It has generally been assumed that any non-neutral mutations within the influenza genome have arisen as selective adaptations and generally help drive influenza toward a stronger and more dangerous pathogen (in terms of either pathenogenicity or transmissibility). This was probably the basis for the extreme caution exhibited during the 2009–2010 H1N1 outbreak. There is a general perception that, given enough time, H1N1 might mutate into a stronger pathogen, and hence might create another catastrophic pandemic, as it did in 1918.
Selection is evident in H1N1 and other influenza genomes. In certain sites of the HA1 genomic segment of H3, non-synonymous substitutions occur at more than twice the rate as in other sites. This is seen as a major signature of adaptive change, but deleterious mutations in the areas not under selection are carried along with the ones under positive selection. In this light, some perceive H1N1 to be a growing threat, with a new outbreak being just a matter of time. Despite this common perception, a more lethal version of H1N1 has not arisen via mutation within the human population during the last 90+ years. This is significant. The two major human influenza pandemics since 1918 did not arise due to mutations within H1N1, but arose via horizontal transmission of new genetic material from bird influenza strains, creating recombinant viruses. They were also less lethal than the 1918 version. It is true that the population had a degree of residual immunity and was not as immunologically naïve as it was in 1917–18, but selection has still not been able to generate a devastating pandemic from the remnants of that which swept the world at the close of WWI.
In this paper, we examine an alternative point of view regarding mutation accumulation within H1N1. We suggest that, while specific adaptive mutations commonly occur within the H1N1 virus, many more deleterious mutations are accumulating than beneficial mutations, even when there is strong selection. Consequently, H1N1 appears to have been in very gradual error catastrophe throughout its history.
Our results strongly confirm the widely recognized fact that all past and present human and swine H1N1 influenza strains derive from the 1918 strain. By extension, this applies to other human influenza strains, including H3N2 and H2N2.
Human influenza has such a high mutation rate that, even within a single host individual, the virus quickly becomes a genetically diverse “swarm”[61, 62]. Yet, globally human H1N1 influenza is monophyletic and all current variation is of recent origin. This is only possible if almost all human influenza lineages rapidly go extinct. Moreover, we present strong evidence that the H1N1 genome has been systematically degenerating since 1918. This is evidenced by continuous, systematic, and rapid changes in the H1N1 genome throughout its history. For example, there was an especially rapid and monotonic accumulation of mutations during a single pandemic (Figure1). Similarly, there was a continuous and rapid accumulation of mutations over the entire history of the virus (Figures2 and3), including a similar steady increase in non-synonymous amino acid substitutions (Figure3). While mutations accumulated in the human H1N1s, there was a parallel accumulation of mutations in the porcine H1N1 lineage (Figure4). Fitch, et al. also showed a linear mutation accumulation curve. The “gnarled trunk” of Ito, et al. should be buried in our data as well, but it will be swamped by the pervasive, genome-wide accumulation of mutations not under active selection.
Within the human lineage, there was a systematic decline in the Ti/Tv ratio (Figure5). In addition, there was a very consistent loss of codon specificity over time (Figures6 and7). We show compelling new evidence supporting the extinction of human H1N1 in the 1950s, its subsequent re-introduction in 1976, and an apparent second extinction event of the human H1N1 lineage in 2009. Strain extinction has often occurred when new strains appeared, including H1N1 replacing the circulating H3-like strains in 1917, H2N2 replacing H1N1 in 1957, and H3N2 replacing H2N2 in 1968. To our knowledge, we are the first to document the replacement of the re-introduced human H1N1 with reassorted swine H1N1 in 2009. All this is consistent with the genetic attenuation hypothesis, and we feel this is the most fundamental explanation for the very smooth, systematic, and exponential decline in H1N1 mortality rates since 1918.
Relevance and potential objections
In light of these findings, what are the medical implications? Does mutation accumulation really have anything to do with virulence? Simonsen, et al. showed mortality statistics for three influenza strains over multiple years (H1N1 from 1918 to 1987; H2N2 from 1958 to 1962; and H3N2 from 1968 to 1995). Even though there is some debate concerning the mortality burden imposed by influenza viruses, there has clearly been a continuous exponential decline in influenza-related mortality over time, and this is true for all three major serotypes. Since there is a strong linear correlation between mutation count and time (Figures2 and3), and since there is also a close correlation between declining virus-related death rates and time, there is obviously also a correlation between mutation count and reduced death rates. Reduction in mortality may be due to many other factors, including herd immunity, advances in medicine, and advances in hygiene, but would these other factors be expected to follow so tightly the time courses seen in Simonsen?
There have been major medical advances since 1918, and these have clearly been a factor in reducing H1N1-related mortalities. Therefore, the correlation between mutation count and reduced H1N1 mortality might be considered spurious by some. However, while it is certainly true that medical intervention has greatly improved in the developed world since 1918, such medical intervention has been much more limited in the rest of the world. Second, the observed decline in mortality is a remarkably smooth curve, while medical advances have occurred in bursts (e.g., the breakthrough in antibiotics, and the more recent development of antivirals). Third, each of the great influenza pandemics (1918, 1956, 1968) involved the emergence of a new viral strain, which then followed its own exponential decline in mortality but within its own timeframe. This uncouples reduction in mortality and stage of medical advance. Finally, the correlation between the exponential decline of H1N1-related mortalities and the linear increase in H1N1 mutations is only one of our evidences for the genetic attenuation of H1N1. Our other evidences include: a) the extinction of all human influenza strains existing prior to the H1N1 strain; b) the apparent extinction of the human lineage of H1N1 in 1956, and then again apparently in 2009; and c) the erosion of H1N1 codon specificity, approaching random codon usage.
It is our contention that all human influenza strains undergo natural attenuation due to mutation accumulation. It is too early to tell if the remaining versions of the 2009–2010 outbreak viruses will do the same, but it is likely given the known history of change in the various influenza genomes.
The decline in codon bias is especially significant for several reasons. First, since the frequency of codon usage is positively correlated with tRNA availability in the cell, the increased use of rare codons is expected to negatively affect protein translation rates. Alternatively, Li, et al. did not notice any decrease in translational efficiency based on codon choice in bacteria, but they did see effects caused by mutation towards other genomic control motifs (i.e., anti-Shine Dalgarno sequences). Part of codon bias deals with a cell’s avoidance of controlling factors that do not directly deal with translation rates (e.g., CG dinucleotides). Thus, there are multiple ways a disruption of codon bias might negatively impact the functionality of a particular stretch of any nucleic acid. Second, even though the codon usage in ducks and humans is similar, although less so for swine, this might affect the ability of a virus to cross species lines (after cellular antigenic recognition is taken into account). Third, Anhlan, et al. entertained several hypotheses, including that the virus was avian in origin but transferred to pigs before it jumped to humans. Our data clearly indicate that all H1N1s studied are more duck-like in their codon usage, although we cannot comment about a pre-human swine intermediate based on our data. Finally, since we see an obvious decay in codon bias over time when compared to codon usage in either human, duck, or pig, it is clear that H1N1 is not evolving toward optimal codon usage in any of these species but is slowing drifting away from optimal translational efficiency. We concur with Anhlan, et al. that, “the issue of codon usage seems to be much more important at least for influenza viruses than previously thought.”
During the last 100 years, the H1N1 influenza genome has diverged from the original genotype by roughly 15%. Might the approximately 1,900 nucleotide substitutions be primarily attributed to the genetic drift of perfectly neutral variations? This seems unlikely for several reasons. First, a viral genome of approximately 13,000 nucleotides does not have room for very much neutral RNA. Not only did 15% of the genome change, but polymorphisms arose across more than 50% of the genome. This strongly points to extreme mutational pressure, high enough, reasonably, to threaten error catastrophe. Second, if some significant portions of the viral genome are neutral, deletions of such portions of the viral genome should be regularly seen, and selection should favor such deletions, rapidly producing smaller genomes. There is no evidence of significantly smaller influenza genomes. Indeed, there is little evidence of deletion in any of the 2009–2010 genomes compared to the 1918 version. The only major indels occurred among the oldest samples (prior to 1948) in the sixth genomic segment (neuraminidase, or NA), but all of these represented deletions compared to the 1918 genome and all later genomes. Third, it is now known that even synonymous mutations are not always neutral. Even though they may not directly affect protein sequence, they can affect RNA stability, RNA architecture, speed of translation, and protein folding. Fourth, there should only be a finite number of nucleotide positions that are perfectly neutral. Because of this, neutral divergence should quickly approach a limit, causing the rate of divergence from the original genotype to slow, but this is not seen. Finally, the extensive genetic changes observed simply do not appear to be phenotypically neutral; they are tightly correlated with rapid fitness decline, attenuation, extinction of most circulating strains, and even more frequent sub-lineage extinction events.
Might the observed divergence be primarily due to adaptive mutations? We feel that the 15% divergence must be primarily non-adaptive because adaptation should occur rapidly and then reach a natural optimum. Yet, we see that divergence increases in a remarkably linear manner. Furthermore, the virus does not seem to be converging on a new optimal genotype since polymorphism remains extreme (over 50%), since many polymorphic sites have more than two alleles, and since codon specificity is declining over time. Codon patterns can inform us about the origin of the virus, and they tell us that H1N1 is not only drifting away from that original codon use, but it is also drifting away from the host codon preferences. When grown in mouse cell culture, 2009 H1N1 viruses exhibited variation in replication rates, virulence, and pathogenicity, but these did not match the severity of clinical symptoms in humans. Thus, at this time, the exact relationship between mutation load and the severity of infection remains unknown. Yet, selective adaptation is limited to only those amino acids that produce significant phenotypic effects, and, since a viral genome in the absence of reassortment is essentially a single linkage block, it is expected that many more than just adaptive mutations occur. Some of the changes might be due to selection, but the majority certainly are not.
Is it feasible that natural selection might fail to remove a large number of deleterious mutations? It is well known that numerous factors can cause a breakdown in the selective removal of deleterious mutations. These factors include a high mutation rate, a high rate of random loss, limited sexual recombination, genetic bottlenecking, and mutations with very small fitness effects. All of these factors should be especially pronounced in an RNA virus such as influenza and all of these are either previously known or documented here. The genetic changes in H1N1 appear non-directional and are distributed quite uniformly across the genome (Additional file3), consistent with an accumulation of low-impact deleterious mutations. Error catastrophe and lethal mutagenesis are already recognized as a threat to any RNA virus. These facts, combined with the dramatic decline in H1N1 mortality and the very high rate of H1N1 strain extinction, all very strongly indicate that most of the genetic divergence from the original H1N1 genotype has been due to fixation of slightly deleterious mutations.
Could H1N1 ever back-mutate into a strain such as the ancestral genotype that caused the catastrophic 1917–1918 pandemic? Given that the modern strains of H1N1 have diverged from the original 1918 strain by nearly 2000 mutations, that many of these mutations should be slightly deleterious, and that natural selection was unable to stop their continuous accumulation in the first place, it is difficult to imagine how mutation/selection might ever restore full virulence. Reassortment might bring in new material, but thus far this has only applied to a limited section of the genome, and reassortment today occurs in a very different mutational/genomic context than that of 95 years ago. It is often thought that a high mutation rate translates to rapid adaptation and evolution, yet the reverse seems more commonly true. Deleterious mutations often interfere with selection for the more rare beneficial mutations[67, 68]. Thus, the rapid accumulation of mutations in all H1N1 lineages should logically lead to their eventual extinction.
The origin of human H1N1 influenza is unknown, but it is generally reasoned that it invaded the human population from a natural reservoir[5, 51], most likely an aquatic waterfowl, with pigs as a possible intermediate host. In light of the strong tendency toward natural genetic attenuation which we document here, we suggest that the natural reservoir most likely involves a very quiescent viral state, as might occur within a host where there is very little viral replication, and hence much lower mutation rates. It would be very interesting to know the rate of influenza mutation accumulation in waterfowl.
Can reassortment explain these findings? Based on our mutation count analysis, there is no evidence for reasortment in the human H1N1 lineage, and it has gone extinct, apparently twice. From other studies, various influenza strains are obviously derived from reassortment, but all this does is set the mutation clock back a little. Any reassortment between a “fresh” virus and a high-mutation-count virus will inevitably lead to, at best, an averaging of the mutational load of the two. The 2009–2010 “swine flu” virus shows evidence of multiple reassortment events in a limited portion of its genome, and it was more robust than the lingering human H1N1 strain that it replaced, but it carries a great number of non-adaptive and presumably deleterious mutations. Reassortment between two viruses of different immunological character might preserve the less degraded genome, but only temporarily.
Might pandemics be shortened by artificially accelerating the rate of genetic attenuation? The continuous and linear accumulation of mutations within a single influenza lineage, as was seen in H1N1 during the 2009–2010 influenza season (in which about 0.3% of all nucleotides mutated), supports the concept that natural genetic attenuation may be an important factor in the natural cessation of influenza pandemics. Thus, the possibility of an artificial acceleration of mutation rate deserves further investigation, and may suggest new avenues of research in terms of pandemic management. It is clear that natural selection is strongly at work in the influenza genome. This can be seen by preservation of all the basic proteins and functions of the virus, in spite of the fact that every possible point mutation happens in every human individual during the course of an infection. A large fraction of all deleterious mutations clearly must be selected away. Likewise, the emergence of major antigenic variants shows that positive selection is operational. It is also clear that genetic drift is strongly in operation, with a major viral bottleneck happening at each transmission from one human host to the next, and perhaps at the start of each local outbreak, ensuring that most unique genotypes are very quickly lost. Yet, in addition to selection and drift, it also appears there is very strong mutational pressure on the influenza genome, potentially leading to lethal mutagenesis in most strains, and a gradual, natural genetic attenuation of human influenza in general.
Read and Taubenberger called the origin of human H1N1 an “enigma” whose riddle was not yet solved. Like them, we see this as an unsolved riddle and we can only hope that our data might bring us one step closer to understanding the origins of this important disease.