Cloning, sequencing and expression of the gene that encodes the major neutralisation-specific antigen of African horsesickness virus serotype 9

Abstract

A marked improvement in the efficiency of cloning the large double stranded RNA (dsRNA) genome segments of African horsesickness virus (AHSV) was achieved when the dsRNA polyadenylation step was carried out with undenatured rather than strand-separated dsRNA. It is a prerequisite to use dsRNA of very high purity because in the presence of even trace amounts of single stranded RNA, the dsRNA appears to be poorly polyadenylated as judged by its effectiveness as a template for oligo-dT-primed cDNA synthesis. The full-length VP2 gene of AHSV-9, cloned by this approach, was sequenced and it was found to show the highest percentage identity (60%) to VP2 of AHSV-6, providing an explanation of why these two serotypes show some cross protection. The VP2 protein was also expressed in Spodoptera frugiperda (Sf9) cells by means of a baculovirus recombinant. The yield of the expressed VP2 was high, but the protein was found to be largely insoluble. Nine smaller, truncated VP2 peptides were subsequently expressed in insect cells, but no significant improvement in solubility of the peptides, as compared to that of the full-sized protein, was observed. A western immunoblot analysis of the overlapping peptides indicated the presence of a strong linear epitope located within a large hydrophilic domain between amino acids 369 and 403.

Introduction

African horsesickness virus (AHSV) is the aetiological agent of African horsesickness, a severe and often fatal viral disease of Equidae that is endemic to sub-Saharan Africa. The virus, of which nine different serotypes have been identified, is classified in the Orbivirus genus of the family Reoviridae and transmitted by arthropods of the genus Culicoides (Verwoerd et al., 1979). As in bluetongue virus (BTV), the prototype member of the Orbivirus genus, the AHSV genome is composed of ten double stranded RNA (dsRNA) segments (Bremer, 1976) encoding seven capsid proteins (VP1–7) and three nonstructural proteins (NS1–3). The segments are encapsidated in an icosahedral core particle surrounded by an outer capsid layer composed of the two major proteins VP2 and 5 (Roy et al., 1994). VP2, encoded by the second largest genome segment of the virus, is the most variable of the different capsid proteins (Bremer et al., 1990) and it is the main determinant of a serotype-specific immune response. Antibodies against VP2 are protective in vivo (Burrage et al., 1993), indicating the important role of the protein in any vaccine development strategy.

The vaccination strategy against AHS currently involves the use of polyvalent live attenuated virus vaccines. However, there are a number of concerns about the efficacy and safety of these vaccines, such as the possibility of the reversion of an avirulent virus to a virulent phenotype when subjected to backpassaging in susceptible hosts (House et al., 1992). To counteract these problems, the development of a non-replicating, VP2-based subunit vaccine for orbiviruses has been the focus of a number of investigations. In the case of BTV, VP2 isolated from purified virus (Huismans et al., 1987) or expressed by means of baculovirus recombinants (Roy et al., 1990) has been demonstrated to elicit protection in sheep against virulent viral challenge. In the case of AHSV-4 protection against AHSV was demonstrated by both inoculation with recombinant VP2 (Roy et al., 1996) and by use of a recombinant vaccinia virus expressing AHSV-4 VP2 (Stone-Marschat et al., 1996).

Most of the studies to locate VP2 neutralisation-specific domains have until recently been focused on BTV. Amino acids 328–335 of BTV-1 VP2 were identified as important serotype-specific determinants (Gould et al., 1988) whereas amino acids in the region of 208–402 were associated with serotype specificity of BTV-10 (DeMaula et al., 1993) and BTV-17 (Pierce et al., 1995). With respect to AHSV-4 the analysis of Escherichia coli expressed truncated VP2 peptides identified a linear region within amino acids 253–413 that was able to elicit neutralising antibodies in rabbits and mice (Martinez-Torrecuadrada and Casal, 1995). No similar studies with other AHSV serotypes have as yet been carried out. However, the sequences of the VP2 genes of AHSV-4 (Iwata et al., 1992b, Sakamoto et al., 1994), AHSV-3 (Vreede and Huismans, 1994) and AHSV-6 (Williams et al., 1998) have been determined. VP2 sequence information of other serotypes has been delayed mainly due to the technical difficulty of cloning genes as large as 3000 bp from virus-specified cDNA.

Some modifications are described to our previous dsRNA cloning strategy that significantly improved the efficiency of cloning genome segments of more than 2500 bp. A large number of full-length copies of the VP2 gene of AHSV-9 were cloned by this approach. The gene was then sequenced and expressed in Spodoptera frugiperda (Sf9) cells. Since the full-length VP2 protein was largely insoluble we expressed a number of smaller overlapping VP2 peptides which were analysed for solubility and the presence of linear epitopes by means of immunoblot analysis.