Fay, Petra Christel
(2018)
Antigenic and phylogenetic relationships of outer capsid protein VP2, from multiple bluetongue virus serotypes.
PhD thesis, University of Nottingham.
Abstract
Bluetongue (BT) is a non-contagious arthropod transmitted virus that infects ruminants including sheep, cattle and goats. Bluetongue virus (BTV) is economically important due to the severity of disease in naïve animals and the financial cost of the development and implementation of effective vaccines and diagnostic tests, and restrictions on animal movements and trade.
Bluetongue virus encapsidates the viral genome, composed of ten linear dsRNA genome-segments, encoding seven structural protein (VP1-VP7) and five non-structural protein (NS1-NS5). The largest-outer-coat and cell-attachment protein of bluetongue virus (BTV), is VP2 (encoded by genome segment-2 (Seg-2)), which elicits protective neutralising antibody (nAb) and total binding antibody (bAb) responses in infected animals. VP2 is the least conserved of the BTV proteins and the nAbs, which are primarily BTV serotype-specific, can be used to identify different serotypes in neutralisation-assays. To-date 27 BTV serotypes have been recognised, with several other putative serotypes currently under investigation. This study investigates the complexity of antigenic cross-reactions between BTV strains belonging to different serotypes, by neutralising and/or total binding antibodies targeting VP2.
VP2s from multiple BTV serotypes were expressed using a novel, plant-based, transient- expression system, in Nicotiana benthamiana. The expressed proteins were purified then used to inoculate IFNAR (-/-) mice, using a homologous prime boost vaccination strategy. The purified proteins were also used to immunise rabbits to obtain polyclonal antisera. Sheep polyclonal reference antisera, raised against reference strains of the different BTV serotypes, were also used in this study. All of these antisera were tested for cross-reactivity in serum neutralisation tests and by indirect ELISA. The antigenic relationships observed were compared and quantified using antigenic cartography. The antigenic data was then compared to the VP2 amino acid phylogenetic sequence data.
The VP2 proteins were shown to raise nAbs and elicit a protective immune response in IFNAR (-/-) mice. A broad range of antigenic cross-reactivities were detected using the two panels of antisera, identifying serological relationships between different serotypes. These cross-reactivities showed similarities but were not identical to the phylogenetic relationships exhibited by VP2. The evaluation of recombinant VP2s and observed antigenic differences/relationships have important implications for disease surveillance and the possibility of developing serological, type-specific, diagnostic tests and cross-reactive vaccines.
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