1 Onderstepoort Veterinary Institute, 0110 Onderstepoort, South Africa
2 Division of Biochemistry, School for Chemistry and Biochemistry, University of Potchefstroom for CHE, 2520 Potchefstroom, South Africa
Correspondence
Albie van Dijk
albievandijk{at}hotmail.com
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ABSTRACT |
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The GenBank accession numbers of the AHSV VP2 gene sequences reported in this paper are: AHSV-1 VP2, AY163329; AHSV-2 VP2, AY163332; AHSV-5 VP2, AY163331; AHSV-7 VP2, AY163330; AHSV-8 VP2, AY163333.
Present address: Institute of Biotechnology and Department of Biosciences, PO Box 56, 00014 Helsinki, Finland.
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INTRODUCTION |
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The availability of a complete set of cloned, sequenced and analysed VP2 genes and proteins for each of the nine AHSV serotypes will be a milestone towards the development of a complete repertoire of recombinant vaccines and molecular diagnostic reagents for AHS. Therefore, the main aim of the work reported in this paper was to generate a first complete set of full-length VP2 gene clones and sequence data for all nine AHSV serotypes. This paper describes the cloning, sequencing and analysis of full-length cDNA copies of the VP2 genes of all nine AHSV reference serotypes. Baculovirus expression of six of the cloned genes verified that they have full open reading frames. By sequencing the cloned VP2 genes of five serotypes, namely 1, 2, 5, 7 and 8, and combining our data with that of serotypes 3, 4, 6 and 9, which have been previously sequenced, we were able to carry out the first analysis of a complete VP2 sequence data set for a species of the Orbivirus genus.
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METHODS |
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Cloning of full-length VP2 cDNA.
The complete set of genome segments for each of the nine AHSV serotypes was amplified using a sequence-independent dsRNA amplification method (Potgieter et al., 2002). The resulting PCR amplicons of the full genomes (all 10 segments) of each serotype were separated on a 1 % agarose gel. The VP2 gene amplicons of dsRNA segment 2 for each of the serotypes was purified from the agarose gel using a Gel Extraction Kit (Qiagen). Resulting full-length VP2 gene amplicons were cloned into the PCR cloning plasmid pCR2.1 (Invitrogen). Positive VP2-gene cDNA clones were identified based on insert size (approximately 3·2 kb) and the respective restriction enzyme profiles of the individual VP2 genes.
Sequencing and sequence analysis.
Separate cloned VP2 cDNAs were digested with several restriction enzymes. The resulting fragments were subcloned into the vector pGEM3Zf+ (Promega). Subclones were sequenced using M13 forward and reverse primers (Promega) and the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 on an ABI PRISM 377 DNA sequencer (both from Perkin-Elmer Applied Biosystems). Sequence assembly and analysis, as well as multiple alignment of deduced amino acid sequences of the cloned VP2 genes and phylogenetic analysis, were performed using DNAMAN Software (Lynnon Biosoft).
Baculovirus expression.
The cloned VP2 genes were cloned into a baculovirus expression vector, pFastBac1 (Life Technologies). Using the Bac-to-Bac expression system (Life Technologies), recombinant viruses were prepared following the manufacturer's specifications. Proteins from cells infected with recombinant viruses were labelled with [35S]methionine for 30 min at 72 h after infection. After labelling, the cells were harvested, washed twice in PBS and their total protein content was separated by SDS-PAGE and analysed by autoradiography. The dried radioactive gels were exposed overnight to Hyperfilm MP X-Ray film (Amersham Life Sciences).
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RESULTS |
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The nine genomes of the AHSV reference strains could each be amplified in a single amplification reaction using a modified, sequence-independent dsRNA cloning procedure. The result for the AHSV-1 genome is shown in Fig. 1. The data for the other eight genomes are not shown. The VP2 amplicons were purified from the complete amplified genomes separated on agarose gels and cloned into pCR2.1. The cloned VP2 genes could easily be identified based on insert size, restriction enzyme patterns, sequencing of the terminal ends of the cloned cDNA and by hybridization. Previously, the identity of these cloned AHSV VP2 genes had been confirmed by using them as digoxygenin-labelled probes, which hybridized in a serotype-specific manner to RNA from the original reference viruses as well as those from field isolates of the same serotypes (Koekemoer et al., 2000
).
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DISCUSSION |
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Phylogenetic analysis of the nine AHSV VP2s grouped together VP2s of serotypes that show serological cross-reaction. Phylogenetic analysis, which included the VP2 amino acid sequences of some other orbiviruses (BTV, EHDV and Chuzan virus), showed very low homology between AHSV VP2 amino acid sequences and the VP2 sequences of these orbiviruses. Chuzan virus was, however, more closely related to AHSV than BTV and EHDV based on the VP2 amino acid sequences (Fig. 4B). Low identity between serotypes was demonstrated for specific regions within the VP2 amino acid sequences that have been shown to be antigenic and play a role in virus neutralization (Martinez-Torrecuadrada et al., 1994
; Bentley et al., 2000
; Venter et al., 2000
).
The multiple alignment of the VP2 amino acid sequences of all nine AHSV serotypes showed that the homology between the different serotypes varied from 47·6 to 71·4 %. The sequence data proves conclusively that VP2 of AHSV is the most variable protein among serotypes. The low homology between the nucleic acid sequences (results not shown) complements published hybridization data showing that partial and full-length VP2 gene probes hybridized in a serotype-specific manner to dsRNA from its cognate serotype (Bremer et al., 1990; Koekemoer et al., 2000
). The NS3 protein of AHSV has recently been shown to be the second most variable protein, with variation of between 1·8 and 36·3 % across serotypes (Van Niekerk et al., 2001
).
The multiple alignment of the nine AHSV VP2 amino acid sequences also showed regions of low identity between VP2 amino acid sequences. It is noteworthy that it is within some of these regions that antigenic regions have been identified on VP2 of AHSV serotypes 3, 4 and 9. Bentley et al. (2000) identified various antigenic regions using recombinant AHSV-3 VP2 phage display libraries with various antisera to AHSV. Antigenic regions were also found using truncated baculovirus-expressed AHSV-9 VP2 proteins and immunoblotting with antiserum to AHSV-9 (Venter et al., 2000
). Pepscan analysis of AHSV-4 VP2 peptides identified antigenic regions of peptides from certain regions that induce neutralizing antibodies in rabbits (Martinez-Torrecuadrada et al., 2001
). Interestingly, most of these antigenic and neutralizing sites were found on the various VP2s between aa 252 and 488. This region not only shows significant low identity between the nine AHSV serotypes but is also mostly hydrophilic, suggesting that these sites could be located on the surface of the virion. Furthermore, within these antigenic regions there is higher identity between the VP2 amino acid sequences of serotypes that show serological cross-reaction. In general, there is also more homology between the VP2s of the serotypes that show serological cross-reaction (Fig. 4A
). This may explain the serological cross-reaction between serotypes, since the VP2 protein determines serotype. However, it should be noted that the abovementioned studies were performed with linear peptides and not with full-length VP2 in its natural conformation. Bentley et al. (2000)
also found a non-continuous epitope on AHSV-3 VP2 using a random peptide library. Since BTV VP2 contains a serotype-specific antigenic region at approximately the same amino acid residues, namely aa 328335 (Gould & Eaton, 1990
) and aa 327402 (Demaula et al., 1993
), it seems possible that antigenic determinants in different orbivirus VP2s might be located in approximately the same region. The use of recombinant peptides from these regions from each serotype for diagnostic applications could, therefore, be an informative area for further investigation.
The importance of the availability of a complete set of full-length AHSV VP2 cDNA clones for each of the nine serotypes for recombinant vaccine development is underscored by the fact that thus far full protection against disease has only been achieved with full-length, soluble baculovirus-expressed AHSV VP2, notably for serotypes 4 and 5 (Roy et al., 1996; Martinez-Torrecuadrada et al., 1996
; Du Plessis et al., 1998
; Scanlen et al., 2002
). We are now developing recombinant VP2-based vaccines for all nine serotypes of AHSV using our set of full-length cDNA copies of the VP2 genes described in this paper.
The value of the diagnostic and epidemiological applications of this first full set of AHSV VP2 clones and sequence data includes the possibility of speeding up and extending procedures for serotyping and topotyping of isolates, serum samples and midge collections. It comprises the development of molecular methods and reagents for serotyping, such as serotype-specific probes and RT-PCR procedures, as well as generating phylogenetic data sets for molecular epidemiology. In fact, the VP2 gene set generated in this study has already enabled us to demonstrate proof of concept for the development of serotype-specific probes (Koekemoer et al., 2000). Using an incomplete VP2 sequence data set, Sailleau et al. (2000)
described the development of a serotype-specific RT-PCR for AHSV. Their method was based on small regions of sequence within the VP2 nucleotide sequences and required eight separate PCR reactions to be performed to determine the serotype of one sample. We envisage that multiple alignment of the full-length nucleic acid sequences of all nine AHSV VP2 genes will enable us to develop a set of primers to amplify a specific region within the VP2 gene of all nine serotypes of AHSV by RT-PCR in a one-step single reaction and generate sequence data sets for the VP2 genes of field strains for phylogenetic and topotyping analyses.
The data described in this paper are the first for a full set of VP2 genes of any orbivirus. The completion of expression, sequencing and phylogenetic analysis of this set of AHSV VP2 genes sets the scene for the development of complete repertoires of new vaccines, the identification and characterization of antigenic regions and the development of molecular diagnostic and epidemiological tools to improve the prevention, control, diagnosis and surveillance of AHS.
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Received 23 October 2002;
accepted 14 January 2003.
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