The 1D nucleotide sequence similarity scan (Figure 1) of the Turkish isolates of the present study reveals two well separated groups for serotype A. The two groups are most similar at position 90 nt with 8% divergence given a windows size of 20 nt and most divergent at position 490 with 31% divergence. The latter region encodes the immuno-dominant GH-loop. The diversity within each of the two serotype A groups is low within a range of 2–4%.

Serotype O, with O1/Manisa/69 as reference sequence, displays one homogeneous group, with a diversity in the range 9%.

The Bayesian tree, based on the 1D nucleotide sequence of serotype A isolates shown in Figure 2 reveals three clusters in Turkey, related to A22, IRN96 and IRN99. The most recent isolate of the A22 cluster, for which sequence information is available was collected in Iraq in 1995. The A22 cluster is distinctly separated from the IRN99 and IRN96 lineages as supported by a clade credibility value of 1.0 and the IRN96 and IRN99 clusters are separated as supported by a clade credibility value of 0.97.

The IRN96 cluster is further subdivided into two temporal divergent groups, I and II. The isolates of group I all originate from outbreaks in 1998, it was two years before this lineage was included in the Turkish FMD vaccine.

The IRN99 related viruses display a single, not further subdivided, phylogenetic structure; with the oldest isolate DQ296550 Igdir/06.99, obtained from an outbreak in June 1999 in the province Igdir, close to the Iranian border. Another member of IRN99 lineage is the isolate A/IRN/22/99# (sequence information kindly provided from IAH Pirbright) obtained from an outbreak in Iran in 1999.

The inferred phylogeny of serotype O isolates (Figure 3), with the vaccine lineage as outgroup, displayed two major clusters A and B, supported by a clade credibility value of 1.0 (Figure 3).

Cluster A is split into the two groups I and II. Group I consists of sequences derived from FMD outbreaks in Pakistan and Iran in addition to a single outbreak in January/February 2004 in the Mersin region of Turkey. The differential success, expressed as correlation to the number of substitutions, with regard to the vaccine lineage, is lowest in the group members obtained from outbreaks in Pakistan in 2002 and 2003, intermediate in the Iranian isolates from 2003 and 2004 and most advanced in the Turkish isolates from 2004.

Group II of cluster A consists of Turkish isolates obtained from outbreaks in the provinces Sivas in 2001 and Igdir in 1998. The latter province is close to the Iranian border.

The majority of Turkish isolates belong to cluster B, which can also be subdivided into two groups I and II.

Cluster I of group B was further subdivided into two lineages. One group consists of isolates, obtained only from outbreaks in Turkey and the other of isolates known as the Panasia lineage. This partition is supported by a clade credibility value of 0.45. The Turkish lineage displays an increasing differential success rate, with regard to the vaccine strain O1/Manisa/69, starting from the DQ296516 Ankara/12.99 isolate, obtained in the Ankara province in 1999, towards the DQ296528 Kutahya/05.04 isolate, obtained in the Kutahya province in 2004.

Cluster II of group B consists of viruses obtained from outbreaks in Turkey, Israel and Lebanon, with the earliest obtained from an outbreak in the Turkish Ankara region in 2001.

The examination of the deduced VP1 amino acid sequences of the serotype O isolates for selective pressures, show no significant (defined as p > 0.5) evidence for positive selection (data not shown). On the contrary, residue 151 of serotype A isolates displays significant evidence (p = 0.29) for positive selection (Figure 4 and additional file 1). Residue 151 is located in the immuno-dominant GH-loop of the VP1 molecule and therefore particularly exposed to the immune system of the host.

By investigating the inferred ancestor history of site 151 (additional file 2 and 3), it can be seen that all but two of the IRN96 isolates, DQ296532 and DQ296533, each collected from the same outbreak, have the common ancestor codon 'AGC' (Ser), whereas IRN99 has most likely the 'GGG' (Gly) codon as precursor.

Comparing the translated amino acid sequences of the whole GH-loop region, located within the residues 140–160 (Figure 5), reveals that IRN99 isolates consistently deviates from IRN96 isolates by a deletion at site 153 as well as in lineage specific conserved amino acids at the sites: 144, 166, 183, 202 and 203. The integrin-binding site Arg-Gly-Asp (RGD) 8, was in all serotype A isolates, but DQ296540, as 'RGDLGAL'-motif present.

To compare the VP1 protein secondary structure we analysed the putative hydrophobic and hydrophilic regions along the length of the VP1 amino acid sequence, using the Kyte and Doolittle algorithm 10. This revealed three different regions for the consensus sequences of the IRN99 and IRN96 lineages (Figure 6a), as well as two major disparate regions for representatives of the serotype O lineages (Figure 7a). The structural alignment of the tertiary structure, based on the consensus sequences of IRN99 and IRN96 (Figure 6b), shown 75% sequence identity, revealed appreciable structural differences between the lineages, within the regions indicated by the hydrophobicity plot. There is evidence that the IRN96 lineage has two β-sheet structures at the amino acid positions 40–55 and 105–124, where IRN99 has none, as well as a different helix-structure at position 140–159 (Figure 6b).

From the hydrophobicity plot, DQ296505 Sivas/02.01 seems to represent the most distinct lineage of the Turkish serotype O isolates (Figure 7a). However, the structural alignment between DQ296505 Sivas/02.01 and the vaccine strain O1/Manisa/69, displays a high similarity of 99% (Figure 7b).

Thirty-three serotype O (Accession numbers: DQ296497 – DQ296531) and twenty serotype A (Accession numbers: DQ296532 – DQ296552) field isolates were collected from 1998 to 2004 from bovine herds widely distributed in Turkey. All isolates were epidermal tissue samples representing vesicular lesions and had been stored in TRIZOL^©-reagent. Province and time (month/year) of collection are indicated with the accession numbers.

Tissue (100–150 mg) was homogenized in 1 ml RNApro™ Solution (Qbiogene, USA) in a Lysing Matrix D tube (Qbiogene, Inc., USA) using a FP 120 Fast Prep™ Cell Disruptor (Qbiogene, USA). Total RNA was extracted using RNeasy-Mini Kit™ (Qiagen, Germany) according to manufacturer's instructions. cDNA synthesis was done using

Ready-To-Go™ You-Prime First-Strand Beads (GE Healthcare Life Sciences, Sweden), employing the primers NV27T and random hexamers pdN₆.

Five μl of the template cDNA were added to 45 μl of the PCR reaction mixture containing 0,2 μM primers (see Table 1), 200 μM each of dATP, dCTP, dGTP and dTTP, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂ and 1 U of AmpliTaq^® Gold DNA polymerase (Applied Biosystems, UK). DNA was amplified with a DNA Thermal Cycler PE9700 (Perkin Elmer) by a two-step cycling reaction as follows: 95°C for 15 min, and five cycles of 94°C for 30 sec, 57°C for 2 min and 72°C for 30 sec, and then 35 cycles of 94°C for 30 sec, 61°C for 30 sec and 72°C for 30 sec, followed by a final extension step of 72°C for 10 min.

The resulting PCR product was examined by electrophoresis, using a 1,2% agarose gel, containing 0,005% ethidium bromide, with a separation time of 1,5 hours at 6,5 V/cm.

Amplicons were extracted and purified from agarosegel with QIAquick Gel Extraction kit (Qiagen, Germany) and cycle-sequencing was then performed by Agowa GmbH, Germany.

Sequence assembling was performed with ContigExpress (VectorNTI^©-software) and multiple alignment was performed by log-expectation comparison, using the MUSCLE (v.3.6) software 13. For the serotype A alignment manual editing at codon position 153 was done.

Similarity plots were performed using Simplot software 14 with the following parameters: For serotype A a windows size of 100 bp (step: 20 bp) with gap-stripping and Jukes-cantor correction was chosen, whereas a window of 200 bp (step:20 bp) with gap-stripping and Kimura (2-parameter) correction was chosen for serotype O.

Models of evolution were determined by hierarchical Likelihood-Ratio test of 24 substitution models, using the programs PAUP*(v. 10) 15 and MrModeltest (v. 2.2) 16.

For serotype A the HKY+G was used and Bayesian analysis was performed using MrBayes (v3.2) 17 with the following settings. The maximum likelihood model employed 2 substitution types ("nst = 2"), with base frequencies set to fixed values ("statefreqpr = fixed"). Rate variation across sites was modelled using a gamma distribution (rates = "gamma"). The Markov chain Monte Carlo search was run with 4 chains for 500000 generations, with trees begin sampled every 100 generations (the first 1000 trees were discarded as "burnin").

For serotype O the GTR+G was used and Bayesian analysis was performed using MrBayes (v3.2) 17 with the following settings. The maximum likelihood model employed 6 substitution types ("nst = 6"), with base frequencies set to variable values ("statefreqpr = dirichlet(1,1,1,1)"). Rate variation across sites was modelled using a gamma distribution (rates = "invgamma"). The Markov chain Monte Carlo search was run with 4 chains for 500000 generations, with trees begin sampled every 100 generations (the first 1000 trees were discarded as "burnin").

Positively selected sites (Codon-specific analyses of dN/dS) was identified using the Single Likelihood Ancestor Counting (SLAC) analysis 18, a modification of the Suzuki-Gojobori method 19, available at the Datamonkey web side 18. For both serotypes, the analysis was done with the HKY85 substitution model on phylogenetic trees inferred using the Neighbour-Joining method with a cut-off p value of 0.5.

Three-dimensional structure of the VP1 protein was obtained using comparative protein modelling 20, employing the CPHmodels 2.0 Server 21 and pairwise structure comparison was done using the DaliLite workbench 22. Molecule Visualization was done using RASMOL 2.7 software 23.

The structural analysis of the A serotype is based on the comparison of the translated amino acid consensus sequences from the IRN96 and IRN99 lineage. Both, IRN99 consensus and IRN96 consensus sequences, used the structure of serotype A-1061 (1ZBA:1; Resolution: 2 Å) 24 as template.

For serotype O viruses the structural analysis was based on the translated amino acid sequences of DQ296505 Sivas/02.01 and the vaccine strain O1/Manisa/69. Both used the structure of strain O-1860 (1FOD:1; Resolution: 2,6 Å) 25 as template.