A decade of sustained selection pressure on two surface sites of the VP1 protein of Enterovirus A71 suggests that immune evasion may be an indirect driver for virulence

Abstract

Enterovirus A71 (EV-A71) is an emerging pathogen in the Enterovirus A species group. EV-A71 causes hand, foot and mouth disease (HFMD), with virulent variants exhibiting polio-like acute flaccid paralysis and other central nervous system manifestations. We analysed all enterovirus A71 complete genomes with collection dates from 2008 to mid-2018. All sub-genotypes exhibit a strong molecular clock with omega (dN/dS) suggesting strong purifying selection. In sub-genotypes B5 and C4, positive selection can be detected at two surface sites on the VP1 protein, also detected in positive selection studies performed prior to 2008. Toggling of a limited repertoire of amino acids at these positively selected residues over the last decade suggests that EV-A71 may be undergoing a sustained frequency-dependent selection process for immune evasion, raising issues for vaccine development. These same sites have also been previously implicated in virus-host binding and strain-associated severity of HFMD, suggesting that immune evasion may be an indirect driver for virulence (154 words).

Figure 1

Phylogenetic relationships within species Enterovirus A. Maximum likelihood phylogenetic tree of reference genomes in the species Enterovirus A, and representative sub-genotype sequences of EV-A71, using the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories, shape parameter = 0.5488). Bootstrap values are displayed at nodes. Red triangle: known virus causing HFMD. Scale: substitutions per site.

Figure 2

TempEst analysis of molecular clocks in genotypes B1, B5, C2, C4 and C5. x-axis: sampling dates; y-axis: root-to-tip distance on a maximum likelihood tree using the GTR + G substitution model. Sub-genotype B1 in inset. See Table 2 for correlation coefficient R values for each sub-genotype.

Figure 3

Bayesian skyline plots for sub-genotypes B5 and C4. Bayesian skylines were plotted in BEAST using the relaxed exponential clock model and the GTR + G substitution model; x-axis: collection date; y-axis: relative genetic diversity.

Figure 4

Substitution rates at the 3 codon positions, relative to the overall substitution rate, of sub-genotypes B5, C2 and C4 since 2008. Substitution rates for all three reading frames were calculated in BEAST using the relaxed exponential clock model and the GTR + G substitution model, then plotted in Tracer. Grey curves: B5; red curves: C2; blue curves: C4.

Figure 5

Positive selection in sub-genotypes B5, C2 and C4 since 2008. For panels (A) (sub-genotype C4), (B) (sub-genotype C2) and (C) (sub-genotype B5), x-axis: amino acid position in polyprotein (colours indicate consecutively the genes for VP4, VP2, VP3, VP1, 2 A, 2B, 2 C, 3 A, 3B, 3 C, 3D); y-axis: omega (dN/dS) calculated for each position in Slr. Positions crossing the statistical significance threshold are indicated and their level of statistical significance shown. Panel D shows the solved structure of the VP1/VP2/VP3 complex of a sub-genotype C4 strain (PDB accession 4YVS); red residues: position 663; yellow residues: position 710. The backbone and surface of the VP1 protein are in black, VP2 backbone in brown and VP3 in blue. Panel E shows the VP1 protein tilted 90 degrees to illustrate the location of residues 663 and 710 in the “mountain” region.

Figure 6

Maximum likelihood unrooted trees using a Poisson distance amino acid alignment showing toggling of the positively selected sites in VP1 during the evolution of sub-genotypes C4 and B5 since 2008. (A) C4 site 663 (VP1–98), black branches are E, red K, mauve G, blue V. (B) B5 site 710 (VP1–145), black branches are E, mauve G, green Q. Scale is amino acid substitutions per site. Site 663 in sub-genotype C4 toggles between amino acids E, K, G and V. Site 710 in sub-genotype B5 toggles between amino acids E, G and Q.