Viral protein seven (VP7) is a major core protein and a group-reactive antigen that can be used for the diagnosis of bluetongue virus. VP7 gene of bluetongue virus serotype 4 was expressed in E. coli. Using phage display technology, anti-VP7st4 scFvs were selected from a chicken scFv library (Nkuku®) following different panning strategies. Polyclonal phage ELISA showed that VP7st4-specific scFvs were enriched after three rounds of panning. Six different scFvs (A1, H2, TA8, TC9, TD12 and SA12) were identified by sequence analysis. Stability of these scFvs was determined by incubation at different temperatures and after several freeze/thaw cycles. The scFvs were also tested in an inhibition ELISA. Inhibition with an anti-bluetongue virus guinea pig serum resulted in a 30% decrease in ELISA signal of A1. No inhibition was obtained with the rest of the scFvs when guinea pig and sheep serum were used. An anti-bluetongue virus chicken IgY inhibited the scFvs by 50% to 86%. A fragmented-gene library displaying peptides of VP7st4 was constructed. The library was subjected to three rounds of affinity selection against the anti-VP7st4 scFvs. Enrichment of clones specific to each scFv was observed. The clones were identified by sequence analyses. The regions on VP7st4 to which the scFvs bind could not be identified since no duplicate clones were selected. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

African horsesickness is an infectious but non-contagious disease caused by an orbivirus belonging to the Reoviridae family. The disease is classified as notifiable by the OlE because of the potential severe economic consequences that can result from outbreaks. Bluetongue, an arthropod-transmitted disease of wild and domestic ruminants, is caused by the bluetongue virus, which is the prototype species of the genus Orbivirus. Bluetongue is also a notifiable disease because it can spread very rapidly in naive populations of susceptible livestock. Strict restrictions have been issued for the trade in animals and animal products from infected areas. In the present study, a duplex one-step real-time RT-PCR using the fluorogenic dye SYBR® Green I was developed for the specific detection and identification of AHSV and BTV in one reaction, using melting temperatures (Tm) to discriminate between the viruses. Two primers pairs were designed to bind to areas of homology within genome segment 7 (VP7) of AHSV and genome segment 5 (NS1) of BTV respectively. The duplex real-time RT -PCR based test utilizes single tube RT -PCR amplification in which AHSV and BTV primers were used simultaneously. The RT-PCR primers amplified 232 bp of genome segment 7 from all nine serotypes of AHSV and 79 bp of genome segment 5 from all 22 BTV serotypes that were tested. When both viruses were present, two melting peaks were simultaneously generated at 76.30°C and 80.04°C representative of BTV and AHSV amplification products respectively. Serogroup-specific products were amplified from dsRNA of field isolates of AHSV and BTV. dsRNA from EHDV and EEV failed to demonstrate either the 232 bp specific AHSV PCR product or the 79 bp specific BTV product. These results indicate that the duplex real-time RT-PCR could be a useful technique for detection of AHSV and BTV from isolated viral dsRNA. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

Vector-borne diseases (VBDs) cause significant harm to humans, plants, and animals worldwide. For instance, VBDs are very difficult to manage, as they are governed by complex interactions. VBD transmission depends on the pathogen itself, vector-host movement, and environmental conditions. Mosquito-borne diseases are a perfect example of how all these factors contribute to changes in VBD dynamics. Although vectors are highly sensitive to climate, modeling studies tend to ignore climate effects. Here, I am interested in the arthropod small vectors that are sensitive to climate factors such as temperature, precipitation, and drought. In particular, I am looking at the effect of temperature on vector traits for two VBDs, namely, dengue, caused by a virus that infects humans and bluetongue disease, caused by a virus that infects ruminants. First, I collect data on mosquito traits' response to temperature changes, this includes adult traits as well as juvenile traits. Next, I use these traits to model mosquito density, and then I incorporate the density into our mathematical models to investigate the effect it has on the basic reproductive ratio R0, a measure of how contagious the disease is. I use R0 to determine disease risk. For dengue, my results show that using mosquito life stage traits response to temperature improves our vector density approximation and disease risk estimates. For bluetongue, I use midge traits response to temperature to show that the suitable temperature for bluetongue risk is between 21.5 °C and 30.7 °C. These results can inform future control and prevention strategies. / Doctor of Philosophy / Infectious diseases are a type of illness that occurs when microorganisms spread between hosts. Some infectious diseases are directly transmitted and some require indirect transmission such as vector-borne diseases (VBDs). Each VBD requires the presence of a vector for the disease to be transmitted. For example, dengue that puts 40% of the world population at risk, requires mosquitoes to transmit the disease between humans. My research aims to investigate how the main climate factor, temperature, influences the spread of VBDs. I develop mathematical and statistical models that explain the effect of temperature on vector traits of a mosquito-borne disease (dengue) and a midge-borne disease (bluetongue) and investigate modeling formulas to improve our estimates for dengue mosquito densities. My results can be used to inform future prevention and control strategies.

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.

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) / Doutor

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

Submitted by ALINE EYKO KAWANAMI null (aline.kawanami@gmail.com) on 2017-01-12T01:13:37Z No. of bitstreams: 1 Tese_ Aline Kawanami.pdf: 4217654 bytes, checksum: 17280c9aecebf3ab6853cdc956889fb3 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-12T18:53:10Z (GMT) No. of bitstreams: 1 kawanami_ae_dr_jabo.pdf: 4217654 bytes, checksum: 17280c9aecebf3ab6853cdc956889fb3 (MD5) / Made available in DSpace on 2017-01-12T18:53:10Z (GMT). No. of bitstreams: 1 kawanami_ae_dr_jabo.pdf: 4217654 bytes, checksum: 17280c9aecebf3ab6853cdc956889fb3 (MD5) Previous issue date: 2016-12-09 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / 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, 90,9% (120/132) dos bovinos, 55,49% (96/173) dos ovinos e 19,56% (9/46) dos caprinos. Pelo ELISA competitivo em fase sólida foram reagentes: 38,46% (30/78) dos cervídeos, 93,18% (123/132) dos bovinos, 60,69% (105/173) dos ovinos e 23,91% (11/46) dos caprinos. Pela técnica de RT-qPCR realizada a partir do concentrado de hemácias dos animais foram positivos: 10,14% (7/69) dos cervídeos, 0,75% (1/132) dos bovinos, 10,65% (18/169) dos ovinos e 9,09% (4/44) dos caprinos. Dos 46 cervídeos que vieram a óbito foi realizado também RT-qPCR de órgãos e 8,69% dos animais (4/46) foram positivos para VLA. Entre 2015 e 2016 foram capturados em armadilhas luminosas insetos hematófagos da família Ceratopogonidae, Psychodidae e Simuliidae, sendo negativos para vírus da LA por RT-qPCR. Quanto aos sorotipos circulantes na região, acometendo os animais, sua identificação ainda está sendo conduzida. Conclui-se que o VLA ocorre na região do município de Jaboticabal, uma vez que os animais estudados apresentaram alta soroprevalência e também foi possível detectar o VLA, tanto em ruminantes domésticos e selvagens saudáveis, quanto em cervídeos que vieram a óbito. Foi possível identificar a presença de mosquitos da família Ceratopogonidae, família a qual pertence o Culicoides, que é conhecidamente o vetor da LA. / 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 were trapped in luminous traps, being negative for BTV by RT-qPCR. The identification of circulating serotypes in the region that affects these animals is still being conducted. It is concluded that the VLA occurs in the region of Jaboticabal county, since the animals studied showed high seroprevalence and it was also possible to detect the VLA in healthy domestic and wild ruminants and in cervids that died. It was possible to identify the presence of mosquitoes of the Ceratopogonidae family, which belongs to Culicoides, which is known as the vector of BT. / FAPESP: 2015/22851-8 / CNPq: 140626/2013-1

Submitted by (edna.saturno@ufrpe.br) on 2016-10-11T16:07:20Z No. of bitstreams: 1 Iagmar Oliveira da Mota.pdf: 349531 bytes, checksum: c6a35db709ead17915cddfd33e74214e (MD5) / Made available in DSpace on 2016-10-11T16:07:20Z (GMT). No. of bitstreams: 1 Iagmar Oliveira da Mota.pdf: 349531 bytes, checksum: c6a35db709ead17915cddfd33e74214e (MD5) Previous issue date: 2009-02-11 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Bluetongue is a non-contagious, infectious disease of ruminants, caused by the Bluetongue virus (BTV), transmitted by insects of the Culicoides genus. The gravity of the clinical signs of the Bluetongue varies according to the species, being the ovine the most severely affected. Serological survey carried out in Brazil indicate that the BT virus is widely diffused among bovine and bubaline; in caprine and ovine, there are few reports of serological survey and two outbreaks with clinical cases. Most publications discuss the epidemiology of the BT in temperate areas that suffer periodic outbreaks. This work was conducted with the objective of estimating the prevalence of antibodies against the BTV in caprine and ovine in the semi-arid region in the State of Pernambuco and to evaluate, preliminarily, the conditions for the maintenance of the Culicoides in semi-arid tropical environments. The serological survey was conducted by probabilistic sampling in two mesoregions of the State of Pernambuco (Semi-arid of Pernambuco and the São Francisco Semi-arid), where sampleswere collected from 41 caprine (n=410) and 40 (n=400) herds. Besides this, information was acquired about the main climate variables (pluviometric precipitation, maximum and minimum temperatures) which interfere in the dynamics of the population and competence of Culicoides to transmit the BTV. Seropositive animals to immunodiffusion in agar gel (IDGA) for the BT were found in 24.39% (11.25≤p≤37.54) of the caprine herds and in 27.5% (13.66≤p≤41.34) of ovines distributed in the mesoregions of Semi-arid region of Pernambuco, which presented 4.84% (2.53≤p≤7.47) of caprine and 4.14% (1.85≤p≤6.43) of seropositive ovine and of the São Francisco of Pernambuco area, 1.00% (0.00≤p≤2.95) of caprine and 4.55% (0.65≤p≤8.44) of seropositive ovine. The estimated prevalence was of 3.90% (2.03≤p≤5.78) and 4,25% (2.27≤p≤6.23) for caprine and ovine, respectively. It was observed that among caprine, positive results were registered in 4.18% (1.87≤p≤6.50) of the sources, 4.88% (0≤p≤11.47) of the reproducers and 2.44% (0≤p≤5.78) of the young animals, while among the ovine 4.29% (1.91≤p≤6.66) of the sources, 5.00% (0≤p≤11.75) of the reproducersand 3.75% (0≤p≤7.91) of the young animals presented positive serology for the BT. No significant differences were reported between positivity for the BT and the mesoregion, species and animal category (χ2; P>0.05). Based on the climatic variables, a rainy period waswell characterized, from December to May, and a dry period, from June to November. The evidence of the presence of antibodies for virus of the BT group distributed equally between herds and mesoregions, indicates that there are epidemiologic conditions for the maintenance of an arboviruses in the semi-arid region, which are discussed in this article. / A língua azul (LA) é uma doença infecciosa, não contagiosa, de ruminantes, causada pelo vírus da LA (VLA), transmitido por insetos do gênero Culicoides. A gravidade dos sinais clínicos da LA varia de acordo com a espécie, sendo os ovinos os mais gravemente afetados. Inquéritos sorológicos realizados no Brasil indicam que o vírus da LA está amplamente difundido entre bovinos e bubalinos; em caprinos e ovinos há poucos relatos de inquéritos sorológicos e descrição apenas de dois surtos com casos clínicos. A maioria das publicações discute a epidemiologia da LA em áreas temperadas que sofrem surtos periódicos. Este trabalho foi conduzido com o objetivo de estimar a prevalência de anticorpos contra o VLA em caprinos e ovinos do sertão do Estado de Pernambuco e de avaliar, preliminarmente, as condições para a manutenção dos Culicoides em ambientes tropicais semiáridos. O inquérito sorológico foi conduzido por amostragem probabilística em duas mesorregiões do Estado de Pernambuco (Sertão Pernambucano e Sertão do São Francisco), onde foram colhidas amostras de 41 criações de caprinos (n=410) e 40 (n=400) de ovinos. Além disso, foram obtidasinformações sobre as principais variáveis climáticas (precipitação pluviométrica, temperaturas máxima e mínima) que interferem na dinâmica da população e competência dos Culicoides para transmitir o VLA. Animais soropositivos à imunodifusão em ágar gel (IDGA) para LA foram encontrados em 24,39% (11,25≤p≤) das criações de caprinos e em 27,5% (13,66≤p≤41,34) de ovinos distribuídas nas mesorregiões do Sertão Pernambucano, que apresentou 4,84%(2,53≤p≤7,47) de caprinos e 4,14% (1,85≤p≤6,43) de ovinos soropositivos e do São Francisco Pernambucano, 1,00% (0,00≤p≤2,95) de caprinos e 4,55% (0,65≤p≤8,44) de ovinos soropositivos. A prevalência estimada foi de 3,90% (2,03≤p≤5,78) e 4,25% (2,27≤p≤6,23) para caprinos e ovinos, respectivamente. Observou-se que dentre os caprinos, foram registrados resultados positivos em 4,18% (1,87≤p≤6,50) das matrizes, 4,88% (0≤p≤11,47) dos reprodutores e 2,44% (0≤p≤5,78) dos animais jovens, enquanto que entre os ovinos 4,29% (1,91≤p≤6,66) das matrizes, 5,00% (0≤p≤11,75) dos reprodutores e 3,75% (0≤p≤7,91) dos animais jovens apresentaram sorologia positiva para LA. Não foram registradas diferenças significativas entre positividade para LA e mesorregião, espécie e categoria animal (χ2; P>0,05). Com base nas variáveis climáticas ficou bem caracterizado um período de chuvas, distribuído nos meses de dezembro a maio, e um seco, de junho a novembro. A constatação da presença de anticorpos para vírus do grupo da LA distribuídos homogeneamente entre os rebanhos e mesorregiões evidencia que existem condiçõesepidemiológicas para manutenção de uma arbovirose no semiárido, que são discutidas no artigo.

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

VP6 is one of the minor structural core proteins of African horsesickness virus. The minor core proteins VP1, VP4 and VP6 are presumed to constitute the dsRNA dependent RNA polymerase transcription complex of the virus. In the Orbivirus prototype bluetongue virus (BTV), VP6 has a helicase activity. The aim of this investigation was to characterize the primary structure and nucleic acid binding function of the inner core protein VP6 of African horsesickness virus (AHSV). To characterize the primary structure of AHSV VP6, VP6 genes of serotypes 3 and 6 were cloned and sequenced. Both genes encode a 369 amino acid polypeptide. A comparison to the VP6 proteins of other Orbiviruses indicated that in all cases the proteins are rich in basic residues and in glycine. The proteins are highly conserved within serogroups but the conservation between serogroups is low. VP6 of AHSV-3 and AHSV-6 have 93.5% identity and 96% similarity in amino acid residues. AHSV-6 VP6 has 27% identical and 46% similar amino acid residues to BTV-10 VP6. Phylogenetic analysis of four orbivirus VP6 genes indicated that AHSV and BTV are most closely related to each other. Motifs characteristic of known helicases were identified by sequence analysis. Glycine rich protein motifs and a N-glycosylation signal were present. No nucleic acid binding motifs identified in other proteins were found in AHSV VP6. To characterize the VP6 protein of AHSV VP6, the genes were expressed using both a baculovirus and a bacterial expression system. Proteins were found to be soluble and the VP6 expressed in insect cells was found to be N-glycosylated. The nucleic acid binding function of AHSV VP6 was investigated. Bacterially expressed VP6 was demonstrated to bind nucleic acids by electrophoretic mobility shift assays. Baculovirus expressed VP6 bound double and single-stranded RNA and DNA in nucleic acid overlay protein blot assays. Competition assays indicated that VP6 may have a preference for binding to RNA rather than DNA. Glycosylation was found to play no direct role in nucleic acid binding but the binding is strongly dependent on the NaCl concentration. A series of truncated VP6 peptides were produced to investigate the importance of localized regions in nucleic acid binding. Two partially overlapping peptides were found to bind dsRNA at pH 7.0, while other peptides with the same overlap did not. Binding appeared to be influenced by charge as reflected by the isoelectric points (pI) of the peptides and experiments indicating the effect of pH on the binding activity. However, only peptides containing amino acid residues 190 to 289 showed binding activity. This region corresponded to the region on BTV VP6 that contains two binding domains. It is proposed that the dsRNA binding domain in AHSV VP6 is a sequence of positively charged amino acids constituting a domain that determines the nucleic acid binding characteristics of the peptide. The mechanism of binding of baculovirus expressed VP6 in a nucleic acid overlay protein blot is proposed to be charge related. / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted