Trap type, time and site influenced the species composition, number collected and female age. Thirty species were collected in the BLTs and 17 in the bait traps. Culicoides biguattatus, C. obsoletus, C. stellifer, C. varipennis, and C. venustus were the most abundant species collected in both traps. Eight new host records were reported in this study. The drop trap collected more midges than did the vacuum trap, and the cattle trap collected more midges than did the sheep trap. Culicoides stellifer began host-seeking activity at SS-30 in bait traps and continued to be collected in the blacklight trap the remainder of the night. Earlier in the evening, nulliparous females were collected in higher percentages than parous females. Time of flight for C. stellifer was delayed when the temperature was above 25°C. Culicoides biguttatus and C. variipennis peaked at SS+30 in the bait traps but continued to be collected in the BLTs after dark. C. venustus peaked in the bait traps at SS + 60 and continued to be collected in the BLTs after dark. The parous rate of C. variipennis was higher in the cow trap than the sheep trap at the CP site. This was also true for C. stellifer at the PP site. A morphometric study of C. variipennis indicated that in Virginia using the present taxonomic characters that only one species is present. Culicoides variipennis males swarmed 15-20 minutes before sunset and 99% of the females that entered the swarm were nulliparous. One mating pair dropped the ground every 10 sec and remain in copula from 30-120 sec. The time spend on the the ground by mating pairs significantly increased successful insemination (p<0.01). Dispersal of C. variipennis was at least 0.89 Km. More midges were collected at 1.83 m and 3.05 m than at 0.61and midges collected increased. C. variipennis was collected at least as high as 9.15m and vertical movement occurred away from the breeding site. There was a positive correlation between numbers collected at different sites. Comparison tests showed that the sticky traps, D-Vac, and the bait traps collected similar percentages of parous individuals. The Marsh BLT collected a higher percentage of parous females than did the above sampling methods. Midge parous rates from the Marsh BLT were also higher than the parous rates from the Totten or Allison BLTs. Gravid females were collected in higher percentages in the Marsh BLT (50%) than on the sticky traps (2%) or in the D-Vac (22%). Though the Marsh BLT was biased for parous and gravid females, other data indicated that nulliparous females moved away from the breeding site. / Ph. D.

Els mosquits del gènere Culicoides actuen com a vectors de malalties importants que afecten tant a humans com a animals domèstics i salvatges. Al llarg de la última dècada aquests mosquits han tingut un paper molt important en l’epidemiologia de la major epizoòtia de Llengua Blava mai detectada a Europa, havent obligat a vacunar a milions de caps de bestiar per evitar les devastadores conseqüències que aquesta malaltia (de declaració obligatòria per la OIE) és capaç de provocar. Recentment, algunes espècies de Culicoides que pertanyen als subgèneres Avaritia i Culicoides han estat descrites com a potencials vectors de la malaltia de la Llengua Blava. En ambdós subgèneres es troben grups o complexes d’espècies que són difícils de diferenciar a nivell morfològic. Òbviament, una identificació correcta i precisa dels vectors resulta fonamental en la vigilància i control de malalties transmeses per artròpodes ja que es poden trobar diferències importants en la capacitat vectorial de les mateixes, inclús entre espècies properes. Per aquesta raó, al llarg del transcurs d’aquesta tesi doctoral s’ha procedit a identificar la presència de grups d’espècies críptiques en els subgèneres Avaritia i Culicoides, tant aquells descrits amb anterioritat com aquells no descrits, i detectar el nombre d’espècies que conformen cada un dels grups a Catalunya. S’ha identificat un total de quatre grups d’espècies críptiques, un en el subgènere Avaritia, l’anomenat grup Obsoletus, i tres en el subgènere Culicoides, els grups Pulicaris, Fagineus i Newsteadi. A continuació s’ha avaluat la validesa dels diferents caràcters morfològics diagnòstics descrits per identificar a les diferents espècies que formen part dels grups d’espècies críptiques en ambdós subgèneres i s’ha contrastat amb els resultats moleculars obtinguts a partir de la seqüenciació d’una regió d’ADN mitocondrial, concretament la subunitat I del gen Citocrom Oxidasa. En ambdós subgèneres ha estat possible detectar que alguns dels caràcters àmpliament utilitzats en la identificació de Culicoides resultaven ser erronis o bé no tenien el nivell de resolució per detectar espècies individuals en certs grups d’espècies. Per aquest motiu s’ha dissenyat i validat una bateria d’oligonucleòtids amb finalitats diagnòstiques que han permès identificar fins a 13 espècies de Culicoides que formaven part de grups d’espècies críptiques. Finalment per facilitar el diagnòstic laboratorial, s’ha dissenyat i validat diferents tipus de PCRs, one tube heminested PCR i multiplex PCR que han permès identificar, en una reacció individual, totes les espècies dins d’un mateix grup prèviament classificades com espècies críptiques. / Biting midges of the genus Culicoides act as vectors for important diseases affecting humans and both wild and domestic animals. In the last decade these midges have played an important role in the epidemiology of the major bluetongue epizootics ever recorded in Europe, forcing several EU countries to perform a mass vaccination campaign for domestic ruminants to avoid the devastating consequences that this disease (being its declaration mandatory for OIE) is able to cause. Recently, some species of Culicoides midges belonging to the subgenus Avaritia and Culicoides have been described as potential vectors for Bluetongue disease. Within both subgenera there are groups or complexes of species which are difficult to distinguish morphologically. Obviously, a correct and precise identification of arthropod vectors is essential for the surveillance and control of arthropod borne diseases, as important differences in the vectorial capacity of these species can be detected, even between closely related species. For this reason, the first part of the dissertation focussed on the detection of cryptic species within the subgenus Avaritia and Culicoides, identifying those species already described as other new species with no previous description. Finally the number of species forming each of the groups in Catalonia was studied. A total of four groups with cryptic species was detected, one in the subgenus Avaritia, named Obsoletus group, and three in the subgenus Culicoides, named Pulicaris, Fagineus and Newsteadi groups. Then, several morphological characters already described and currently in use for diagnostic purposes were assessed for their validity to identify the species present within those groups of cryptic species detected in both subgenera. The results obtained for morphological characters were compared with molecular results obtained by sequencing a region in the mitochondrial DNA, the subunit I of the gene cytochrome oxidase (COI). Within both subgenera, it was possible to detect some of the characters widely used for routine Culicoides species identification were not valid diagnostic characters in fact, as they were just not diagnostic or they did not have the necessary resolution level to detect and distinguish individual species within certain groups of species. Thus, a group of oligonucleotides with diagnostic purposes were designed and validated through a battery of single PCRs. These oligonucleotides can be used to properly detect and identify each of the thirteen species of Culicoides which were part of the detected groups of cryptic species. Finally, to facilitate the laboratorial diagnostic assay, different types of PCR were designed and validated, one tube heminested PCR and three multiplex PCRs. Each of these diagnostic PCR assays allowed to identify, in an individual PCR reaction, all the species detected within a specific group previously classified as cryptic species.

African horsesickness virus (AHSV) belongs to the Orbivirus genus within the family Reoviridae. AHSV is transmitted to vertebrates by Culicoides midges and causes an acute disease in horses with a high mortality rate. The virion consists of an outer layer composed of proteins VP2 and VP5, which surround an icosahedral core containing two major proteins, VP3 and VP7, three minor proteins, VP1, VP4 and VP6, and ten segments of double-stranded (ds)RNA. The VP7 protein is not only important in maintaining the structural integrity of the virus particles, but has been reported to play a key role in Culicoides cell entry. The phenomenon of RNA interference (RNAi), which can be used to selectively silence homologous genes post-transcriptionally, has revolutionized approaches to study gene function and it is also projected as a potential tool to inhibit virus replication. In mammalian cells, RNAi can be triggered by the direct introduction of 21-23 nucleotide duplexes of small interfering RNA (siRNA) that specifically and potently inhibits endogenous and heterogeneous gene expression. Consequently, the aims of the investigation were to develop RNAi assays whereby the expression of the AHSV-9 VP7 gene could be suppressed in Spodoptera frugiperda insect cells and in mammalian BHK-21 cell culture, as well as to determine whether gene-specific siRNAs may prevent AHSV-9 infection in cell culture. To investigate RNAi-mediated silencing of an enhanced green fluorescent protein (eGFP) in S.frugiperda cells, the bacmid expression system was used. Transfection of the insect cells with an eGFP-specific siRNA prior to infection with the recombinant bacmid, inhibited eGFP protein expression by 75%, as quantified by fluorometry. Although the results suggest that RNAi could potentially be used as tool to study the function of an expressed transgene in insect cells, the lack of complete inhibition, coupled with the highly cytolytic nature of the bacmid, may complicate interpretation of the gene interference results. To investigate whether siRNAs targeting the AHSV-9 VP7 mRNA is able to silence VP7 protein expression, two siRNAs were designed that targeted different regions on the VP7 mRNA. siVP7-336 and siVP7-441, directed at nucleotides 336-356 and 441-461 on the VP7 coding strand, respectively, were chemically synthesized. The effect of these siRNAs on VP7 protein expression was evaluated by cotransfection of BHK-21 cells with the respective siRNAs and the VP7 expression plasmid pCMVVP7- eGFP. The results indicated that siVP7-336 and siVP7-441 inhibited VP7-eGFP expression by 88% and 75%, respectively. BHK-21 cells were subsequently transfected with the respective siRNAs in separate experiments followed by viral infection. The VP7 mRNA quantities were measured by quantitative reverse transcription PCR and the effect of the siRNAs on viral replication was evaluated by plaque assays. Of the two siRNAs, siVP7-336 was found to be the most effective inhibitor of VP7 transcription and suppressed VP7 mRNA by 93%. The exposure of BHK-21 cells to the VP7 genespecific siRNA, siVP7-336, also led to a 84% reduction in progeny virions, as measured by a plaque assay. Taken together, the results demonstrate that siRNA-mediated gene silencing is an efficient approach for reducing the level of VP7 transcripts and proteins and for inhibiting virus propagation. / Dissertation (MSc (Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Please read the abstract in the dissertation / Dissertation (MSc Agric)--University of Pretoria ,1967. / gm2014 / Zoology and Entomology / unrestricted

A survey of the localised distribution of Culicoides obsoletus and Culicoides pulicaris was performed on a dairy and sheep farm in south-west England. Culicoides obsoletus and C. pulicaris have both been confirmed as vector species for the transmission of bluetongue virus in Europe. Sampling was done using motorised black-light suction insect traps. Seventeen traps were set around the farmyard and animal housing and five traps were set in varying pasture locations. Sampling was carried out on eight occasions between mid-September and mid-October 2008. The trapped Culicoides were counted, speciated, sexed and the reproductive stages of the females were recorded. Culicoides obsoletus, C. chiopterus, C. scoticus, C. dewulfi and C. pulicaris (group) were identified during the study. The trap sites were selected to examine the Culicoides populations associated with a wide range of microclimates. The selected sites included manure stores; forage feed stores; yard areas and sites surrounding as well as inside the animal housing. Comparisons were made between Culicoides numbers trapped from different directional sides of the animal buildings and the numbers found inside compared to numbers found outside the buildings. Culicoides numbers collected from the animal areas were compared to the non-animal areas and to the manure and forage sites. The field sites consisted of a marsh area; stream; water trough; open field site and a group of trees in a hedge field boundary. Culiccompared catch sizes from the field trap sites were compared to each other and to the farm holding sites. The highest number of Culicoides trapped were at the farm holding sites, apart from one catch on one occasion from a single field site. Weather changes, particularly high wind speeds with direction changes appeared to reduce the catch sizes during some of the trapping occasions. A greater number of C. obsoletus were collected from both the farm and field sites although a higher relative proportion of C. pulicaris was collected from the field sites. Of the C. obsoletus group, C. dewulfi was only found in farm holding catches, not at any of the field sites. The remaining three sibling species were found in both the farm and field catches. Relatively high numbers of Culicoides were found within the animal housing, with external numbers apparently influencing those found within the housing. An increase in numbers of Culicoides trapped inside the buildings may have been associated with a small shed size and possibly with straw bedding. A relative shift in the Culicoides population into the buildings appeared associated with prolonged high wind speeds. Widely varying female life stages found at all of the farm trap sites suggested possible dispersal of the Culicoides populations between these sites. Populations appeared to remain localised around the farm holding, but possibly dispersed over greater distances from the pasture locations. A wide distribution of breeding sites was suspected around the farm holding. A ranking system was used to identify specific areas associated with increased numbers of female Culicoides collected from these sites. Three sites surrounding a straw bedded cow shed were highlighted as higher risk Culicoides exposure sites; two sites adjacent to a cubicle shed; inside the calf housing; the manure store area and the silage store area. A field site with trees in a hedge boundary was the only high-risk field site identified. Multilevel modelling was used to examine for possible factors influencing Culicoides numbers. Factors examined included wind, temperature and humidity variables; distance from manure, forage, water and trees and livestock variables such as: time of contact, time since contact and distance from sheep and cattle. The model suggested wind speed at light trap setting and an increased time since contact with cattle both appeared significantly associated with reduced Culicoides numbers. Culicoides obsoletus numbers also appeared significantly reduced with increasing distance from manure. From an on-farm risk assessment point of view the farm holding area of a dairy farm as a whole should generally be considered a high-risk site for Culicoides exposure and specific pasture sites can periodically become high exposure sites. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Veterinary Tropical Diseases / unrestricted

This investigation was concerned with determining the relationships of Culicoides to poultry in Virginie while studying their role as potential vectors of infectious synovitis. In addition, a survey of Culicoides in Virginia was made to study their distribution, bionomics and life histories. The insects were trapped in the field, collected as larvae, and reared in the laboratory. Using light traps in chicken coops at Ferrum, Virginia in 1959, it was found that the following species of Culicoides were present; arboricola, crepuscularis, debilipalpis, guttipennis, haematopotus, obsoletus (most abundant), ousairani, stellifer, travisi, variipennis, venustus, and villosipennis. During the same summer, traps at Poplar Camp took many of the above species plus biguttatus and the piliferus group. That same year a single specimen of C. paraensis was taken while biting the author. Light trapping in 1960 was done at Blacksburg about three nights a week for six months. Other trapping was done at Elkton, Newport and Saltville. At Blacksburg and Elkton traps were operated both in the woods and in poultry houses. The 1960 trapping activities revealed the following species were present: arboricola, baueri, biguttatus, crepuscularis, chiopterus, debilipalpis, guttipennis, haematopotus, obsoletus, ousairani, piliferus group, snowi, spinosus, stellifer, travisi, variipennis, venustus, and villosipennis. All but chiopterus, debilipalpis, snowi, and spinosus have been taken in poultry houses. In 1960 crepuscularis was the most abundant species taken in poultry houses and travisi was the most numerous species in the woods. It was found that more Culicoides were taken in poultry houses located near a woods than in ones farther from a woods. Trapping revealed that Culicoides were most abundant at Blacksburg in June, with May being the next most favorable month. The following species were found every month: haematopotus, obsoletus, stellifer, and variipennis. All specimens were preserved in 70 per cent ethyl alcohol and latex mounted in phenol-balsam on microscope slides. Apparently the weather had little influence on the trapping results. During 1959 and 1960 larvae were collected in a number of situations, but mostly from rot hole: in trees and soft mud. Tree-hole collections produced specimens of C. arboricola, guttipennis, and namis. Mud and muddy water produced C. variipennis. The latter were extremely abundant in saline mud at Saltville, there being found as many as 2000 larvae per 500 cc. of mud. These were used in the infectious synovitis transmission experiments. The larvae were reared in pans and crocks containing their original substrate. After emergence they were held in cages fashioned from round ice cream cartons, and kept in an incubator at 75° F. and approximately 40 per cent relative humidity. Under these conditions the adults lived an average of 27.7 days after emergence with the longest time being 96 days. The adults emerged in largest numbers four to eight days after collection. In 1959 attempts were made to transmit infectious synovitis to healthy chickens by injecting them with macerated, engorge. Culicoides which had been live-trapped in poultry houses containing chickens infected with the disease. These attempts were unsuccessful. In 1960 specimens of C. variipennis were induced to feed upon chickens injected with the agent of infectious synovitis. These were later macerated at varying intervals and injected into healthy chickens. No transmission was demonstrated. It was found that the insects fed most readily on the heads of chickens when the insects were two to four days old. / Ph. D.

African horsesickness (AHS) is one of the most deadly diseases of horses, with a mortality rate of over 90% in horses that have not been exposed to any African horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are primarily transmitted to their mammalian hosts through certain haematophagous midge vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2 by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans, 1982). It is believed that this cleavage affects the ability of the virus to infect cells of the mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a truncated VP2. Upon further investigation, this strain was also shown to be more infective than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012). Therefore, through proteolytic cleavage of these viral particles, the ability of the adult Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel et al., 2011). Based on these findings, it is important to investigate the factors that influence the capability of arthropod-borne viruses to infect their insect vectors, mammalian hosts and their known reservoirs. In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C. imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were separated on SDS-PAGE and yielded several protein bands, one of which also had a molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel. The activity of the protein of interest was also confirmed to be a trypsin-like serine protease with the use of class-specific protease inhibitors. A recombinant trypsin-like serine protease of C. sonorensis was generated using the pColdIII bacterial expression vector. The expressed protein was partially purified with nickel ion affinity chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the expressed protein was classified as a serine protease. It was also proposed that incubation of purified AHSV4 with the recombinant protease would result in the cleavage of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after which the virus was incubated with the recombinant protease. Since not enough virus sample was obtained, the outcome of VP2 digestion was undetermined. In the last part of this study, it was postulated that C. imicola and C. sonorensis have the same trypsin-like serine protease responsible for the cleavage of VP2 based on the protease activity visualised in the whole midge homogenate. Since the genome of C. imicola is not yet sequenced, the sequence of this likely protease is still unknown. Therefore, we attempted to identify this C. imicola protease through polymerase chain reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA fragment was amplified. However, sequence alignment and the basic local alignment software tool (BLAST), revealed that DNA did not encode with any other known proteins or proteases. From the literature it seems that there is a correlation between the proteases in the vector and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al., 2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The 29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in future investigations on how proteolytic viral modifications affect infectivity between different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Les invasions biologiques constituent une source de préoccupation majeure du fait des conséquences écologiques, économiques et sanitaires dont elles sont responsables. Déterminer et comprendre les facteurs sous-jacents au succès invasif des espèces envahissantes permet de prédire de nouvelles invasions et de mettre en place des stratégies de contrôle. Culicoides imicola est un vecteur majeur d’Orbivirus d’intérêt vétérinaire incluant le virus de la fièvre catarrhale ovine (FCO). Suite à l’émergence de la FCO dans le bassin méditerranéen, les populations de C. imicola ont été découvertes dans des territoires où elles étaient considérées comme absentes, caractérisant alors cette présence comme la résultante d’une expansion récente de l’espèce. Cette thèse décrit un ensemble de travaux visant à comprendre l’histoire de la colonisation du bassin méditerranéen par C. imicola. L’utilisation d’une approche multi-marqueurs combinant des analyses de génétique de populations, des inférences basées sur la méthode Approximate Bayesian Computation (ABC) et la simulation mathématique de la dispersion atmosphérique de l’espèce, a permis (i) de déterminer l’origine des populations installées au Maghreb et au Moyen Orient et de décrire les routes de colonisation et la chronologie de ces évènements, (ii) de définir les caractéristiques démographiques, évolutives et temporelles de la colonisation du sud de l’Europe et (iii) de caractériser les principaux facteurs expliquant le succès d’expansion géographique des populations installées. Les principaux résultats de cette thèse permettent de proposer des hypothèses pour expliquer le succès de l’installation des populations de C. imicola dans le bassin méditerranéen / Biological invasions are of major concern because of their environmental, economic and health consequences. Determining and understanding the factors underlying the invasion success of species allow predicting potential other biological invasions, and developing vector control strategies. Culicoides imicola is a major vector species of Orbivirus, including the bluetongue virus (BTV) which affects domestic ruminants. Following BT emergence in the Mediterranean basin, C. imicola populations were recorded in territories where the species was considered to be absent, and consequently was described as expanding its range expansion on a short period. This Phd work describes a set of studies aiming at understanding the colonization history of the Mediterranean basin by C. imicola. The use of a multi-loci approach combining population genetics analyses, Approximate Bayesian Computation (ABC) methods and mathematical simulations of the atmospheric dispersion of the species enabled to (i) determine the origin of the established populations in the Maghreb and the Middle-East and describe the routes of colonization and the chronology of such events, (ii) define the demographic, evolutionary and temporal characteristics of south-western Europe colonization and (iii) characterize the main factors explaining the successful range expansion of the established populations. The main results of this thesis allow suggesting hypotheses to explain the successful establishment of C. imicola populations in the Mediterranean basin.

African horsesickness (AHS) is one of the most deadly diseases of horses, with a mortality rate of over 90% in horses that have not been exposed to any African horsesickness virus (AHSV) serotype previously (Howell, 1960; Darpel et al., 2011). The Orbiviruses, African horsesickness virus (AHSV) and Bluetongue virus (BTV), are primarily transmitted to their mammalian hosts through certain haematophagous midge vectors (Culicoides spp.) (Erasmus, 1973). The selective cleavage of BTV and AHSV VP2 by trypsin-like serine proteases (Marchi et al., 1995) resulted in the generation of subsequent infectious sub-viral particles (ISVP) (Marchi et al., 1995; van Dijk & Huismans, 1982). It is believed that this cleavage affects the ability of the virus to infect cells of the mammalian and vector host (Darpel et al., 2011). Darpel et al (2011) identified a trypsinlike serine protease in the saliva of Culicoides sonorensis (C. sonorensis), which also cleaves the serotype determinant viral protein 2 (VP2) of BTV. And, a similar cleavage pattern was also observed by van Dijk & Huismans (1982) and Marchi et al (1995) with the use of trypsin and chymotrypsin. Manole et al (2012) recently determined the structure of a naturally occurring African horsesickness virus serotype 7 (AHSV7) strain with a truncated VP2. Upon further investigation, this strain was also shown to be more infective than the AHSV4 HS32/62 strain, since it outgrew AHSV4 in culture (Manole et al., 2012). Therefore, through proteolytic cleavage of these viral particles, the ability of the adult Culicoides to transmit the virus might be significantly increased (Dimmock, 1982; Darpel et al., 2011). Based on these findings, it is important to investigate the factors that influence the capability of arthropod-borne viruses to infect their insect vectors, mammalian hosts and their known reservoirs. In this study, we postulated that one of the vectors for AHSV, Culicoides imicola (C. imicola), has a protease similar to the 29 kDa C. sonorensis trypsin-like serine protease identified by Darpel et al (2011). Proteins in the total homogenate of C. imicola were separated on SDS-PAGE and yielded several protein bands, one of which also had a molecular mass of around 29 kDa. Furthermore, proteolytic activity was observed on a gelatin-based sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE) gel. The activity of the protein of interest was also confirmed to be a trypsin-like serine protease with the use of class-specific protease inhibitors. A recombinant trypsin-like serine protease of C. sonorensis was generated using the pColdIII bacterial expression vector. The expressed protein was partially purified with nickel ion affinity chromatography. Zymography also confirmed proteolytic activity. With the use of the protease substrates containing fluorescent tags and class specific protease inhibitors, the expressed protein was classified as a serine protease. It was also proposed that incubation of purified AHSV4 with the recombinant protease would result in the cleavage of AHSV4 VP2, resulting in similar VP2 digestion patterns as observed in BTV by Darpel et al (2011) or the truncated VP2 of AHSV7 by Manole et al (2012). BHK-21 cell cultured AHSV4 was partially purified through Caesium chloride gradient ultracentrifugation after which the virus was incubated with the recombinant protease. Since not enough virus sample was obtained, the outcome of VP2 digestion was undetermined. In the last part of this study, it was postulated that C. imicola and C. sonorensis have the same trypsin-like serine protease responsible for the cleavage of VP2 based on the protease activity visualised in the whole midge homogenate. Since the genome of C. imicola is not yet sequenced, the sequence of this likely protease is still unknown. Therefore, we attempted to identify this C. imicola protease through polymerase chain reaction (PCR) amplification. Total isolated ribonucleic acid (RNA) of C. imicola was used to synthesize complementary deoxyribonucleic acid (cDNA). The cDNA was subjected to PCR using C. sonorensis trypsin-like serine protease-based primers. An 830 bp DNA fragment was amplified. However, sequence alignment and the basic local alignment software tool (BLAST), revealed that DNA did not encode with any other known proteins or proteases. From the literature it seems that there is a correlation between the proteases in the vector and the mammalian species that succumb to AHS (Darpel et al., 2011, Wilson et al., 2009, Marchi et al., 1995). Based on the work performed in the study, a proteolytically active protein similar to the 29 kDa protein of C. sonorensis is present in C. imicola. The 29 kDa protease of C. sonorensis can also be expressed in bacteria which could aid in future investigations on how proteolytic viral modifications affect infectivity between different host species. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

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