06 × 10−2/site/year (95% HPD 9.53 × 10−3 to 1.05 × 10−2). This is find more similar to the report (1.12 × 10−2/site/year) for VP1 sequences of A-Iran-05 viruses [13]; but higher than those reported by others [26], [27], [28], [29], [30], [31] and [32]. The high evolutionary rate of serotype A viruses in the ME is resulting in emergence of new variants in the region. An unbiased analysis of capsid sequences of the 51 A-Iran-05 viruses revealed 692 nt substitutions at 637 sites distributed

across the region (Fig. 1B). Out of these, 80.05% of nt substitutions were found to be synonymous (silent) and 19.95% were non-synonymous (non-silent). Forty seven sites were identified to have been substituted twice and four were substituted three times. At one site (VP2-134) the

first two bases of the codon were mutated encoding 5 different aa (P->T/S/L/H). This residue is located very close to residues VP2-132 and 133 that were reported as critical by mar-mutant studies for A10 virus [9]. In addition, the residue at this position has been reported to strongly influence the binding of antigenic site-2 mAbs in serotype O viruses [16]. Out of the four Ibrutinib sites with three nt substitutions (encoding 2–4 aa residues), three were present in VP3 and one in VP1 (Table 1A). The analysis of the capsid aa residues of A-Iran-05 viruses revealed 140 substitutions at 101 sites across the capsid (Fig. 2A) with some sites having 2–5 alternate aa (Table 1B). Interestingly, sequences for VP1-204 encoded five different aa and exhibited nt changes at all the three positions within the codon as did VP1-196, with changes at all the three positions of the codon giving rise to four alternative aa. In addition, the non-synonymous nt substitutions were not equally distributed across the capsid coding regions: there were several local areas where the dN/dS ratio was higher than in other parts of the sequence alignment

(Fig. 2B). One region in VP3 (57–65), two in VP2 (75–76 and 130–134) and eight regions in VP1 (52–53, 83–84, 92–105, 131–132, 137–141, 145–152, 168–171 and 192–204) had dN/dS ratio of >1 indicative of sites under strong positive selection. Investigation of aa variability GBA3 across the capsid of the A-Iran-05 viruses revealed VP4 to be highly conserved and VP1 least conserved (Fig. 3A); similar to an earlier report [13]. The residues with a score greater than 0.75 (3 in VP2, 6 in VP3 and 12 in VP1) are shown in Fig. 3B-D indicating that over 50% of the residues with very high variability scores were present in VP1 (Fig. 3A). All these residues were found to be surface-exposed, except one residue in the N-terminus of VP1 (position 28) and one in N-terminus of VP3 (position 8) (Fig. 3C and D).