Simultaneous amino acid substitutions at antigenic sites drive influenza A hemagglutinin evolution

Abstract

The HA1 domain of HA, the major antigenic protein of influenza A viruses, contains all of the antigenic sites of HA and is under continual immune-driven selection. To resolve controversies on whether only a few or many residue sites of HA1 have undergone positive selection, whether positive selection at HA1 is continual or punctuated, and whether antigenic change is punctuated, we introduce an approach to analyze 2,248 HA1 sequences collected from 1968 to 2005. We identify 95 substitutions at 63 sites from 1968 to 2005 and show that each substitution occurred very rapidly. The rapid substitution and the fact that 57 of the 63 sites are antigenic sites indicate that hitchhiking plays a minor role and that most of these sites, many more than previously found, have undergone positive selection. Strikingly, 88 of the 95 substitutions occurred in groups, and multiple mutations at antigenic sites sped up the fixation process. Our results suggest that positive selection has been ongoing most of the time, not sporadic, and that multiple mutations at antigenic sites cumulatively enhance antigenic drift, indicating that antigenic change is less punctuated than recently proposed.

Fig. 1.

Frequency diagrams of six sites. (a and b) Frequency changes at residue sites 156 (a) and 145 (b) were highly dynamic. (c and d) Sites 138 (c) and 194 (d) were identified as under positive selection by Bush et al. (8) but did not undergo major frequency change over time. (e–h) Sites 83 (e), 155 (f), 172 (g), and 189 (h) each underwent three or more substitutions but were not identified by Fitch et al. (9) or Bush et al. (8) as positively selected sites.

Fig. 2.

Frequency switches. Numbers of effective and ineffective residue frequency switches are shown. Gray bars represent the effective switches, and open bars represent the ineffective ones.

Fig. 3.

Transition times. Shown is the distribution of transition times for substitutions from 1987 to 2005.

Fig. 4.

A temporal map of amino acid frequencies on sites of the detected substitutions. The detected substitutions are grouped in rows by the year of fixation; the transition of each site is given in the second column. Information on antigenic clusters, extracted from Smith et al. (17), is included as marked on the top of the columns according to the year of isolation, and the antigenic transition between two clusters is marked in the third column; the unmarked areas in the third column represent isolates devoid of antigenic cluster information in the Smith et al. study (17). The yearly frequency (year on top of each column) of the emerging amino acids in transit (amino acid residues of each site are shown on the left of each row in the second column) is presented as proportional frequency of 0∼1. The original or transited amino acids of three antigenic transitions are different between our samples and those in the Smith et al. study (17): N137S, K145N, and S193D, as indicated by †, ‡, and ∗ in the figure, respectively.