kann. / Bluetongue disease (Bluetongue, BT) is a globally widespread infectious disease of ruminants. The causative agent is the bluetongue virus (BTV), an Orbivirus that is transmitted by living vectors. Its distribution was originally limited to regions between the 35th southern and the 40th northern latitude, however the BT virus spread from the Mediterranean and manifested in southern parts of Europe. In late summer of 2006, BTV was detected in Central Europe (Belgium, Netherlands and Germany) for the first time and spread to other European countries in the following years. Before, the detected serotype 8 (BTV 8) was limited to areas south of the Sahara, as well as Central and South America. The importance of wild ruminants in the epidemiology of BT in Germany and Central Europe was unknown up to this point. If native wild ruminants (red deer (Cervus elaphus), fallow deer (Dama dama), mouflon (Ovis aries musimon) and roe deer (Capreolus capreolus)) are susceptible to BTV, they could play a role as a virus reservoir and must be considered in an effective control strategy. From April 2008 to March 2011, a monitoring for the detection of BTV was carried out in Thuringia. The role of indigenous wild ruminants in the epidemiology of BT during the BTV-8 epidemic was to be investigated. In the course of the study period 2535 blood and 3922 muscle samples from 4204 fenced and free ranging wild ruminants (red deer, fallow deer, mouflon, roe deer and sika) were examined for BTV. For red deer, fallow deer and mouflon, the proportion of the tested samples on the hunting bag in the season 2008/09 was 3.7 %, 4.1 % and 24.3 % and decreased in the following hunting seasons to 0.4 %, 0.3 % and 5.3 %, respectively. In the case of roe deer, the proportion was between 0.8 % and 1.7 % in all hunting seasons. From the hunting seasons 2008/09 to 2010/11, the detected seroprevalences in mouflons (n=354) decreased from 4.1 % to 1.3 %, in fallow deer (n=1721) from 0.7 % to 0.0 % and in red deer (n=283) from 3.2 % to 0.0 %. These findings show the susceptibility and the exposure of these species to BTV in Thuringia. No antibodies against BTV could be detected in blood samples of roe deer (n=132) over the entire period of observation. BT virus genome was not detected in any of the 2535 investigated blood samples by RT-qPCR. The declining seroprevalences in red deer, fallow deer and mouflon and the absence of viral genome indicate that there were no new infections and no persistence of BTV-8 in the wild ruminant population. It can be assumed that there was no circulation of BTV-8 in the wild ruminant population in Thuringia during the study period, which would have been comparable to the 'wild cycle' reported from Spain. Roe deer appears to be far less susceptible to BTV-8 infection than other wild ruminants, therefore its importance for the spread of BTV-8 is negligible. To detect the reoccurrence of BTV infections in Germany or Central Europe as soon as possible, for outbreak control, to estimate the risk of infection in the wild ruminant population and to assess if a wild ruminant cycle has been established, the investigation of wild ruminant species (red deer, fallow deer and mouflon), especially the sampling of farmed wild ruminants, is recommended in addition to monitoring programs in the livestock sector. The Data of this study shows that EDTA blood is a suitable sample matrix for both, antibody and genome detection of BTV in wild ruminants, which can be easily collected during the ante and post mortem inspection.

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.

<p>Вирус катаралне грознице оваца наноси велике штете сточарској производњи због чега су правовремено препознавање болести и брза дијагноза есенцијални предуслови за адекватну реакцију ветеринарске службе. Из тих разлога, предмет ове докторске дисертације су анализе патолошких и хистолошких промена код животиња, серолошких и молекуларних метода потребних за идентификацију и ближу карактеризацију узрочника што је од примарног значаја за ефективност предузетих мера у борби против катаралне грознице оваца.<br />Серолошки, током активног надзора, пргледано је 944 узорка крвног серума оваца и 953 узорка крвног серума говеда, а потом, током пасивног надзора, 114 узорака крвног серума говеда, 302 узорка крвног серума оваца и 22 узорка крвног серума коза. Коришћена је компетитивна имуноензимска ELISA метода, којом је доказано присуство специфичних антитела код говеда, оваца и коза. Из пуне крви серопозитивних јединки, ланчаном реакцијом полимеразе уз коришћење Pan-BTV rRT-PCR протокола заснованог на публикацији Toussiant и сар., (2007) је доказано присуство секвенце вирусне РНК. У случају позитивних резултата спроведена је и конвенционална PCR метода, употребом One step PCR кита, уз коришћење два пара прајмера за серотип BTV-4 чиме је доказано да је овај серотип узрочник обољења у Црној Гори. Секвенцирањем и филогенетском анализом изолата утврђено је да припада западном топотипу BTV-4 и да је сродан, готово идентичан са изолатима земаља из окружења.<br />Од 302 овце, којима је узета крв за серолошку анализу, уочене су каратеристичне макроскопске и микроскопске промене код свих угинулих од катаралне грознице оваца. Испитивањем је обухваћено 20 угинулих оваца које су претходно, током испитивања за живота, поред манифестне клиничке слике, имале позитивне резултате серолошких и молекуларних испитивања. Након угинућа, овце су обдуковане при чему су констатоване и сликане макроскопске промене те издвојена ткива за хистолошке анализе. Ткива су фиксирана у 10% пуферизованом неутралном формалину, а потом уклапљена у парафин. Парафински исечци дебљине 5 микрометара су бојени хематоксилин-еозин методом. На промењеним органима доминирају едем, хиперемија и крварења. Хистолошки се запажају интензивне хеморагичне инфилтрације свих промењених органа, а поред тога и местимични некротични процеси у срцу и језику као и периваскуларни едеми лимфних органа са израженом<br />лимфоцитном деплецијом у лимфним чворовима, слезини и тимусу.</p> / <p>Virus kataralne groznice ovaca nanosi velike štete stočarskoj proizvodnji zbog čega su pravovremeno prepoznavanje bolesti i brza dijagnoza esencijalni preduslovi za adekvatnu reakciju veterinarske službe. Iz tih razloga, predmet ove doktorske disertacije su analize patoloških i histoloških promena kod životinja, seroloških i molekularnih metoda potrebnih za identifikaciju i bližu karakterizaciju uzročnika što je od primarnog značaja za efektivnost preduzetih mera u borbi protiv kataralne groznice ovaca.<br />Serološki, tokom aktivnog nadzora, prgledano je 944 uzorka krvnog seruma ovaca i 953 uzorka krvnog seruma goveda, a potom, tokom pasivnog nadzora, 114 uzoraka krvnog seruma goveda, 302 uzorka krvnog seruma ovaca i 22 uzorka krvnog seruma koza. Korišćena je kompetitivna imunoenzimska ELISA metoda, kojom je dokazano prisustvo specifičnih antitela kod goveda, ovaca i koza. Iz pune krvi seropozitivnih jedinki, lančanom reakcijom polimeraze uz korišćenje Pan-BTV rRT-PCR protokola zasnovanog na publikaciji Toussiant i sar., (2007) je dokazano prisustvo sekvence virusne RNK. U slučaju pozitivnih rezultata sprovedena je i konvencionalna PCR metoda, upotrebom One step PCR kita, uz korišćenje dva para prajmera za serotip BTV-4 čime je dokazano da je ovaj serotip uzročnik oboljenja u Crnoj Gori. Sekvenciranjem i filogenetskom analizom izolata utvrđeno je da pripada zapadnom topotipu BTV-4 i da je srodan, gotovo identičan sa izolatima zemalja iz okruženja.<br />Od 302 ovce, kojima je uzeta krv za serološku analizu, uočene su karateristične makroskopske i mikroskopske promene kod svih uginulih od kataralne groznice ovaca. Ispitivanjem je obuhvaćeno 20 uginulih ovaca koje su prethodno, tokom ispitivanja za života, pored manifestne kliničke slike, imale pozitivne rezultate seroloških i molekularnih ispitivanja. Nakon uginuća, ovce su obdukovane pri čemu su konstatovane i slikane makroskopske promene te izdvojena tkiva za histološke analize. Tkiva su fiksirana u 10% puferizovanom neutralnom formalinu, a potom uklapljena u parafin. Parafinski isečci debljine 5 mikrometara su bojeni hematoksilin-eozin metodom. Na promenjenim organima dominiraju edem, hiperemija i krvarenja. Histološki se zapažaju intenzivne hemoragične infiltracije svih promenjenih organa, a pored toga i mestimični nekrotični procesi u srcu i jeziku kao i perivaskularni edemi limfnih organa sa izraženom<br />limfocitnom deplecijom u limfnim čvorovima, slezini i timusu.</p> / <p>The Bluetongue virus (BTV) incurs great damage to the production of livestock, due to which timely recognition of the resultant disease and rapid diagnosis are essential prerequisites for an adequate reaction by the veterinary service. For these reasons, the subject of this doctoral dissertation are the analyses of pathological and histological alterations in animals, the serological and molecular methods necessary for identifying and closely characterizing the causative agent which is of primary concern for the efficacy of the measures taken in the fight against bluetongue disease.<br />Serologically, during active surveillance, 944 samples of sheep blood serum as well as 953 samples of cattle blood serum were examined, followed by 114 samples of cattle, 302 samples of sheep and 22 samples of goat blood serum during passive surveillance. Competitive ELISA was utilized to detect the presence of specific antibodies in cattle, sheep and goats. The presence of viral RNA sequences was confirmed in whole blood samples of seropositive individuals using the Pan-BTV rRT-PCR protocol as described in Toussiant et al. (2007). In the case of positive results conventional PCR analysis was also performed, using the One Step PCR kit with two primer pairs specific to the BTV- serotype, which showed that this serotype is the cause of the disease in Montenegro. Sequencing and phylogenetic analysis of the isolate determined that it belongs to the western topotype of BTV-4 and that it is related and practically identical to the isolates from neighbouring countries.<br />This work describes the macroscopic and microscopic changes found in sheep which died due to affliction with bluetongue disease. The study investigated a sample of 20 sheep for which premortem analysis, apart from the manifested clinical features, had confirmed positive serologic and molecular test results. The animals were dissected postmortem during which macroscopic alterations were identified and imaged, followed by tissue isolation for histological analysis. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections of 5&mu;m thickness were cut and stained with haematoxylin/eosin. The predominant organ changes identified included edema, hyperaemia and bleeding. Histologically intensive hemorrhagic infiltrations of all affected organs were observed as well as sporadic necrotic processes in the heart and tongue accompanied by perivascular edemas of the lymphoid organs with pronounced lymphocyte depletion within the lymph nodes,<br />spleen and thymus.</p>

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.

O vírus da mancha das orquídeas ("Orchid fleck virus" - OFV), transmitido pelo ácaro Brevipalpus californicus, causa manchas cloróticas e necróticas em orquídeas de vários gêneros e foi relatado em diversos países. O diagnóstico de "orchid fleck", doença causada pelo OFV, tem sido feito através da análise dos sintomas, sorologia, observação de cortes ultrafinos de tecido infectado em microscópio eletrônico de transmissão ou RT-PCR. No entanto, apesar de testes moleculares serem freqüentemente mais eficientes e específicos que outros métodos, os "primers" disponíveis na literatura nem sempre detectam o vírus em baixas concentrações no tecido vegetal, ou amplificam regiões da planta sadia. Com base nas seqüências nucleotídicas da capa protéica viral depositadas no GenBank foram desenhados novos "primers", que amplificam um fragmento de 326 pb. Esses "primers" foram utilizados para a detecção do OFV por RT-PCR e para a marcação com digoxigenina de sondas para hibridização. A variabilidade de um fragmento do gene da capa protéica deste vírus foi estudada por polimorfismo conformacional de fita simples ("Single strand conformational polymorphism" - SSCP) e seqüenciamento de nucleotídeos. Quarenta e oito amostras de 18 gêneros de orquídeas foram coletadas no Brasil, Costa Rica e Austrália. As análises dos padrões de SSCP resultaram em seis haplótipos diferentes e em agrupamentos baseados na origem geográfica das amostras. Amostras representando cada um desses padrões foram seqüenciadas e comparadas com aquelas disponíveis no GenBank. A análise de SSCP provou ser eficiente para fornecer informações preliminares sobre a variabilidade do OFV. No entanto, apenas através do seqüenciamento de nucleotídeos das amostras foi possível determinar a real variabilidade das mesmas. / Orchid fleck virus (OFV), transmitted by the mite Brevipalpus californicus, causes chlorotic and necrotic ringspots in many orchid genera and was reported in several countries. The diagnosis of the Orchid fleck disease has been performed by symptomatology, transmission electon microscopy, serology or RT-PCR. Even though the molecular tests are usually more efficient and specific than other methods, the available primers did not always detect the OFV in low concentrations or sometimes amplified healthy plant samples. Based on the nucleotide sequences of the coat protein gene (cp) available in the GenBank, new primers were designed. These primers amplified a 326 pb specific OFV fragment and were used for RT-PCR and as hybridization probes. The variability of a fragment of the cp of this virus was investigated by "single strad conformational polymorphism (SSCP)" and nucleotide sequencing. Forty eight samples of 18 genera of orchids were collected from Brazil, Costa Rica and Australia. The SSCP analysis resulted in six different haplotypes and demonstrated a clustering in samples based on geographical origin. Samples representing the different SSCP patterns were sequenced and compared with those available in the GenBank. The SSCP analysis proved to be efficient to provide preliminary information about OFV variability. However, only through nucleotide sequencing it was possible to determine the actual variability amongst the samples.

O vírus da mancha das orquídeas (“Orchid fleck virus” - OFV), transmitido pelo ácaro Brevipalpus californicus, causa manchas cloróticas e necróticas em orquídeas de vários gêneros e foi relatado em diversos países. O diagnóstico de "orchid fleck", doença causada pelo OFV, tem sido feito através da análise dos sintomas, sorologia, observação de cortes ultrafinos de tecido infectado em microscópio eletrônico de transmissão ou RT-PCR. No entanto, apesar de testes moleculares serem freqüentemente mais eficientes e específicos que outros métodos, os "primers" disponíveis na literatura nem sempre detectam o vírus em baixas concentrações no tecido vegetal, ou amplificam regiões da planta sadia. Com base nas seqüências nucleotídicas da capa protéica viral depositadas no GenBank foram desenhados novos “primers”, que amplificam um fragmento de 326 pb. Esses “primers” foram utilizados para a detecção do OFV por RT-PCR e para a marcação com digoxigenina de sondas para hibridização. A variabilidade de um fragmento do gene da capa protéica deste vírus foi estudada por polimorfismo conformacional de fita simples ("Single strand conformational polymorphism" – SSCP) e seqüenciamento de nucleotídeos. Quarenta e oito amostras de 18 gêneros de orquídeas foram coletadas no Brasil, Costa Rica e Austrália. As análises dos padrões de SSCP resultaram em seis haplótipos diferentes e em agrupamentos baseados na origem geográfica das amostras. Amostras representando cada um desses padrões foram seqüenciadas e comparadas com aquelas disponíveis no GenBank. A análise de SSCP provou ser eficiente para fornecer informações preliminares sobre a variabilidade do OFV. No entanto, apenas através do seqüenciamento de nucleotídeos das amostras foi possível determinar a real variabilidade das mesmas. / Orchid fleck virus (OFV), transmitted by the mite Brevipalpus californicus, causes chlorotic and necrotic ringspots in many orchid genera and was reported in several countries. The diagnosis of the Orchid fleck disease has been performed by symptomatology, transmission electon microscopy, serology or RT-PCR. Even though the molecular tests are usually more efficient and specific than other methods, the available primers did not always detect the OFV in low concentrations or sometimes amplified healthy plant samples. Based on the nucleotide sequences of the coat protein gene (cp) available in the GenBank, new primers were designed. These primers amplified a 326 pb specific OFV fragment and were used for RT-PCR and as hybridization probes. The variability of a fragment of the cp of this virus was investigated by “single strad conformational polymorphism (SSCP)” and nucleotide sequencing. Forty eight samples of 18 genera of orchids were collected from Brazil, Costa Rica and Australia. The SSCP analysis resulted in six different haplotypes and demonstrated a clustering in samples based on geographical origin. Samples representing the different SSCP patterns were sequenced and compared with those available in the GenBank. The SSCP analysis proved to be efficient to provide preliminary information about OFV variability. However, only through nucleotide sequencing it was possible to determine the actual variability amongst the samples.