Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

Determining the epitope dominance on the capsid of a serotype SAT2 foot-and-mouth disease virus by mutational analyses.

UNLABELLED: Monoclonal-antibody (MAb)-resistant mutants were used to map antigenic sites on foot-and-mouth disease virus (FMDV), which resulted in the identification of neutralizing epitopes in the flexible ßG-ßH loop in VP1. For FMDV SAT2 viruses, studies have shown that at least two antigenic sites exist. By use of an infectious SAT2 cDNA clone, 10 structurally exposed and highly variable loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of SAT2/Zimbabwe (ZIM)/7/83 (topotype II) and replaced with the corresponding regions of SAT2/Kruger National Park (KNP)/19/89 (topotype I). Virus neutralization assays using convalescent-phase antisera raised against the parental virus, SAT2/ZIM/7/83, indicated that the mutant virus containing the TQQS-to-ETPV mutation in the N-terminal part of the ßG-ßH loop of VP1 showed not only a significant increase in the neutralization titer but also an increase in the index of avidity to the convalescent-phase antisera. Furthermore, antigenic profiling of the epitope-replaced and parental viruses with nonneutralizing SAT2-specific MAbs led to the identification of two nonneutralizing antigenic regions. Both regions were mapped to incorporate residues 71 to 72 of VP2 as the major contact point. The binding footprint of one of the antigenic regions encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 48 to 50 of VP1, and the second antigenic region encompasses residues 71 to 72 and 133 to 134 of VP2 and residues 84 to 86 and 109 to 11 of VP1. This is the first time that antigenic regions encompassing residues 71 to 72 of VP2 have been identified on the capsid of a SAT2 FMDV. IMPORTANCE: Monoclonal-antibody-resistant mutants have traditionally been used to map antigenic sites on foot-and-mouth disease virus (FMDV). However, for SAT2-type viruses, which are responsible for most of the FMD outbreaks in Africa and are the most varied of all seven serotypes, only two antigenic sites have been identified. We have followed a unique approach using an infectious SAT2 cDNA genome-length clone. Ten structurally surface-exposed, highly varied loops were identified as putative antigenic sites on the VP1, VP2, and VP3 capsid proteins of the SAT2/ZIM/7/83 virus. These regions were replaced with the corresponding regions of an antigenically disparate virus, SAT2/KNP/19/89. Antigenic profiling of the epitope-replaced and parental viruses with SAT2-specific MAbs led to the identification of two unique antibody-binding footprints on the SAT2 capsid. In this report, evidence for the structural engineering of antigenic sites of a SAT2 capsid to broaden cross-reactivity with antisera is provided.

Challenges and prospects for the control of foot-and-mouth disease: an African perspective.

The epidemiology of foot-and-mouth disease (FMD) in Africa is unique in the sense that six of the seven serotypes of FMD viruses (Southern African Territories [SAT] 1, SAT2, SAT3, A, O, and C), with the exception of Asia-1, have occurred in the last decade. Due to underreporting of FMD, the current strains circulating throughout sub-Saharan Africa are in many cases unknown. For SAT1, SAT2, and serotype A viruses, the genetic diversity is reflected in antigenic variation, and indications are that vaccine strains may be needed for each topotype. This has serious implications for control using vaccines and for choice of strains to include in regional antigen banks. The epidemiology is further complicated by the fact that SAT1, SAT2, and SAT3 viruses are maintained and spread by wildlife, persistently infecting African buffalo in particular. Although the precise mechanism of transmission of FMD from buffalo to cattle is not well understood, it is facilitated by direct contact between these two species. Once cattle are infected they may maintain SAT infections without the further involvement of buffalo. No single strategy for control of FMD in Africa is applicable. Decision on the most effective regional control strategy should focus on an ecosystem approach, identification of primary endemic areas, animal husbandry practices, climate, and animal movement. Within each ecosystem, human behavior could be integrated in disease control planning. Different regions in sub-Saharan Africa are at different developmental stages and are thus facing unique challenges and priorities in terms of veterinary disease control. Many science-based options targeting improved vaccinology, diagnostics, and other control measures have been described. This review therefore aims to emphasize, on one hand, the progress that has been achieved in the development of new technologies, including research towards improved tailored vaccines, appropriate vaccine strain selection, vaccine potency, and diagnostics, and how it relates to the conditions in Africa. On the other hand, we focus on the unique epidemiological, ecological, livestock farming and marketing, socioeconomic, and governance issues that constrain effective FMD control. Any such new technologies should have the availability of safe livestock products for trade as the ultimate goal.

Mapping of antigenic determinants on a SAT2 foot-and-mouth disease virus using chicken single-chain antibody fragments.

Recombinant single-chain variable fragments (scFvs) of antibodies make it possible to localize antigenic and immunogenic determinants, identify protective epitopes and can be exploited for the design of improved diagnostic tests and vaccines. A neutralizing epitope, as well as other potential antigenic sites of a SAT2 foot-and-mouth disease virus (FMDV) were identified using phage-displayed scFvs. Three unique ZIM/7/83-specific scFvs, designated scFv1, scFv2 and scFv3, were isolated. Further characterization of these scFvs revealed that only scFv2 was capable of neutralizing the ZIM/7/83 virus and was used to generate neutralization-resistant virus variants. Sequence analysis of the P1 region of virus escaping neutralization revealed a residue change from His to Arg at position 159 of the VP1 protein. Residue 159 is not only surface exposed but is also located at the C-terminal base of the G-H loop, a known immunogenic region of FMDV. A synthetic peptide, of which the sequence corresponded to the predicted antigenic site of the VP1 G-H loop of ZIM/7/83, inhibited binding of scFv2 to ZIM/7/83 in a concentration-dependent manner. This region can therefore be considered in the design of SAT2 vaccine seed viruses for the regional control of FMD in Africa.