Questioning the evidence for genetic recombination in the 1918 "Spanish flu" virus.

Genetic Recombination in the 1918 “Spanish Flu” Virus Influenza viral sequences have been obtained from preserved tissues of victims of the “Spanish flu” pandemic that killed over 20 million people from 1918 to 1919 (1, 2). Phylogenetic analysis of hemagglutinin (HA) gene sequences has indicated that the 1918 Spanish flu virus was more closely related to the human lineage than to the swine or avian influenza lineages of the H1N1 subtype (2). In a recent reanalysis of the 1918 HA gene, however, Gibbs et al. (3) proposed that recombination had occurred such that the majority of the globular domain (HA1) in the Spanish flu virus was acquired from the swine lineage, but its stalk region (HA2) was derived from the human lineage. Gibbs et al. also speculated that this intragenic recombination resulted in the increased virulence associated with the Spanish flu pandemic. We find no evidence for recombination in the 1918 HA gene, however. Rather, the apparent recombination described in (3) results from a difference in the rate of evolution between HA1 and HA2—a difference present only in human influenza A viruses. In contrast to previous analyses (1, 2), Gibbs et al. (3) did not use the avian lineage to root their phylogenetic trees, and they suggested that the position of the bird lineage depends on the substitution model used. However, using avian influenza to distinguish the human and swine lineages results in phylogenetic tree topologies (Fig. 1) that are almost identical for both putative recombinant regions proposed by Gibbs et al. (3). In particular, the 1918 strain is placed on the human lineage not just for the putative human region but, crucially, for the supposed swine region as well (with 96% bootstrap support). Thus, maximum likelihood trees incorporating the avian lineage provide a robust, informative, and necessary test of the recombination hypothesis, whereas the midpoint rooting employed by Gibbs et al. is affected by differences in the rate of molecular evolution between the HA1 and HA2 gene regions. The phylogenies (Fig. 1) show the same relative depth of the swine influenza clades for HA1 versus HA2, illustrating a nearly constant rate of evolution across the entire HA in this lineage. In contrast, for the human lineage, the HA2 region evolves at a considerably lower rate than HA1. Pairwise comparisons among the human lineage strains confirm that rate difference, with uncorrected distances roughly twice as high for the HA1 region as for the HA2 region. Plots presented by Gibbs et al. [figures 1A and B in (3)] give the strong impression that the 1918 strain is more similar to the swine