The aim of this investigation was to clone, characterize and express the gene that encodes the outer capsid protein, VP2, of African horsesickness virus (AHSV), with a view to the evaluation of this protein as a subunit vaccine. The VP2 gene of AHSV serotype 3 (AHSV-3) was cloned as incomplete cDNA fragments of the genome segment 2 double-stranded (ds)RNA, sequenced in its entirety and compared with previously published cognate sequences of AHSV-4, Epizootic hemorrhagic disease virus (EHDV)-l and various bluetongue virus (BTV) serotypes. AHSV-3 genome segment 2 was shown to be 3221 nucleotides in length, encoding a protein of 1057 amino acids with a 50.5% identity to AHSV-4 VP2. Two areas of high variability (approximately 65%) were identified adjacent to the conserved termini. The N-proximal region (amino acids 128-309) exhibited significant hydrophilicity, suggesting a possible role in the determination of the serotype-specific immune response. Orbivirus interserogroup comparisons of VP2 amino acid sequences revealed extreme variability, although an overall structural conservation was demonstrated. Oligonucleotide primers derived from the AHSV-3 genome segment 2 terminal nucleotide sequences were used for PCR amplification and cloning of full length segment 2 cDNA. The cloned gene was expressed in a baculovirus expression system and the expressed VP2 protein was shown to react specifically with anti AHSV-3 serum in Western blots. Although the yields of VP2 in the baculovirus system were low, due to a possible toxic effect on the host cells, sufficient antigen was obtained for further future investigations into the efficacy of VP2 as a possible subunit vaccine against AHSV. / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted

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