Spatial distribution of foot-and-mouth disease (FMD) outbreaks in South Africa (2005-2016).

Foot-and-mouth disease (FMD) is a transboundary animal disease that has negative socioeconomic consequences including impacts on food security. In South Africa, FMD outbreaks in communal farming communities cause major livestock and human livelihood concerns; they raise apprehensions about the effectiveness of FMD control measures within the FMD protection areas. This study aimed to identify high-risk areas for FMD outbreaks at the human/domestic animal/wildlife interface of South Africa. Cuzick-Edwards tests and Kulldorff scan statistics were used to detect spatial autocorrelation and spatial-temporal clusters of FMD outbreaks for the years 2005-2016.Four high-risk clusters were identified and the spatial distribution of outbreaks in cattle were closer to game reserve fences and consistent with wildlife contacts as a main contributor of FMD occurrence. Strategic allocation of resources, focused control measures, and cooperation between the affected provinces are recommended to reduce future outbreaks. Further research is necessary to design cost-effective control strategies for FMD.

Characterization of SAT2 foot-and-mouth disease 2013/2014 outbreak viruses at the wildlife-livestock interface in South Africa.

The Southern African Territories (SAT)-type foot-and-mouth disease viruses (FMDV) are endemic to the greater Kruger National Park (KNP) area in South Africa, where they are maintained through persistent infections in African buffalo. The occurrence of FMDV within the Greater KNP area constitutes a continual threat to the livestock industry. To expand on knowledge of FMDV diversity, the genetic and antigenic relatedness of SAT2-type viruses isolated from cattle during a FMD outbreak in Mpumalanga Province in 2013 and 2014 were investigated. Cattle from twelve diptanks tested positive on polymerase chain reaction (PCR), and molecular epidemiological relationships of the viruses were determined by VP1 sequencing. Phylogenetic analysis of the SAT2 viruses from the FMD outbreak in Mpumalanga in 2013/2014 revealed their genetic relatedness to other SAT2 isolates from topotype I (South Africa, Zimbabwe and Mozambique), albeit genetically distinct from previous South African outbreak viruses (2011 and 2012) from the same topotype. The fifteen SAT2 field isolates clustered into a novel genotype with ≥98.7% nucleotide identity. High neutralization antibody titres were observed for four 2013/2014 outbreak viruses tested against the SAT2 reference antisera representative of viruses isolated from cattle and buffalo from South Africa (topotype I) and Zimbabwe (topotype II). Comparison of the antigenic relationship (r1 values) of the outbreak viruses with reference antisera indicated a good vaccine match with 90% of r1 values > 0.3. The r1 values for the 2013/2014 outbreak viruses were 0.4 and above for the three South African vaccine/reference strains. These results confirm the presence of genetic and antigenic variability in SAT2 viruses and suggest the emergence of new variants at the wildlife-livestock interface in South Africa. Continuous characterization of field viruses should be performed to identify new virus strains as epidemiological surveillance to improve vaccination efforts.

A novel method for performing antigenic vaccine matching for foot-and-mouth disease in absence of the homologous virus.

Foot-and-mouth-disease (FMD) is a highly contagious transboundary animal disease that has negative consequences on regional and international trade. Vaccination is an important approach for FMD control and an essential consideration is the degree of cross-protection conferred by the vaccine against currently circulating field viruses. The objective of this study was to evaluate a new vaccine matching technique that does not require knowledge concerning the homologous vaccine virus. As a proof of concept, the vaccine-match was assessed for 41 FMD field viruses isolated from southern Africa over a 25-year period. A diverse group of 20 SAT1 and 21 SAT2 FMDV isolates collected from cattle and wildlife during 1991-2015 were selected for this study. Virus neutralization tests were performed against two sets of pooled sera for each serotype: vaccinated cattle sera (4-16 weeks post-vaccination) and convalescent cattle sera (3 weeks post-experimental challenge). Novel r1-values were calculated as the ratio of the titre of the vaccinated sera to the titre for convalescent cattle sera. A validation r1-value was calculated based on an assumption concerning the true homologous vaccine virus. There was a strong positive correlation between r1-values for the novel and the validation methods for SAT1 viruses (Spearman's rho = 0.84, P < 0.01) and a very strong correlation for SAT2 viruses (Spearman's rho = 0.90, P < 0.01). In addition, there was moderate to good agreement between the novel and validation methods for both serotypes based on a r1-value cut-off of 0.3, which is presumed to represent a good vaccine-match. The agreement between methods using prevalence-adjusted and bias-adjusted kappa (PABAK) was 0.67 and 0.84 for SAT1 and SAT2 viruses, respectively. The new r1-value method provides a feasible, alternative vaccine matching approach that could benefit FMD control in southern Africa.

Tracking the Antigenic Evolution of Foot-and-Mouth Disease Virus.

Quantifying and predicting the antigenic characteristics of a virus is something of a holy grail for infectious disease research because of its central importance to the emergence of new strains, the severity of outbreaks, and vaccine selection. However, these characteristics are defined by a complex interplay of viral and host factors so that phylogenetic measures of viral similarity are often poorly correlated to antigenic relationships. Here, we generate antigenic phylogenies that track the phenotypic evolution of two serotypes of foot-and-mouth disease virus by combining host serology and viral sequence data to identify sites that are critical to their antigenic evolution. For serotype SAT1, we validate our antigenic phylogeny against monoclonal antibody escape mutants, which match all of the predicted antigenic sites. For serotype O, we validate it against known sites where available, and otherwise directly evaluate the impact on antigenic phenotype of substitutions in predicted sites using reverse genetics and serology. We also highlight a critical and poorly understood problem for vaccine selection by revealing qualitative differences between assays that are often used interchangeably to determine antigenic match between field viruses and vaccine strains. Our approach provides a tool to identify naturally occurring antigenic substitutions, allowing us to track the genetic diversification and associated antigenic evolution of the virus. Despite the hugely important role vaccines have played in enhancing human and animal health, vaccinology remains a conspicuously empirical science. This study advances the field by providing guidance for tuning vaccine strains via site-directed mutagenesis through this high-resolution tracking of antigenic evolution of the virus between rare major shifts in phenotype.

Analysis of SAT type foot-and-mouth disease virus capsid proteins and the identification of putative amino acid residues affecting virus stability.

Foot-and-mouth disease virus (FMDV) initiates infection by adhering to integrin receptors on target cells, followed by cell entry and disassembly of the virion through acidification within endosomes. Mild heating of the virions also leads to irreversible dissociation into pentamers, a characteristic linked to reduced vaccine efficacy. In this study, the structural stability of intra- and inter-serotype chimeric SAT2 and SAT3 virus particles to various conditions including low pH, mild temperatures or high ionic strength, was compared. Our results demonstrated that while both the SAT2 and SAT3 infectious capsids displayed different sensitivities in a series of low pH buffers, their stability profiles were comparable at high temperatures or high ionic strength conditions. Recombinant vSAT2 and intra-serotype chimeric viruses were used to map the amino acid differences in the capsid proteins of viruses with disparate low pH stabilities. Four His residues at the inter-pentamer interface were identified that change protonation states at pH 6.0. Of these, the H145 of VP3 appears to be involved in interactions with A141 in VP3 and K63 in VP2, and may be involved in orientating H142 of VP3 for interaction at the inter-pentamer interfaces.

Analysis of SAT1 type foot-and-mouth disease virus capsid proteins: influence of receptor usage on the properties of virus particles.

The three SAT serotype viruses, endemic in Africa, are well known for their difficulty to adapt to cell culture. The viral mechanism involved in foot-and-mouth disease virus (FMDV) tissue tropism and cell-entry is not well understood. A recombinant, small plaque-forming virus (vSAT1tc), derived from a tissue culture-adapted SAT1 virus (SAR/9/81tc), revealed four amino acid substitutions (VP3 Asp192→Tyr; VP3 Ser217→Ile; VP1 Ala69→Gly and VP1 Asn110→Lys) in the capsid, compared to the SAR/9/81wt isolate collected from infected impala epithelium. One substitution added a positively charged lysine residue to the short ßF-ßG loop of VP1. Furthermore, vSAT1tc displayed a high affinity for CHO-K1 cells possibly via interaction with negatively charged sulphated polysaccharides while SAT1 impala strain relied strongly on α(V)ß6 integrin receptors for cell entry. The cell culture adaptation and small plaque phenotype of vSAT1tc was accompanied by differences in particle aggregation and significant differences in acid stability. Based on limited cross neutralization data, the antigenic features seem to be unchanged. Thus, acquisition of positively charged residues in the virion may be beneficial for adaptation of SAT type field strains to cell culture.

Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective immune responses in pigs.

Chimeric foot-and-mouth disease viruses (FMDV) of which the antigenic properties can be readily manipulated is a potentially powerful approach in the control of foot-and-mouth disease (FMD) in sub-Saharan Africa. FMD vaccine application is complicated by the extensive variability of the South African Territories (SAT) type viruses, which exist as distinct genetic and antigenic variants in different geographical regions. A cross-serotype chimeric virus, vKNP/SAT2, was engineered by replacing the external capsid-encoding region (1B-1D/2A) of an infectious cDNA clone of the SAT2 vaccine strain, ZIM/7/83, with that of SAT1 virus KNP/196/91. The vKNP/SAT2 virus exhibited comparable infection kinetics, virion stability and antigenic profiles to the KNP/196/91 parental virus, thus indicating that the functions provided by the capsid can be readily exchanged between serotypes. As these qualities are necessary for vaccine manufacturing, high titres of stable chimeric virus were obtained. Chemically inactivated vaccines, formulated as double-oil-in-water emulsions, were produced from intact 146S virion particles of both the chimeric and parental viruses. Inoculation of guinea pigs with the respective vaccines induced similar antibody responses. In order to show compliance with commercial vaccine requirements, the vaccines were evaluated in a full potency test. Pigs vaccinated with the chimeric vaccine produced neutralizing antibodies and showed protection against homologous FMDV challenge, albeit not to the same extent as for the vaccine prepared from the parental virus. These results provide support that chimeric vaccines containing the external capsid of field isolates can be successfully produced and that they induce protective immune responses in FMD host species.

Sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus.

Identifying when past exposure to an infectious disease will protect against newly emerging strains is central to understanding the spread and the severity of epidemics, but the prediction of viral cross-protection remains an important unsolved problem. For foot-and-mouth disease virus (FMDV) research in particular, improved methods for predicting this cross-protection are critical for predicting the severity of outbreaks within endemic settings where multiple serotypes and subtypes commonly co-circulate, as well as for deciding whether appropriate vaccine(s) exist and how much they could mitigate the effects of any outbreak. To identify antigenic relationships and their predictors, we used linear mixed effects models to account for variation in pairwise cross-neutralization titres using only viral sequences and structural data. We identified those substitutions in surface-exposed structural proteins that are correlates of loss of cross-reactivity. These allowed prediction of both the best vaccine match for any single virus and the breadth of coverage of new vaccine candidates from their capsid sequences as effectively as or better than serology. Sub-sequences chosen by the model-building process all contained sites that are known epitopes on other serotypes. Furthermore, for the SAT1 serotype, for which epitopes have never previously been identified, we provide strong evidence--by controlling for phylogenetic structure--for the presence of three epitopes across a panel of viruses and quantify the relative significance of some individual residues in determining cross-neutralization. Identifying and quantifying the importance of sites that predict viral strain cross-reactivity not just for single viruses but across entire serotypes can help in the design of vaccines with better targeting and broader coverage. These techniques can be generalized to any infectious agents where cross-reactivity assays have been carried out. As the parameterization uses pre-existing datasets, this approach quickly and cheaply increases both our understanding of antigenic relationships and our power to control disease.

Mapping of amino acid residues responsible for adhesion of cell culture-adapted foot-and-mouth disease SAT type viruses.

Foot-and-mouth disease virus (FMDV) infects host cells by adhering to the alpha(V) subgroup of the integrin family of cellular receptors in a Arg-Gly-Asp (RGD) dependent manner. FMD viruses, propagated in non-host cell cultures are reported to acquire the ability to enter cells via alternative cell surface molecules. Sequencing analysis of SAT1 and SAT2 cell culture-adapted variants showed acquisition of positively charged amino acid residues within surface-exposed loops of the outer capsid structural proteins. The fixation of positively charged residues at position 110-112 in the beta F-beta G loop of VP1 of SAT1 isolates is thought to correlate with the acquisition of the ability to utilise alternative glycosaminoglycan (GAG) molecules for cell entry. Similarly, two SAT2 viruses that adapted readily to BHK-21 cells accumulated positively charged residues at positions 83 and 85 of the beta D-beta E loop of VP1. Both regions surround the fivefold axis of the virion. Recombinant viruses containing positively charged residues at position 110 and 112 of VP1 were able to infect CHO-K1 cells (that expresses GAG) and demonstrated increased infectivity in BHK-21 cells. Therefore, recombinant SAT viruses engineered to express substitutions that induce GAG-binding could be exploited in the rational design of vaccine seed stocks with improved growth properties in cell cultures.