Foot-and-mouth disease (FMD) is a highly contagious, economically important disease of cloven-hoofed animals (3). The causative agent, foot-and-mouth disease virus (FMDV), belongs to the
Although vaccines for FMD have been available for seven decades, disease outbreaks have still occurred in many regions of the world (30). One of the main problems in controlling FMD is the broad genetic diversity of the virus, which makes the prevention of the disease difficult (32). Within each FMDV serotype there are multiple antigenic variants. No cross-protection between the serotypes was recorded for vaccines and also within the same serotype vaccine-derived protection can be limited (13).
Understanding the FMDV molecular epidemiology lineage is mainly dependent on the phylogenetic relationship of different mutations of VP1, which is a highly variable capsid protein accommodating the relevant antigenic domains (22, 23). VP1 phylogeny determines the serotype, topotypes, and sub-lineages of the virus based on the percentage of sequence identity between different iterations of this gene, which determines the genetic distances and the phylogenetic clustering of the viruses (9, 15, 16, 24, 27, 38).
FMD virus has a quasispecies nature; its evolutionary change rate is high, and it proceeds through natural selection rapidly as it is associated with high mutation and substitution rates and deficient repair mechanisms, which leads to new strains of viruses arising that differ completely from the circulating strain and render the vaccine strains ineffective. Estimates of the mutation rates of the virus and the nucleotide substitutions in the VP1 gene between closely related FMDV isolates are important not only for understanding the virus’ evolution and estimation of the interval between isolates but also for selection of the appropriate vaccine (10, 28, 32, 40).
In Egypt, routine prophylactic vaccination for FMD is usually conducted with local serotypes of the newly emerging strains, and recently a trivalent vaccine (A, O, and SAT2) was used as reported in the WRLFMD-Egypt database. The aim of the study was to trace the different FMDV outbreaks in Egypt to understand their epidemiology and evolution and to understand the situation of the vaccine strains compared with the circulating serotypes by using the data available on Egyptian FMD outbreaks from GenBank and WRLFMD.
A meta-analysis was conducted on VP1 sequence datasets of A, O, and SAT2 circulating in Egypt. The data was obtained from GenBank from 2006–2018 (supplementary Table 1), and complemented only by one sequence for serotype O identified in 1993 (Eu553840.1 FMD-O/EGY/3/93). The short sequences were excluded, the nucleotide sequences were manually adjusted to equal numbers to avoid any variation arising from different nucleotide numbers, and they were also adjusted to the coding frame. The sequences used numbered 45 for serotype O (390 nucleotides starting from amino acid 1), 14 for serotype A (408 nucleotides starting from amino acid 79) and 25 for SAT2 (504 nucleotides starting from amino acid 49). The data of different outbreaks in Egypt were obtained from the WRLFMD report for Egypt and include the vaccine strains used (for which data were recorded from 2012) (supplementary Table 1).
Year distribution of O, A, and SAT2 serotypes of FMDV and vaccine strains in Egypt based on the data in GenBank and WRLFMD
SAT2 | O | A | Vaccine | ||||
---|---|---|---|---|---|---|---|
GenBank | WRL | GenBank | WRL | GenBank | WRL | ||
2006 | 0 | 0 | 0 | 1 | 0 | 5 | |
2007 | 0 | 0 | 0 | 8 | 0 | 3 | |
2008 | 0 | 0 | 0 | 5 | 0 | 0 | |
2009 | 5 – the serotype recorded in GenBank and missing in WRL | 0 | 6 | 19 | 0 | 9 | |
2010 | 0 | 0 | 2 – the serotype recorded in GenBank and missing in WRL | 0 | 0 | 3 | |
2011 | 0 | 0 | 0 | 3 | 1 | 4 | |
2012 | 17 | 19 | 1 | 12 | 0 | 4 | O, A, SAT2 |
2013 | 2 – the serotype recorded in GenBank and missing in WRL | 0 | 1 – the serotype recorded in GenBank and missing in WRL | 0 | 2 | 10 | A |
2014 | 1 | 6 | 4 | 21 | 3 | 4 | O, A, SAT2 |
O, SAT2 | |||||||
2015 | 0 | 0 | 0 | 6 | 3 – the serotype recorded in GenBank and missing in WRL | 0 | No vaccine |
2016 | 0 | 1 | 9 | 12 | 3 | 1 | O |
O, A, SAT2 | |||||||
2017 | 0 | 1 | 19 | 22 | 0 | 1 | O, A |
2018 | 0 | 6 | 0 | 1 | 2 | 2 | O, A, SAT2 |
The VP1 sequences of FMDV were aligned using MUSCLE in MEGA X (19) on translated sequences, and amino acids were aligned and substituted using this application. An unrooted maximum likelihood phylogenetic tree including four rate categories was constructed using MEGA X, the robustness of the tree topology was assessed with 1,000 bootstrap replicates, and all parameters were estimated from the data. Gamma distribution with invariant sites (G+I) was used to model evolutionary rate differences among sites. The phylogeny and molecular evolution were simultaneously estimated also using MEGA X. The GTR model of nucleotide substitution was applied with gamma-distributed rates among sites and a proportion of invariant sites.
A comparison of the GenBank database and that of the WRL revealed the absence of the SAT2 serotype in 2009 and serotype O in 2010 from the WRL database and its inclusion in GenBank. Similarly, in 2013 serotypes SAT2 and O were only reported in GenBank. Also, in 2015, serotype A was only reported in GenBank and serotype O only in the WRL records, and no vaccine strain was recorded (Table 1, supplementary Table 1).
Virus strains recorded within the same year also varied genetically as Fig. 1 shows; the overall distance among FMD serotype O VP1 sequences of 2009 is 0.1, there are 22 amino acid substitutions among them, and they include two subclades. In the 2014 sequences, the distance is 0.01, five amino acid substitutions are evident, and they comprise two subclades separating the vaccine strain from the circulating virus strains. In 2016, the distance between them is 0.01 and 10 amino acid differences are apparent, and they devolve into two subclades. In 2017, the distance is 0.01 with five amino acid differences among them. Interestingly, no repeats occur in the substituted amino acids among the circulating viruses except for amino acid 59 in 2009 and 2017 and amino acid 107 in 2009 and 2014.
Diversity was also seen in serotype A VP1 sequences as Fig. 2 diagrammatises; although the number of sequences included in the tree is small and the overall distance among the virus genotypes circulating from 2013 to 2018 is 0.03, amino acid substitutions within the same year were recorded. These were especially prevalent in 2015 outbreak strains, where 18 amino acid substitutions were noted, and there were four in 2018, two in 2014, and only one in 2016 sequences.
The SAT2 strain VP1 gene also showed itself to be varied, and Fig. 3 presents its diversity. The overall distance between the virus genotypes from 2012 to 2018 is 0.02; two major clusters exist for SAT2 separating the viruses of 2012 from those of 2014 and indicating great genetic variation among the circulating virus strains within the same year. The location of one vaccine strain (JX570617.1FMD-SAT2-EGY/2/2012) is far from the circulating virus and is placed in a different subclade. The number of amino acid substitutions among the viruses in 2012 is 25, and in 2014 it is 26.
Maximum likelihood estimate of substitution matrix-A among O, A, and SAT2 serotypes circulating in Egypt
O serotype | A serotype | SAT2 serotype | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
A | T/U | C | G | A | T/U | C | G | A | T/U | C | G | |
- | - | - | ||||||||||
- | - | - | ||||||||||
- | - | - | ||||||||||
- | - | - |
Bold letters indicate transitional substitutions,
N.B. The substitution matrix covered the virus isolates from 1993 to 2018 for the O serotype, from 2009 to 2018 for the A serotype, and from 2012 to 2018 for the SAT2 serotype
Mean evolutionary rates of the O, A, and SAT2 serotypes
O | A | SAT2 | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
From/To | A | T | C | G | A | T | C | G | A | T | C | G | |
Mean evolutionary rates | 0.03 | 0.25 | 0.82 | 2.90 | 0.01 | 0.11 | 0.61 | 3.28 | - | 3.16 | 3.16 | 18.67 |
For successful control of FMD in developing countries many challenges must be overcome. There is always a variable gap between the actual state of the disease and countries’ announcements to the World Organisation for Animal Health (OIE), members of which most countries are (33). Discrepancy between the Egyptian FMD data WRL and GenBank in some years caused the actual vaccine serotypes to be missed, as observed in 2013 and 2015 (supplementary Table 1), and subsequent more extensive virus dissemination led to infection of more varied hosts or reservoirs, the emergence of new variants of the virus and ensuing difficulty in the control of the disease, as described by Parthiban
The sequence coding for VP1 is 627–657 nucleotides (according to the serotype) and this has been used in molecular epidemiology and investigation of the evolution of FMDV. The virus has a high mutation rate of between one and eight nucleotides per replication. Within the VP1 gene, there is a highly variable nucleotide sequence with a higher mutation rate that varies among the subtypes (codons 130–171); this region called the G-H loop represents an important immunogenic site of the virus (42). In our study, SAT2 has the highest amino acid substitution per year at this site, serotype A having less, and serotype O the least. Similarly, Lycett
The maximum likelihood estimate of the substitution matrix of VP1-coding segments is highest in serotype SAT2 and lowest in serotype O. The number of mutations accumulate over one year with altered amino acid residues, especially at the immunogenic site, which leads to diversity of serotype and antigenic shift. This shift could be associated with a widening host range and an ability of the virus to infect animals with a small dose, and could lead to invalid vaccination as described by Kitching (3). Consequently, a different strategy is required for selection of the vaccine strain of the three circulating serotypes. Strategy formulation is a contributor to the difficulty of FMD outbreak control when the virus remains in circulation in the vaccinated population (31).
The study of the evolutionary history and dynamics of FMD viruses along boundaries, in different countries or at continent level is important for better understanding of the basic epidemiological aspects of the virus and the geographical basis of the functional divergence of the virus serotypes (3, 14, 17, 18, 21, 35, 36). However, with recovery of live virus 28 days after acute infection and the persistence of FMDV in cattle in the oropharynx for years (3, 26), local strains of the viruses evolve with different immunological identity. Additionally, Arzt