Prediction and characterization of novel epitopes of serotype A foot-and-mouth disease viruses circulating in East Africa using site-directed mutagenesis.
Bottom Line: Mutations at four different positions, namely VP1-43, VP1-45, VP2-191 and VP3-132, led to significant reduction in VN titre (P value = 0.05, 0.05, 0.001 and 0.05, respectively).This is the first time, to our knowledge, that the antigenic regions encompassing amino acids VP1-43 to -45 (equivalent to antigenic site 3 in serotype O), VP2-191 and VP3-132 have been predicted as epitopes and evaluated serologically for serotype A FMDVs.This identifies novel capsid epitopes of recently circulating serotype A FMDVs in East Africa.
Affiliation: The Pirbright Institute, Ash Road, Woking, Surrey, GU24 0NF, UK.Show MeSH
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Mentions: The impact of the amino acid substitutions on sero-reactivity was assessed by VN test using the pooled post-vaccination serum (bovine) raised against rO1K/A-EA-2007 antigen. The main goal was to quantify the reduction in neutralization following mutations in the capsid of FMDV. Therefore, it was crucial to determine the VN titre of the sera against all the mutant viruses at a fixed virus dose (100 TCID50). Therefore, a two-dimensional micro-neutralization test (2D-VNT) was carried out using five different doses of the virus encompassing 100 TCID50 for this purpose. The resultant VN titres at each virus dose were used to calculate the serum titre at 100 TCID50 by regression analysis. Because getting consistent results was very important for the evaluation of the mutant viruses, each test was conducted in duplicate and repeated at least eight times. Test results showing evidence of a reduction in serum titre after mutagenesis were repeated eight more times for further confirmation. Among the 12 mutants generated in this study, only five mutants, i.e. rO1K/A-EA-2007M1 (VP1-L43A), rO1K/A-EA-2007M4 (VP1-L45P), rO1K/A-EA-2007M5 (VP2-T191A), rO1K/A-EA-2007M6 (VP2-T191D) and rO1K/A-EA-2007M10 (VP3-T132S), exhibited significant reductions in serum titre (Fig. 3). The substitution of threonine at VP2-191 to alanine or aspartic acid exhibited relatively greater (15 % and 12.5 %, respectively) reductions in serum titre as compared with the parent virus. This agrees with the report of Crowther et al. (1993) who reported ~15 % reduction in serum titre as a result of a single amino acid change. In line with this, recently, Asfor et al. (2014) evaluated this epitope for serotype O FMDV using a cDNA clone and reported ~ 30 % reduction in serum titre. Hence this residue could represent a novel epitope across several serotypes. The residues VP1-43 to -45 are in an equivalent position to antigenic site 3 in serotype O (Kitson et al., 1990). Though mar-mutant studies have been carried out in several type A viruses, this region has never been reported to be of antigenic significance. However, VP1-45 has been indicated to impact on the antigenic nature of the serotype A viruses from the Middle East (Jamal et al., 2011; Upadhyaya et al., 2014). In addition, analysis of 115 serotype A capsid sequences revealed amino acids VP1-42 to -46 to be highly variable (data not shown). The substitution of threonine at position VP3-132 led to significant reduction in serum titre whereas substitution to alanine did not have much impact, indicating certain residue changes are more powerful than others. Mutations in epitopes may also have the opposite effect, i.e. neutralizing titres may increase after mutation of capsid residues. In fact, Opperman et al. (2014) reported significantly higher VN titres in SAT epitope-replaced mutants that were related to higher avidity index. However, we did not observe significantly higher VN titre in the mutant viruses in this study. In our previous study on serotype O epitope mutants (Asfor et al., 2014) we also did not observe higher VN titre than the homologus virus. This could be due to a different serotype or strain of the virus.
Affiliation: The Pirbright Institute, Ash Road, Woking, Surrey, GU24 0NF, UK.