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.

Bluetongue (BT) is a non-contagious disease of mainly sheep but other ruminants like cattle, goats, and wild ruminants like alpacas, African antelopes and deer can also be affected. It is transmitted by Culicoides midges and its occurrence is seasonal, especially after good rains. The disease is subsiding when temperatures drop. The virus is distributed throughout the world in the tropical, subtropical and temperate areas where there are culicoides vectors which can transmit it (Tabachnick et al., 2011). This includes most countries in Africa, the Middle East, India, China, Australia, the United States of America, Canada and Mexico. Until 2008 24 BTV serotypes were known, but from 2008, data on the 25th serotype was published and recently, the 26th serotype has been identified (Hofmann et al., 2008; Maan et al., 2012a). In Africa 21 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze–dried live attenuated polyvalent BTV vaccine. Currently the vaccine used in the Southern African Development Community (SADC) region is produced by Onderstepoort Biological Products (OBP). The vaccine is constituted of fifteen serotypes of the bluetongue virus (BTV) divided into three separate bottles. Each bottle contains five serotypes. The inoculation procedures are that bottle B is given three weeks after bottle A and bottle C three weeks after bottle B. The full immunity is established three weeks after the last bottle. The vaccine is effective and it induces both humoral and cellular immune response (Dungu et al., 2004). However, the challenge with the vaccine is that during outbreaks, sheep might not have nine full weeks to develop protection against the disease; and the farmer loses money on treatment and death of animals. Hence the purpose of the study is to determine whether the number of serotypes in the vaccine can be reduced without affecting efficacy; thus shorten the time taken for the full development of immunity after vaccination of animals. This study is based on previously reported cross-neutralization of specific BTV serotypes in in vitro studies by Howell et al. (1970) and Dungu et al. (2004). Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control), 1, 8 (unrelated serotypes) 9, 10 and 11 in sheep using the serum neutralization test (SNT). The unvaccinated animals in all groups reacted to the challenge material. The animals vaccinated with and challenged with BTV-4, showed good immune response. Those animals that were vaccinated with BTV-4 and challenged with BTV-1 which is not directly related to BTV-4 (Howell et al., 1970), only 20% of the group was completely protected and did not show clinical signs other than a temperature reaction. The rest showed clinical signs, however the reaction was not as severe as the unvaccinated animal. The animals challenged with BTV-9 and 11 had good protection while those challenged with BTV-10, some showed good protection, some got very sick while others had mild clinical signs. The results showed that BTV serotype 4 do not only develop a specific immune response but can also protect against other serotypes. Future studies should be done looking at more serotypes but also look at the specific titres used per serotype in the vaccine. The development of cellular immunity should also be taken in consideration. With further studies it should be possible to develop a vaccine with fewer serotypes without compromising the immunity against the disease. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

The spread of bluetongue virus (BTV) to previously disease-free regions which prohibit the use of the current BTV live-attenuated vaccine has highlighted the need for a new generation of vaccines (Ferrari, De Liberato et al. 2005; Veronesi, Hamblin et al. 2005). Subunit vaccines are one of the attractive alternative strategies. Subunit vaccines against BTV would target the outercapsid protein VP2, the main neutralization-specific antigen (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). A subunit vaccine based on the use of BTV-VP2 may be achieved by either using VP2 by itself or by means of virus-like particles (VLPs) on which VP2 proteins are exposed. In VLPs, the VP2 is co-expressed with other capsid and core proteins to form a particle that resembles the intact BTV. The BTV-VLP vaccine strategy is advantageous since it presents the neutralizing epitopes of more than one viral protein in a more authentic manner as found on the virus itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However there are difficulties associated with large scale production and a decrease in the stability of the particles over time (Berg, Difatta et al. 2005; Wang, Zhao et al. 2006). Studies have already demonstrated the vaccine potential of BTVVP2 by itself (Huismans, van der Walt et al. 1987; Roy, Urakawa et al. 1990; Roy, French et al. 1992; Roy, Bishop et al. 1994). However if BTV-VP2 is to be used by itself as a single subunit vaccine, it is important that the protein is expressed under conditions where it is correctly folded and soluble. Solubility refers to the capacity of the expressed antigen to fold into an ordered tertiary structure that authentically exposes the neutralizing epitopes to the immune system (Dinner, Sali et al. 2000; Dobson 2003). However non-native interactions within and between in vitro synthesized viral proteins such as BTV-VP2 often leads to protein aggregation or insolubility. The immune response against aggregated or insoluble proteins is generally very poor. This problem of aggregation and insolubility may be alleviated to an extent by generating truncated versions of the protein from which hydrophobic regions that promote aggregation have been deleted leaving only the major neutralizing epitopes of the antigen (Fukumoto, Xuan et al. 2003; Bonafe, Rininger et al. 2009; Liu, Zeng et al. 2009; Seo, Pyo et al. 2009). The focus of the research presented in this dissertation was to evaluate the solubility of full-length BTV(10)-VP2 and truncated versions thereof after expression in a prokaryotic and baculovirus-Sf9 expression system. The full-length BTV(10)-VP2 (956 amino acids) gene and genes encoding truncated versions of BTV(10)-VP2 i.e. BTV(10)-VP2(aa450) (amino acid 1 to 450) and BTV(10)- VP2(aa650) (amino acid 1 to 650) were cloned into the bacterial expression vector pET160-DEST and the baculovirus expression vector pDEST™8. The C-terminal hydrophobic regions which might contribute to aggregation or insolubility of the protein when expressed in vitro were deleted from these truncated BTV(10)-VP2 proteins. The truncated proteins however still contained BTV neutralizing epitopes that were predicted from literature. The prokaryotic expression of the full-length BTV(10)-VP2 and the other truncated recombinant BTV(10)-VP2 proteins was carried out in E. coli BL21 Star DE3 expression strain. The initial pilot expression study confirmed high level expression of the recombinant proteins. The study also revealed that these proteins were insoluble. The optimization of the prokaryotic expression in order to increase the yield of soluble proteins by means of differential inducer concentrations, fermentation temperature and harvesting times did not produce soluble BTV(10)-VP2 and truncated BTV(10)-VP2 proteins. Previous studies have demonstrated the role of L-arginine in the recovery of soluble proteins from aggregation by reversing aggregation (Tsumoto, Umetsu et al. 2003). However in the current study, arginine treatment of the inclusion body and bacterial lysate containing the BTV(10)- VP2 and truncated recombinant proteins did not release soluble proteins. No soluble recombinant BTV(10)-VP2 proteins were detected when the recombinant proteins were expressed in BL21 host cells over-expressing heat-shock proteins (hsps) and chemical chaperones. However when the different recombinant proteins were co-expressed with the molecular chaperones dnaK-dnaJ-GrpE, it resulted in a fraction of soluble recombinant BTV(10)-VP2 proteins. In particular, approximately 50% of the total expressed BTV(10)-VP2(aa450) protein was soluble while approximately 20% of the total expressed BTV(10)-VP2(aa650) and full-length BTV(10)-VP2 were found soluble when coexpressed with dnaK-dnaJ-GrpE chaperones. These recombinant proteins could be eluted from a nickel affinity column further confirming that these proteins are in fact soluble. Interestingly the coexpression of the BTV(10)-VP2(aa450) protein with the above chaperones in combination with chaperones groEL-groES or only groEL-groES did not produce any soluble proteins. Baculovirus-insect expression of the aforementioned BTV(10)-VP2 recombinant proteins was carried out in Spodoptera frugiperda 9 (Sf9) cells. High level expression of the recombinant proteins was confirmed by an initial pilot expression study conducted at 42 hours post infection (p.i.). The pilot study also revealed that the recombinant proteins were insoluble. Arginine treatment of the lysate released a small fraction of soluble BTV(10)-VP2(aa450) and BTV(10)-VP2(ORF) proteins only detectable with immunoblot analysis using the anti-BTV(10) IgY antibodies. The amount of solubilized proteins was however too small to justify the cost associated with this expression system. / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted

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

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

Bluetongue virus belongs to the Orbivirus genus from the Reoviridae family. It infects predominantly domestic and wild ruminants and is economically significant worldwide. Bluetongue virus VP7 forms the intercepting layer between the outer capsid (VP2 and VP5) and VP3 which surrounds the genomic material. BL21(DE3), NiCo21(DE3), C43(DE3) pLysS and KRX Escherichia coli cells were transformed with a pET28a plasmid with the cDNA sequence encoding Bluetongue virus VP7. Expression of Bluetongue virus VP7 was tested at post induction temperatures between 16˚C and 37 ˚C, at inducer concentrations between 0.1 mM and 1.0 mM isopropyl-β-D-thiogalactopyranoside in BL21(DE3), NiCo21(DE3) and C43(DE3) pLysS cells and 0.05 % and 0.15 % rhamnose for KRX cells, in two types of growth media (LB and 2xYT) and post-induction growth times between two and 16 hours. Under all conditions tested; Bluetongue virus VP7 expression was found to be predominantly in the insoluble fraction (pellet). BL21(DE3) and NiCo21(DE3) cells were chosen and grown for five hours post induction, induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside and grown at a post-induction temperature of 37 ˚C. Bluetongue virus VP7 in bacterial cell inclusion bodies was solubilised using urea and a freeze-thaw step. Solubilisation was tested with urea concentrations between 2 M and 8 M, with solubilisation efficiency not increasing past 5 M urea. Solubilized Bluetongue virus VP7 was purified using nickel-affinity chromatography. Purified Bluetongue virus VP7 was then probed with far-UV circular dichroism and intrinsic fluorescence in several buffer conditions including different urea and guanidinium chloride concentrations as well as in the presence of glycerol and sodium chloride. Guanidinium chloride was able to cause Bluetongue virus VP7 unfolding, and the unfolding transition had 94 % and 89 % reversibility at 218 nm and 222 nm respectively. Bluetongue virus VP7 was shown to contain a native-like structure in 20 % glycerol and in up to 8 M urea and was found to be stable till at least 55 ˚C, even in the presence of 5 M urea. Glycerol and sodium chloride influenced the conformation of the protein resulting in different unfolding transitions. Thermal unfolding of Bluetongue virus VP7 was found to be irreversible. / Life and Consumer Sciences / M. Sc. (Life Sciences)

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.