Doctor of Philosophy / Department of Entomology / Ludek Zurek / The larval stages of Culicoides sonorensis Wirth and Jones, a confirmed vector of bluetongue and epizootic hemorrhagic disease viruses affecting ruminants in North America, have been observed to occur typically in animal waste enhanced muds. In this dissertation, I studied the larval development (first instar to adult stage) and oviposition (four-choice assays) of C. sonorensis on sterilized mud (autoclaved) enriched with manure of different farm animal species (dairy cattle, beef cattle, sheep, goats, pigs, horses, white-tailed deer, and chicken). In addition, to determine why only some manure-polluted sites are colonized by C. sonorensis even when they are in close proximity to each other, I examined the moisture levels and microbial concentrations (mud) and physicochemical characteristics (standing water) of a manure-overflow pond site producing C. sonorensis and compared them to nearby cattle stock pond site(s) that produced different Culicoides species. Finally, as the first step in examining the role of microbiome in various physiological functions of C. sonorensis and other suspected/potential vector Culicoides species, I assessed the bacterial communities in field-collected adult females of C. sonorensis, C. crepuscularis, C. haematopotus, and C. stellifer (Illumina sequencing of 16S rRNA gene). In larval development experiments, the proportion of adults emerged and development time to adult stage varied with manure type and its concentration present in the substrate. Mud supplemented with chicken manure did not support C. sonorensis development, mud enriched with white-tailed deer manure poorly supported midge development, while C. sonorensis development in mud enhanced with manure of sheep, goats, beef cattle, dairy cattle, pigs, and horses varied. In oviposition experiments, colonized females preferred to deposit eggs on substrates without animal manure over substrates with animal manure. In subsequent studies, the manure-overflow pond site that produced mainly C. sonorensis contained significantly higher total aerobic culturable bacteria, pH, salinity, total dissolved solids, and conductivity levels than cattle stock pond sites that produced different Culicoides species. Finally, bacterial composition of field-collected C. sonorensis adult females comprised mainly of the phyla Proteobacteria and Firmicutes, while the majority of bacterial taxa identified from C. crepuscularis, C. haematopotus, and C. stellifer belonged to Proteobacteria. An unidentified bacterial genus (related to Tumebacillus), Propionibacterium, and Curvibacter were detected commonly across all four midge species. These results suggest that manure of several farm animal species can contribute to C. sonorensis development in the field. However, oviposition preferences remain uncertain, as colonized females appeared to show aversion to animal manure, which is in contradiction to the typical presence of C. sonorensis larvae in animal waste enhanced muds. Nonetheless, variations in microbial and/or physicochemical conditions in the larval habitats likely play a role in the differential emergence of C. sonorensis from various manure-polluted sites. Moreover, some bacterial taxa are associated commonly with C. sonorensis and other suspected/potential vector Culicoides species. Future studies are needed to examine oviposition preferences of field-collected females, life history traits of adults emerging from various manure-enriched substrates, developmental requirements of larvae, and the role of microbiome in various physiological functions of the host including vector competence for orbiviruses.

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 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

Doctor of Philosophy / Division of Biology / Kristin Michel / The biting midge, Culicoides sonorensis, vectors orbiviruses of economic importance, such as epizootic hemorrhagic disease virus (EHDV). Due to the limitations in available molecular tools, critical Culicoides-orbivirus interactions underlying vector competence remain unclear. To provide a foundation for the study of midge-EHDV interactions, RNA interference (RNAi) was developed as a reverse genetic tool, and EHDV-2 infection dynamics were determined within C. sonorensis. To develop RNAi, exogenous double-stranded RNA (dsRNA) was injected into C. sonorensis adults specific to the C. sonorensis inhibitor of apoptosis protein 1 (CsIAP1) ortholog (dsCsIAP1). A significant decrease in CsIAP1 transcripts was observed in whole midges, with highest reduction in the midgut. In addition, dsCsIAP1-injected midges had increased mortality, a loss of midgut tissue integrity, and increased caspase activity. The longevity and midgut phenotypes were partially reversed by the co-injection of dsRNA specific to the C. sonorensis initiator caspase Dronc ortholog and CsIAP1. These results demonstrated that RNAi can be achieved in the midge midgut through injection of target dsRNAs into the hemolymph. Furthermore, the time course of EHDV-2 infection within C. sonorensis was characterized. EHDV-2 infection was observed in the midgut and secondary tissues, including the salivary glands, by 5 days post-feeding (dpf). These data are consistent with dissemination of EHDV-2 to secondary susceptible tissues throughout the midge via the hemolymph and indicate that virus transmission by C. sonorensis may occur as early as 5 dpf. This work provides a foundation for the future study of Culicoides-orbivirus interactions, including the antiviral role of RNAi at the midgut barrier.