African horsesickness virus (AHSV), a member of the Orbivirus genus in the family Reoviridae, is the aetological agent of an economically important disease affecting equids. One of the outer coat proteins of the virus, VP5, has the important function of interacting with the conserved core proteins as well as adapting to facilitate changes in the major outer coat protein, VP2. VP5 plays a role in destabilizing the endosomal membrane following viral attachment to the host cell, thereby allowing access of the viral core into the host cytoplasm. Furthermore, VP5 contributes to the immune response in infected animals and has relevance to recombinant vaccine development. The main aim of this study was to compare the genetic variation between VP5 proteins of field isolates of AHSV, isolated from dead animals, to that of laboratory strains, likely to be avirulent. This comparison was done in order to determine if any significant/common amino acid changes could be linked to the virulence phenotype. The VP5 genes of six AHSV isolates of serotypes 3, 6, 8 and 9 respectively were cloned, sequenced and the amino acid sequences deduced. This study represents the first report on the sequencing analysis of the VP5 gene and protein of an AHSV serotype 8. All sequences were aligned and compared to those of published laboratory/vaccine strains, and other available sequence data. Interserotype amino acid identities of between 95% and 99% were observed between isolates of serotypes 3, 6 and 9 (apart from a laboratory strain of AHSV9), indicating significant conservation between these serotypes. The VP5 protein of AHSV8 showed less homology and differed from the other serotypes by between 5.2 and 7%. The AHSV4 protein differed significantly from the rest, showing between 15 and 19% amino acid differences. The identity between the VP5 proteins of different isolates/strains of the same serotype was very high in most instances, at approximately 97% for different AHSV3 strains and 94 % for different AHSV6 strains. The VP5 protein of a laboratory adapted strain of AHSV9, however, differed from an AHSV9 field isolate by nearly 10%. In the analyses of these amino acid differences, no common amino acid change, indicative of a virulence marker, could be identified between the assumedly virulent field isolates and their respective laboratory adapted strains. Furthermore, no distinctive differences in regions supposedly involved in membrane destabilization, or particular patterns of variation were observed. The results indicate that virulence of a particular AHSV serotype is unlikely to be influenced by a common amino acid change in VP5, and more likely to be multi-faceted. The second aim of this study was to investigate whether VP5 has a cytotoxic effect when expressed in a bacterial system, in light of similar effects reported for the analogous protein of bluetonguevirus (BTV), a related orbivirus. Computer analyses were performed and indicated that amphipatic helices found in the N-terminus of BTV VP5, proposed to be responsible for cytotoxicity, were also present in AHSV VP5. A full-length copy of the AHSV4 VP5 gene was cloned into the pET41 transfer plasmid and expression induced in bacterial cells. Optical density measurements of the bacterial cultures, indicative of cell growth, were taken at time intervals post induction. The expression of AHSV VP5 clearly had a severe negative effect on cell density (hence growth) over time, in contrast to a control where the cell density continued to increase. The results therefore clearly indicated that expression of AHSV VPS was cytotoxic to bacterial cells. Lastly, a neutralizing epitope present on VPS was expressed on the surface of a particulate display system, based on crystalline particles formed when the VP7 protein of AHSV9 is expressed in insect cells. Expression of the VP7/epitope A chimeric protein was confirmed by polyacrylamide gel electrophoresis and partially purified by sucrose gradient separation. Hexagonal crystals with a unique surface structure, but morphologically quite similar to VP7 crystals, were observed by scanning electron microscopy. The VP7 display vector could therefore tolerate insertion of the epitope without losing its ability to form crystals. The chimeric particles are now available for further evaluation as to its immunological properties. The results of this study should contribute to the development of a possible vaccine strategy against AHS in future. / Dissertation (MSc (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted

The viral determinants of African horsesickness virus (AHSV) cytopathology are not well understood. Several AHSV proteins may play a role, including non-structural protein NS3, a cytotoxic membrane protein that localises to sites of virus release and plasma membrane disorganisation in infected cells. AHSV NS3 is highly variable and clusters into three phylogenetic groups, termed α, β and γ. In chapter 2 we examined the role of NS3 in determining the phenotypic characteristics observed during AHSV infection of cells. Three AHSV strains, AHSV-2 (γ NS3), AHSV-3 (β NS3) and AHSV-4 (α NS3), were shown to have quantitatively different phenotypes in Vero cells. To investigate the contribution of NS3 to these differences, reassortants were generated between these strains in which the S10 genome segment encoding NS3 was exchanged, alone or in combination with other segments. Exchange of NS3 resulted in changes in virus release and membrane permeability, indicating an important role for NS3 in these viral properties. The cytopathic effect and decreased viability of infected cells was not associated with NS3 alone and it is likely that a number of viral and host factors contribute to these complex phenotypes. In chapter 3 the cytolytic effect of the NS3 proteins of the orbiviruses AHSV, bluetongue virus (BTV) and equine encephalosis virus (EEV) were compared. Inducible expression in Escherichia coli (E. coli) showed differences in cytotoxicity, with EEV NS3 having a greater lytic effect than than AHSV and BTV NS3. Cytotoxicity was linked to increased membrane permeability of the cells as confirmed by an increased uptake of membrane impermeant compounds. When expressed in insect cells however all three NS3 proteins caused a marked but equivalent decrease in cell viability. Although the orbivirus NS3 proteins have similar predicted secondary structures, differences could lie in structural stability and association with membranes of specific cell types, which impacts on cytotoxicity. To determine the regions within AHSV NS3 that mediate cytotoxicity, a series of truncated mutants of NS3 where constructed and expressed in E. coli. The combined presence of both hydrophobic domains of AHSV NS3 was found to be critical for membrane permeabilisation and cytotoxicity. In chapter 4 the AHSV-2, AHSV-3 and AHSV-4 NS3 proteins (from the γ, β and α NS3 clades)were compared to examine the impact of sequence variation in NS3 on structure and function. The proteins were expressed in the baculovirus expression system as both wild-type proteins and C-terminal eGFP (enhanced green fluorescent protein) fusions. Exogenous addition of the baculovirus expressed proteins to Vero cells resulted in different permeabilisation levels that could be linked to that induced by the AHSV strains. Cell viability and membrane association assays in insect cells showed that all three proteins were equivalently cytotoxic and membrane associated. The subcellular localisation of the eGFP-NS3 fusion proteins was examined by confocal fluorescen imaging of live cells. NS3 localised to the plasma membrane, and as distinct punctuate foci in the perinuclear region. This suggests localisation to the internal membrane systems of cells and has important implications for the function of this membrane permeabilising protein. / Thesis (PhD)--University of Pretoria, 2011. / Genetics / unrestricted

Non-structural protein, NS1 of African horse sickness virus is a hydrophobic protein of 63 kDa that spontaneously assembles into highly distinct tubular structures when expressed in mammalian or insect cells. The spontaneous assembly of these proteins into a predictable multimeric structure, high levels of expression and ease of purification make this protein an ideal candidate for the immune display of foreign peptides. The potential of such a display system has been investigated for BTV NS1 that is able to successfully elicit both a humoral and a cellular immune response against inserted peptides. The aims of this study were to investigate both the stability of the AHSV NS1 particulate structure after insertion of peptides as well as the antigenicity and immunogenicity of the peptides presented in this system. Two overlapping regions consisting of 40 and 150 amino acids, and which correspond to a neutralising region identified within the AHSV major neutralising protein VP2, were inserted into an internal site in NS1. This site offered the best surface display of inserted peptides on the tubular structures. An enhanced green fluorescent protein, 240 amino acids long, was also inserted into the NS1 protein. Sucrose gradient analysis of the recombinant proteins indicated that the majority of the baculovirus expressed chimeric proteins formed particulate structures with a sedimentation value similar to that of the native NS1 protein. This was confirmed by transmission electron microscopic analysis, which clearly showed that all the chimeric proteins assembled into tubular structures similar to those observed for AHSV NS1 proteins. Furthermore, fluorescence analysis of sucrose gradients of NS1/eGFP also showed high levels of fluorescence that corresponded directly to particle formation. Not only do the inserts remain functional but are also presented successfully on the surface of the intact NS1 tubule structure. The potential of the NS1 vector to efficiently present peptides to the immune system was subsequently investigated. The serums generated against these chimeric proteins in guinea pigs were tested against chimeric constructs, the baculovirus expressed inserts (for eGFP) and the inserts presented on other presentation vectors. Western blot analysis showed that most of the serums generated against the chimeric proteins contained antibodies not only against the chimeric proteins but antibodies that reacted specifically with the inserted peptides on their own or on another presentation system. Preliminary immune studies seem to indicate that the humoral immune response elicited by the chimeric NS1 proteins is predominantly against the inserts. The inserts are successfully presented to the immune system on the surface of the NS1 vector and are able to elicit the production of antibodies with the potential to provide a protective immune response. / Dissertation (MSc (Genetics))--University of Pretoria, 2010. / Genetics / unrestricted

The aim of this study was to standardize and validate an immunohistochemical test for the routine diagnosis of African horsesickness in horses. Hamblin developed the primary anti- African horsesickness virus serum that I used and the avidin-biotin complex detection system was employed. During the standardization process I demonstrate that lung, heart and spleen samples are the most reliable. I also show that it is not necessary to take multiple samples per organ, because the AHSV-positive signal is generally widespread throughout the lung and heart, in particular. In order to validate the technique, samples from 118 negative and 128 positive horse cases, including all nine known serotypes, were immunostained. All of the positive cases were confirmed by means of virus isolation. Negative horse samples were obtained from countries where African horsesickness does not occur. None of the negative cases stained positive and all the positive cases were correctly identified. Therefore, there was 100 % concordance between immunohisto chemistry (when applied to formalin-fixed, paraffin-embedded heart and/or lung and/or spleen tissues from positive horse cases that had been archived for less than 10 years) and virus isolation results. Heart and lung had consistently more positive signal than spleen. The Hamblin antiserum did not cross-react with closely-related orbiviruses (specifically equine encephalosis virus and bluetongue virus) in selected horse and sheep tissues, respectively. Characteristic positive staining was observed in lung, heart and spleen samples from two dogs that died of African horsesickness. Positive signal was not affected by long-term storage in formaldehyde (up to 365 days). Also, specific positive staining could be detected in heart and/or lung and/or spleen samples in more than 95 % of positive horses where tissue blocks had been stored for between 10 and 83 years. The principal target cells in the horse and dog cases were microvascular endothelial cells, intravascular monocyte-macrophages and, to a lesser extent, interstitial macrophages in lung, spleen and liver, in particular. Positive staining is intracytoplasmic with a bead/dot and/or granular character. Beads, dots or granules may occur singly or in clusters. Occasionally, linear deposits of positive signal delineate segments of capillary vessels. The veterinary pathologist must look for characteristic positive signal in target cells, because, occasionally, certain bacteria (Rhodococcus equi and Helicobacter sp.) cross-react with the Hamblin antiserum. Clearly, the test is highly sensitive, specific and robust, sufficiently so for the routine diagnosis of African horsesickness virus. / Dissertation (MSc)--University of Pretoria, 2008. / Paraclinical Sciences / unrestricted

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

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

There have been numerous reports of vaccinated horses that contract fatal African horse sickness due to African horse sickness virus (AHSV) serotypes that are included in the current commercial vaccine used in southern Africa, which emphasizes the importance of thorough characterization of the equine immune response to AHSV. In particular, there are concerns about possible interference between vaccine strains in the polyvalent vaccine which led us to hypothesise that the administration of individual AHSV serotypes could induce a better immunity to individual serotypes than that achieved with the current polyvalent vaccines. There is also little published information describing the half-life of maternally derived neutralising antibody in foals to the nine AHSV serotypes. This is important for revising and developing vaccination protocols for foals of vaccinated mares. Given the lethality of both natural and experimental AHSV infections in horses, several aspects of immunity induced by different types of AHSV vaccines were evaluated. The neutralising antibody response of horses (foals) immunized with a commercial modified live virus (MLV) AHSV vaccine was evaluated and compared to the immune response elicited to monovalent MLV AHSV serotypes. Foals were immunized with either the polyvalent AHSV vaccine, or one of four monovalent vaccines containing individual AHSV serotypes 1, 4, 7 and 8. There were marked differences in the immunogenicity of individual virus serotypes contained in the vaccine. Foals more consistently seroconverted to AHSV 1 and responses to other serotypes were highly variable, and often weak or not detected. The serotype-specific responses of foals given the monovalent MLV vaccines were similar to those of foals given the polyvalent preparation suggesting that there is no apparent enhanced immune response through the administration of a monovalent vaccine as opposed to the polyvalent vaccine. Furthermore, the immunogenicity of individual AHSV serotypes contained in the commercial MLV vaccine varies remarkably. Neutralising antibody titres to the 9 known serotypes of AHSV were determined in a cohort of brood mares that were regularly vaccinated with the MLV AHSV vaccine, and the passive transfer and rate of decay of maternal antibody to the individual virus serotypes in their foals were measured. Similar to the data obtained from immunized foals, there was marked variation in the neutralising antibody response of the mares to individual AHSV serotypes even after repeated vaccination. This was mirrored in the duration of maternally-derived antibodies in their respective foals. In an effort to further characterize cellular immune (CMI) responses to AHSV, the immunity in horses induced by an experimental canarypox virus vectored recombinant (ALVAC®-AHSV4) vaccine was characterised. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme-linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC®-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. Flow cytometry demonstrated that this vaccine induced mainly CD8+ T-cells, able to recognize multiple T-cell epitopes throughout all of VP2 and only the N-terminus portion of VP5. In summary, the antibody and cellular response of horses to different AHSV vaccines was evaluated and compared. The results are relevant to the design of more efficacious AHSV vaccines and to identification of protective immunity in horses to this virus. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted

African horse sickness (AHS) is a non-contagious viral disease transmitted by an arthropod vector and is endemic to sub-Saharan Africa. The disease affects all equine species, but is more severe in horses and other equid species not native to Africa. Vaccination is the only demonstrated means of its prevention. The horse-owning public provides much anecdotal evidence of prophylactic strategies, such as repellents, stabling, alternate hosts, traps, paraffin, blankets, smoke or fans. The present study investigated the relationship of these strategies to the incidence of AHS, and evaluated alternate hosts, wind speed and repellents on the activity of males and females of the different Culicoides species.. Cypermethrin and citronella-containing repellents repelled the most female midges. Sheep and cattle offer an alternate blood meal to gravid and nulliparous female midges. Fans are very effective in keeping midges away from horses. Methods are summarised for the horse owner to implement in addition to vaccination to prevent AHS. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

African horse sickness virus causes a non-contagious, infectious disease of equids. It is epizootic to sub-Saharan Africa and parts of the Middle East. The epizootics caused by the virus have caused widespread devastation amongst equids worldwide. Fortunately no epizootic has lasted more than 5 years outside of sub- Saharan Africa. It is vectored by species of Culicoides midges (Diptera: Ceratopogonidae) and most importantly by the two Avarita species of C. imicola Keiffer and C. bolitinos Meiswinkel. The literature pertaining to the study and research of the virus, the disease and the vectors is reviewed. Models allowing prediction of future possible outbreaks as well as details of control strategies and findings of researchers are presented and discussed. The virus needs a long term reservoir host in which to overwinter and various theories are discussed. Control measures in South Africa are suggested so that outbreaks of the disease can be reduced. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermarizburg, 2008.

African horse sickness (AHS) was reported in the south-western Khomas Region, central Namibia (22° 24.063´ S, 17° 01.791´ E; 23° 32.617´ S, 15° 53.870´ E), contrary to expectations that the arid conditions in the area would limit its occurrence. This prompted investigation into the occurrence of AHS in horses, a possible reservoir animal, the Hartmann’s mountain zebra (Equus zebra. hartmannae) and the occurrence of the Culicoides midge vector (Diptera: Ceratopogonidae) of the disease in the area. Questionnaires were used to explore the geographic characteristics of the study area, the occurrence of an expected AHS virus reservoir animal, E. z. hartmannae and AHS in horses in the study area. According to the questionnaire, rainfall patterns seem to follow topography of the area, where the north-east formed the higher rainfall (420 mm/a) high-ground and the south-western formed the lower rainfall (120 mm/a) pediment zone in the south-west. Cases of AHS occurred mostly in mid-rainfall zones. E. z. hartmannae were present throughout the area. They migrated from the southwest towards the north-eastern high-grounds during droughts, presumably along ephemeral river beds. E. z. hartmannae were sampled for blood and tissues and analysed for evidence of African Horse Sickness Virus (AHSV) infection by indirect ELISA, RT-PCR and virus isolation techniques. All useable samples tested positive for anti-AHSV antibodies. Viral RNA was demonstrated in 26% of all the zebra sampled. No viable viruses were isolated from these samples, however further research is required, as difficult sampling conditions may have yielded false-negatives. From 6 July to 21 September 2009, Culicoides midges were collected during the dry winter season in suction UV-light traps installed at five selected sites along a rainfall gradient. In 38 collections, a total of 9091 Culicoides individuals, representing 25 species were collected. The dominance of the proven vector of AHSV, Culicoides imicola Kieffer, varied in dominance from 94% near Windhoek with high altitude and relatively higher annual rainfall, to 12% at the site situated farthest southwest, with the lowest altitude and annual rainfall. From what was observed of the occurrence of AHS in horses, E. z. hartmannae and the distribution and abundance of the AHSV vector (Culicoides spp.), it was concluded that AHS can be maintained in the south-western Khomas Region even in the lowest mean annual rainfall zones. / Thesis (MSc (Environmental Sciences))--North-West University, Potchefstroom Campus, 2012