Alpacas were first introduced into South Africa during the year 2000. They are valuable because of the fine quality wool they produce which has much better insulation properties than that of merino wool fibres. Alpacas are also used to act as guards of sheep herds against predators. During 2008, blood samples from an alpaca that died acutely with severe lung oedema, respiratory distress and froth exuding from the nose were received at Elsenburg Veterinary Laboratory. The alpaca was from a herd of 23 alpacas of a British veterinarian in the Montagu district in the western Cape. Virus isolation attempts on the blood produced infrequent embryo mortalities. Embryonated chicken egg (ECE) material was send to the Virology Department at the Onderstepoort Veterinary Institute (OVI). A bluetongue virus (BTV) PCR performed at the diagnostic PCR laboratory at OVI on the ECE material was positive. Further intra-venous (IV) inoculations in ECE produced embryo mortalities on two consecutive days, the 8th and 9th November. The dead embryos were harvested separately and named and treated as two separate virus samples, Alp8 and Alp9 which were further passaged on baby hamster kidney (BHK) cells. The BTV virus neutralisation tests (VNT) performed at the Office International des Epizooties (OIE) Laboratory on both Alp8 and Alp9 were negative. Because of the close serological relationship between BTV and epizootic haemorrhagic disease virus (EHDV), an EHDV VNT was also performed and was also negative. In the light of the negative VNT and the positive BTV PCR results, more in-depth molecular analyses were performed. RNA was purified from tissue culture material and agarose gel electrophoresis (AGE) performed. Both Alp8 and Alp9 had a typical orbiviral electrophoretic profile, but their respective profiles were different. A sequence-independent reverse transcriptase PCR amplification method generated ample complementary DNA (cDNA) of both samples for sequencing. Sanger sequencing was used to partially sequence genome segments 5 (NS1) and 2 (VP2). BLAST analysis of the partial information of the genome segments 5 (NS1) of Alp8 confirmed it as being a BTV and Alp9 as being an EHDV. BLAST analysis of the deduced amino acid sequence generated of VP2 of both Alp8 and Alp9 established that these samples were possibly new serotypes of BTV and EHDV respectively. The complete genome of both Alp8 and Alp9 was sequenced with next generation 454 Pyrosequencing. This confirmed the partial sequencing results. BLAST analysis of the complete sequence of S2 (VP2) of Alp8 showed that it has 73 % nucleotide and 77 % deduced amino acid identity to BTV15. In contrast the nucleic acid sequence of genome segment S2 (VP2) of Alp9 had no nucleotide sequence identity to any virus, but its deduced amino acid sequence had 71 % amino acid identity to EHDV2. Hyper immune guinea pig (GP) serum prepared against the putative new BT (Alp8) and EHD (Alp9) virus serotypes were tested for serological cross-reactivity against the 24 OIE reference antigen strains of BTV and the 8 OIE reference antigen strains of EHDV. Alp8 had a neutralising antibody (NAb) titre of > 32 against BTV15. Alp9 did not cross react with any of the OIE BTV and EHDV strains. Six out of the remaining 22 alpacas on the farm had NAbs to a greater or lesser extend against Alp8 (BTV) and Alp9 (EHDV) viruses, which confirmed that the viruses were also present in other alpacas in the herd. Very few cases of EHDV in alpacas have ever been reported in literature. A small scale pilot vector susceptibility study showed that vector competence of C. imicola for both Alp8 and Alp9 was low, below 2 %. The fact that neutralising antibodies to Alp8 and Alp9 were detected in other alpacas in the herd raises the question as to whether there are other Culicoides species circulating in the area that could vector the viruses. In conclusion, the results from the serological and virological analyses as well as the nucleic acid sequence data of the genomes of two virus samples, Alp8 and Alp9, from an alpaca that died in the Montagu district in the western Cape identified Alp9 as a definite new serotype of EHDV and Alp8 as a possible new serotype of BTV most closely related to BTV15. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Alpacas were first introduced into South Africa during the year 2000. They are valuable because of the fine quality wool they produce which has much better insulation properties than that of merino wool fibres. Alpacas are also used to act as guards of sheep herds against predators. During 2008, blood samples from an alpaca that died acutely with severe lung oedema, respiratory distress and froth exuding from the nose were received at Elsenburg Veterinary Laboratory. The alpaca was from a herd of 23 alpacas of a British veterinarian in the Montagu district in the western Cape. Virus isolation attempts on the blood produced infrequent embryo mortalities. Embryonated chicken egg (ECE) material was send to the Virology Department at the Onderstepoort Veterinary Institute (OVI). A bluetongue virus (BTV) PCR performed at the diagnostic PCR laboratory at OVI on the ECE material was positive. Further intra-venous (IV) inoculations in ECE produced embryo mortalities on two consecutive days, the 8th and 9th November. The dead embryos were harvested separately and named and treated as two separate virus samples, Alp8 and Alp9 which were further passaged on baby hamster kidney (BHK) cells. The BTV virus neutralisation tests (VNT) performed at the Office International des Epizooties (OIE) Laboratory on both Alp8 and Alp9 were negative. Because of the close serological relationship between BTV and epizootic haemorrhagic disease virus (EHDV), an EHDV VNT was also performed and was also negative. In the light of the negative VNT and the positive BTV PCR results, more in-depth molecular analyses were performed. RNA was purified from tissue culture material and agarose gel electrophoresis (AGE) performed. Both Alp8 and Alp9 had a typical orbiviral electrophoretic profile, but their respective profiles were different. A sequence-independent reverse transcriptase PCR amplification method generated ample complementary DNA (cDNA) of both samples for sequencing. Sanger sequencing was used to partially sequence genome segments 5 (NS1) and 2 (VP2). BLAST analysis of the partial information of the genome segments 5 (NS1) of Alp8 confirmed it as being a BTV and Alp9 as being an EHDV. BLAST analysis of the deduced amino acid sequence generated of VP2 of both Alp8 and Alp9 established that these samples were possibly new serotypes of BTV and EHDV respectively. The complete genome of both Alp8 and Alp9 was sequenced with next generation 454 Pyrosequencing. This confirmed the partial sequencing results. BLAST analysis of the complete sequence of S2 (VP2) of Alp8 showed that it has 73 % nucleotide and 77 % deduced amino acid identity to BTV15. In contrast the nucleic acid sequence of genome segment S2 (VP2) of Alp9 had no nucleotide sequence identity to any virus, but its deduced amino acid sequence had 71 % amino acid identity to EHDV2. Hyper immune guinea pig (GP) serum prepared against the putative new BT (Alp8) and EHD (Alp9) virus serotypes were tested for serological cross-reactivity against the 24 OIE reference antigen strains of BTV and the 8 OIE reference antigen strains of EHDV. Alp8 had a neutralising antibody (NAb) titre of > 32 against BTV15. Alp9 did not cross react with any of the OIE BTV and EHDV strains. Six out of the remaining 22 alpacas on the farm had NAbs to a greater or lesser extend against Alp8 (BTV) and Alp9 (EHDV) viruses, which confirmed that the viruses were also present in other alpacas in the herd. Very few cases of EHDV in alpacas have ever been reported in literature. A small scale pilot vector susceptibility study showed that vector competence of C. imicola for both Alp8 and Alp9 was low, below 2 %. The fact that neutralising antibodies to Alp8 and Alp9 were detected in other alpacas in the herd raises the question as to whether there are other Culicoides species circulating in the area that could vector the viruses. In conclusion, the results from the serological and virological analyses as well as the nucleic acid sequence data of the genomes of two virus samples, Alp8 and Alp9, from an alpaca that died in the Montagu district in the western Cape identified Alp9 as a definite new serotype of EHDV and Alp8 as a possible new serotype of BTV most closely related to BTV15. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014