Homologous recombination is very rare or absent in human influenza A virus

Abstract

To determine the extent of homologous recombination in human influenza A virus, we assembled a data set of 13,852 sequences representing all eight segments and both major circulating subtypes, H3N2 and H1N1. Using an exhaustive search and a nonparametric test for mosaic structure, we identified 315 sequences (approximately 2%) in five different RNA segments that, after a multiple-comparison correction, had statistically significant mosaic signals compatible with homologous recombination. Of these, only two contained recombinant regions of sufficient length (>100 nucleotides [nt]) that the occurrence of homologous recombination could be verified using phylogenetic methods, with the rest involving very short sequence regions (15 to 30 nt). Although this secondary analysis revealed patterns of phylogenetic incongruence compatible with the action of recombination, neither candidate recombinant was strongly supported. Given our inability to exclude the occurrence of mixed infection and template switching during amplification, laboratory artifacts provide an alternative and likely explanation for the occurrence of phylogenetic incongruence in these two cases. We therefore conclude that, if it occurs at all, homologous recombination plays only a very minor role in the evolution of human influenza A virus.

FIG. 1.

ML phylogenetic trees for nucleotide positions 202 to 2189 (minor segment) (top) and 1 to 201 and 2190 to 2347 (major segment) (bottom) of the PB2 data set of 1086 A/H3N2 sequences. All bootstrap values greater than 50% are shown. The tree is midpoint rooted for purposes of clarity only, and all branch lengths are drawn to a scale of nucleotide substitutions per site. The red sequence is the candidate recombinant sequence, with the yellow box identifying three additional sequences that cluster with A/New York/11/2003 in both phylogenies. The blue sequence is the candidate major parent (“Parent P”), and the green sequence is the candidate minor parent (“Parent Q”).

FIG. 2.

ML phylogenetic trees for nucleotide positions 98 to 1454 (minor segment) (top) and 1 to 97 and 1455 to 1570 (major segment) (bottom) of the NP data set of 1,256 A/H3N2 sequences. All bootstrap values greater than 50% are shown. The tree is midpoint rooted for purposes of clarity only, and all branch lengths are drawn to a scale of nucleotide substitutions per site. The red sequence is the candidate recombinant sequence, with the yellow box identifying eight additional sequences that cluster with A/Christchurch/14/2004 in both phylogenies. The blue sequence is the candidate major parent (“Parent P”), and the green sequence is the candidate minor parent (“Parent Q”).