A new look at an old virus: patterns of mutation accumulation in the human H1N1 influenza virus since 1918

Abstract

Background: The H1N1 influenza A virus has been circulating in the human population for over 95 years, first manifesting itself in the pandemic of 1917-1918. Initial mortality was extremely high, but dropped exponentially over time. Influenza viruses have high mutation rates, and H1N1 has undergone significant genetic changes since 1918. The exact nature of H1N1 mutation accumulation over time has not been fully explored.

Methods: We have made a comprehensive historical analysis of mutational changes within H1N1 by examining over 4100 fully-sequenced H1N1 genomes. This has allowed us to examine the genetic changes arising within H1N1 from 1918 to the present.

Results: We document multiple extinction events, including the previously known extinction of the human H1N1 lineage in the 1950s, and an apparent second extinction of the human H1N1 lineage in 2009. These extinctions appear to be due to a continuous accumulation of mutations. At the time of its disappearance in 2009, the human H1N1 lineage had accumulated over 1400 point mutations (more than 10% of the genome), including approximately 330 non-synonymous changes (7.4% of all codons). The accumulation of both point mutations and non-synonymous amino acid changes occurred at constant rates (μ = 14.4 and 2.4 new mutations/year, respectively), and mutations accumulated uniformly across the entire influenza genome. We observed a continuous erosion over time of codon-specificity in H1N1, including a shift away from host (human, swine, and bird [duck]) codon preference patterns.

Conclusions: While there have been numerous adaptations within the H1N1 genome, most of the genetic changes we document here appear to be non-adaptive, and much of the change appears to be degenerative. We suggest H1N1 has been undergoing natural genetic attenuation, and that significant attenuation may even occur during a single pandemic. This process may play a role in natural pandemic cessation and has apparently contributed to the exponential decline in mortality rates over time, as seen in all major human influenza strains. These findings may be relevant to the development of strategies for managing influenza pandemics and strain evolution.

Figure 1

Mutation accumulation within the 2009 A/H1N1 influenza strain during the course of the 2009/2010 flu season. The earliest sequenced genotype (California/04/2009) was used as the baseline for comparison with all subsequent genotypes (mutation counts reflect divergence from that starting sequence). The vertical red line indicates the date the reference sequence was collected. This sequence is not included because it, by definition, has a mutation count of zero and ignores contemporaneous sequence variation.

Figure 2

Mutation accumulation in H1N1 in deeper time. The published Brevig Mission strain from 1918 was used as the baseline for comparison with all available human H1N1 genomes. There are two distinct trend lines in the data. The 2009–2010 outbreak samples and additional samples from 2011–2012 are circled.

Figure 3

Mutation accumulation in human H1N1, using the 1918 Brevig Mission strain as a reference. Sequences derived from the 1976-reintroduction (red symbols) were moved back in time by 21 years to adjust for an apparent period of dormancy, resulting in highly-linear mutation accumulation curves. Filled symbols = nucleotide changes. Hollow symbols = non-synonymous amino acid changes.

Figure 4

Mutation accumulation in porcine H1N1. Solid line = porcine regression. Dashed line = the human regression line from Figure 3. There is considerably more variability among the porcine versions of H1N1, and there was a significant difference in the two slopes (ANCOVA, p=0.0001).

Figure 5

Changes in the transition/transversion ratio over time for the direct 1918 human H1N1 lineage viruses,after adjusting the “frozen” portion of the lineage (1976–2009)for a 21-year dormancy period.

Figure 6

Average normalized codon scores for the direct 1918 human H1N1 lineage viruses, after adjusting the “frozen”portion of the lineage (1976–2009)backwards in time by 21 years. The red line at 1.812 indicates the score that would be obtained from a series of proteins with the same amino acid content as those produced by H1N1, but with randomized codons.

Figure 7

Relative synonymous codon usage in the direct 1918 human H1N1 lineage for the four alanine codons,after adjusting the “frozen”portion of the lineage (1976–2009)by 21 years. Note that the two decreasing in frequency over time are exactly matched by the two increasing over time (i.e., non-synonymous changes are having a negligible effect). Also, three of these lines (GCA, GCC, and GCG) indicate these codons are drifting even farther away from the human average.