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.