Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes

Abstract

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana, based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

Keywords: BTV; Bluetongue virus; VP2; antigenic cartography; cross-serotype antibodies; orbivirus; orbivirus serotypes; plant expressed proteins.

Figure 1

Phylogenetic tree illustrating relationships between VP2 aa sequences of different BTV serotypes. Figure 1: A neighbour-joining phylogenetic tree constructed with aa sequences of VP2(OC1) of BTV-1 to BTV-27 depicting phylogenetic groupings. The tree was generated using the p-distance algorithm (pairwise deletion) implemented in the MEGA X software program. VP2/Seg-2 nucleotypes are indicated as previously reported [41] although with novel serotypes BTV-25, -26 and -27 are included in nucleotype (K).

Figure 2

Three-dimensional antigenic maps illustrating cross-relationships between BTV VP2 proteins. Figure 2: Computational multidimensional scaling, with multiple repeat simulations, were used to position the seventeen rVP2 protein antigens derived from different BTV strains and topotypes, in 3-dimensional maps, based on their antigenic relationships as determined by I-ELISA, using either (A): the ten rabbit anti-BTV-rVP2 sera (Table 2) or (B): the 24 sheep anti-BTV reference sera. The coloured spheres representing rVP2 proteins from different BTV serotypes and topotypes (as indicated) are colour-coded by nucleotype (see colour key). The grey cubes represent either the ten rabbit sera (panel A) or the 24 sheep sera (panel B). The scale bar represents one antigenic unit (AU), equivalent to a two-fold change or difference in antibody titre (Tables S2 and S4, for the rabbit and sheep sera respectively). The proteins showing closer antigenic relationships, are therefore positioned closer to each other in the maps. ‘Front’ and ‘Back’ (rotated 180°) 3D views are shown.

Figure 3

Antigenic relationships between BTV serotypes: The BTV serotype antigenic map, originally developed by Erasmus (1990) [35] to summarise relationships between BTV serotypes, has been modified to include two-way cross-reactions in SNT, detected here with the rabbit anti rVP2 sera (in red) and sheep reference-antisera (in blue). A strong, two-way cross-reaction is shown by a thick black connecting line in the original map, weak reactions by a thin black connecting line and very weak reactions by a dotted black connecting line. The novel BTV serotypes BTV-25, -26 and -27 are included as nucleotype K (red box). The red arrows indicate one-way reactions by rabbit anti-rVP2 sera with BTV-26. Several other one way cross-reactions were also detected in the current study with the rabbit or sheep sera but are not shown in this figure.