BACKGROUND
viruses are the cause of several deadly diseases such as yellow fever, dengue, hepatitis or seasonal influenza.
the etiologic agent of the latter, the influenza virus, can cause mild to severe illnesses depending on the influenza type and strain.
the case of the  <dig> h1n <dig> outbreak, first detected in humans in early  <dig>  <cit> , was caused by a antigenically novel strain that led the world health organization to declare the outbreak as the first influenza pandemic of the 21st century.
the emergence of such viruses in the human population has since attracted intense scrutiny, with a particular focus on two of their properties: virulence and interspecies transmission  <cit> .
the h1n <dig> virus is an influenza a virus that belongs to the family of orthomyxoviruses, and has a segmented negative single-stranded rna genome made of eight segments that each encode 1- <dig> proteins necessary for virus attachment to host cells and spread of viral infection.
by approximate order of decreasing sizes, these genes code for polymerase subunits , the hemagglutinin  and neuraminidase  antigens, a nucleoprotein , a ribonucleoprotein exporter , an interferon antagonist , an ion channel protein  and a matrix protein .
two other proteins, pb2-f <dig>  <cit>  and pb1-n <dig>  <cit> , whose roles are now emerging, have also been characterized.
this segmented genome is constantly evolving either by accumulating mutations, which generally lead to small antigenic differences  or by exchanging genomic segments, a process termed reassortment, which, when occurring between different subtypes, can lead to dramatic changes in antigenic properties, also called "antigenic shift" .
the actual changes that may have led to the emergence of past pandemics start to become clearer thanks to a number of studies.
for instance, the first pandemic of the 20th century in  <dig>  also known as 'spanish flu', was caused by an h1n <dig> virus, which was isolated and sequenced from a casualty preserved in the alaskan permafrost  <cit> .
structural and genetic studies have shown that this particular  <dig> virus lacked a cleavage site in ha  <cit> , that virulence was determined by several proteins including ha, the replication complex, ns <dig> and pb1-f <dig>  <cit> , while ha and pb <dig> played an important role in viral transmissibility  <cit> .
the precise origin of this  <dig> virus is however difficult to trace back in time due to the absence of genetic information on the viruses circulating before the 20th century.
the emergence of the  <dig> h1n <dig> pandemic is, on the other hand, not as well understood.
structural information revealed that the  <dig> ha protein had a striking similarity to its  <dig> counterpart  <cit> .
in a landmark study, smith and collaborators showed that the etiologic agent of the  <dig> pandemic had three key features:  the polymerase genes as well as ha, np and the ns genes emerged from triple reassortant north-american swine viruses while the na and m genes originated from avian-like swine viruses,  that the pandemic viruses diversified about a year before the onset of the pandemic and  that a long branch separated the diversification of these pandemic viruses from their first emergence  <cit> .
these authors suggested that the long branch leading to the diversification of the pandemic viruses both reflects a long unsampled history and mild evidence for positive selection, but they did not fully characterize the adaptive nature of the pandemic.
it is also unclear whether the actual host-switch events from non-human animals to humans have an adaptive nature.
here we revisit the adaptive nature of the  <dig> h1n <dig> pandemic with a detailed analysis of the role of selection in  the emergence of this virus, and  its adaptation to human hosts.
on the basis of an extended data set compared to  <cit> , we show that while the acquisition of efficient human-to-human transmission was driven by positive selection, the emergence of the  <dig> h1n <dig> pandemic was essentially nonadaptive, and resulted from stochastic processes, which in turn are expected to make the prediction of such dramatic events difficult.
RESULTS
phylodynamics of the  <dig> h1n <dig> pandemic
we downloaded  <dig> complete influenza a genomes of the h1n <dig> subtype in north america collected between year  <dig> and  <dig>  and selected only the gene sequences with at most  <dig> % similarity for each of the ten "canonical" protein-coding genes .
this clustering step allowed us to performed all phylogenetic analyses in a reasonable timeframe while conserving most of the sequence diversity present in the original data set.
in order to reconstruct rooted phylogenetic trees for each of these genes, we used the 'relaxed molecular clock' approach implemented in beast  <cit>  , where tip dates were set to the collection year of each virus.
a calibration scheme at a finer time-scale was not used because the information about the collection month was missing from some of the sampled genomes.
the substitution models selected by the akaike information criterion  <cit>  were all gtr + Γ + i, except for pb <dig> , m <dig>  m <dig> and ns <dig>  and ns <dig> .
since tvm-based models are not implemented in beauti, we employed the next best aic model which in all cases was based on gtr for the relaxed clock analyses with beast.
the results show that h1n <dig> sequences across all ten genes have very similar histories .
if we assume that the ancestral h1n <dig> genome is of swine or other non-human origin  <cit> , there were a minimum of three host-switch events to human: two occurred on internal  branches, one of which led to the  <dig> pandemic.
this particular host-switch event was placed on the long branch sustaining the  <dig> clade rather than on the short branch leading to the mexico-swine- <dig> genome because the position of this latter genome within the  <dig> clade is weakly supported .
the third host-switch event occurred on a terminal branch of the tree .
it is notable that only one of the two internal host-switch events led to a pandemic, which suggests that the two processes of host-switch event and 'pandemicity' are not tightly coupled, as already suggested by the  <dig> h5n <dig> viruses.
in the rest of the text, we will denote the part of the tree that leads to the human  <dig> pandemic sequences as the "pandemic clade", while all the other human sequences are part of the "non-pandemic clade" .
the relaxed clock analyses also allow us to derive three additional results on  the population dynamics of the different segments and genes,  their rates of evolution and  their coalescence times.
first, the results of the skyline analyses show that the population dynamics of the different segments and genes exhibit two very contrasted trajectories .
while most segments followed similar and downwards dynamics in the past, a decoupling event or a series of such events took place at most five years before  <dig>  ca.
<dig>  when seven of the ten genes underwent a rapid expansion suggestive of a selective sweep.
the time resolution of our analyses is too low for us to derive more accurate dates, but the suddenness of this expansion suggests that it would have been difficult to forecast as it represents a dramatic departure from the previously decreasing trend.
these "expanding genes" include one of the polymerase genes , the two antigenic determinants , and the genes on the last two segments, m and ns.
on the other hand, two of the polymerase genes  as well as the nucleoprotein  underwent a steady decrease in terms of scaled effective population size .
note first that these estimates are relative to the viral population, not to the host's dynamics, and therefore represent the incidence of the virus rather than its prevalence  <cit> .
second, segment dynamics are not linked to the origin of the segments or genes, as pa and np, which come form a north american avian and a classical avian source, respectively  <cit> , still exhibit similar dynamics .
yet, such a decoupling of segment dynamics is not atypical in influenza a viruses .
one notable difference with the latter study however is that our reconstruction goes  <dig> years back in time before the  <dig> outbreak without encountering any of the oscillations reconstructed over a  <dig> year period for h3n <dig> viruses  <cit> .
a potential explanation is that the pattern observed here is due to our smaller effective sample size , and/or to the lower temporal resolution of our analysis.
in spite of these potential confounding factors, the lack of oscillations detected in our results might also reflect the lack of evidence for seasonality in h1n <dig> dynamics, which is consistent with the dominant incidence of h3n <dig> viruses in the human population between  <dig>  and  <dig>  <cit> .
while in the face of the  <dig> pandemic it makes sense that neτ for both the ha and na antigens increased, it is unclear  why neτ decreased for some segments and  why a decoupling is inferred within the polymerase genes, setting pb <dig>  which has a role in host restriction , apart.
this decoupling of segments cannot be due to our sequence clustering that eliminated highly similar sequences, but under-sampling of genomes cannot be ruled out .
second, the posterior distributions of the absolute rates of evolution are summarized in figure  <dig>  these rates are similar to those estimated in previous studies , and our results suggest that there is extensive rate heterogeneity between the different segments of the influenza a genomes of h1n <dig> viruses, and even within segments as demonstrated in particular by the posterior estimates for m <dig> and m <dig> .
post-hoc comparisons of rates sampled from their posterior distributions, either by means of tukey hsd or pairwise t tests, show significant differences at the α =  <dig>  level, even under the very conservative bonferroni correction.
therefore, influenza a viruses of different subtypes evolve at different rates as reviewed before  <cit> , and each of their protein-coding genes, even on the same segment, exhibit significant rate heterogeneity.
summarizing rates of evolution of influenza a viruses and possibly other segmented rna viruses by a single number might therefore not give a realistic picture of the extensive rate variation found in these viruses.
third, table  <dig> shows that the pandemic and non-pandemic h1n <dig> protein-coding genes analyzed here coalesced on average  <dig> years before  <dig>  that is, around  <dig> .
ns <dig> and in particular pb <dig> have both been circulating for much longer periods of time .
keeping in mind that the accuracy of the estimated dates depends on the density of sampled genomes, three points can be made here:  these dates are much deeper than in  <cit> , where time to the most recent common ancestor  of the sampled sequences goes back to ~  <dig>  due to the lower breadth of their sampling strategy.
the inclusion of the  <dig> brevig genome a/brevig mission/1/ <dig>  <cit>  for instance would only pull this root age further back in time;  we also detected variation of coalescence times within segments: the protein-coding genes on segment  <dig> and  <dig>  m2-m <dig> on the one hand and ns2-ns <dig> on the other hand, coalesced at slightly different dates, although their 95% hpds overlap - which might be due to a combination of short sequences and small numbers of variable sites in the overlapping genes on segments  <dig> and 8;  the observation that different segments share the same coalescence times has already been documented and interpreted as evidence for the correlated evolution and co-transmission of segments  <cit> , so that there would be genetic linkage between segments.
however,  <cit>  found that coalescence times were shared by the pb2/pb1/pa/np/m segments, while our table  <dig> suggests that pb2/pa/ha/na/ns <dig> have similar root age and therefore could be linked.
the different linkage groups or constellations might be a characteristic of the different viruses studied .
however, it is also possible that such gene constellations are highly labile both in time and across subtypes.
all dates are in years before  <dig>
notes--hpd: highest posterior density; mrca: most recent common ancestor.
a way to test this lability hypothesis is to estimate the tmrca of the pandemic sequences.
unlike the tmrca of the sampled h1n <dig> sequences, the emergence of the pandemic sequences shows a very consistent date across all segments and genes at  <dig>  years before  <dig>  that is during the last semester of  <dig> .
this date is slightly older than previous estimates that put the tmrca of pandemic sequences sometime between mid- <dig>  <cit>  to early january  <dig>  <cit> ; this difference can be due to relaxation of selective constraints that are not directly accounted for here  <cit> , slight differences in model specifications  and to our generally broader  sampling of genomes.
based on the synchrony argument used above and in  <cit> , this result suggests the formation of a new gene constellation in the late  <dig>  the emergence of this constellation could be the consequence of a selective sweep, as suggested in the case of avian influenza viruses  <cit> , but it could also be due to a demographic bottleneck in the viral population or other nonadaptive processes.
the lability hypothesis has a corollary that is easily testable: although the reassortment events that led to the emergence of the pandemic strain have a history that goes back to the early 1990's  <cit> , consistently with the tmrca estimated here , the coalescence times of the genomes analyzed here occurred only shortly before the pandemic.
the most recent common ancestor of the pandemic clade  has a mean age of  <dig>  years before  <dig> , which corresponds to the end of  <dig>  the  <dig>  years  gap separating this mrcapandemic from the pandemic clade represents a long period of time when sequences leading to the  <dig> pandemic were not sampled  <cit> .
but the long branch leading to the pandemic clade  could also be due to the simultaneous action of positive selection.
test of positive selection for the  <dig> h1n <dig> pandemic
to test the hypothesis that the long branch leading to the  <dig> pandemic might represent the action of positive selection, we performed a branch-site test of positive selection along this branch in all ten protein-coding genes of the h1n <dig> influenza a genome.
the results, presented in table  <dig> demonstrate quite dramatically that none of the ten protein-coding genes shows any evidence for positive selection, hereby suggesting that this long branch reflects exclusively a period of  <dig> years of unsampled history, and hence a dramatic failure of the current surveillance system of circulating influenza viruses  <cit> .
results of the mcdonald-kreitman test , tajima's d test , as well as log-likelihood values , parameter estimates and p-values for the ten standard protein-coding genes of  <dig> h1n <dig> influenza a genomes.
h <dig> is the null branch-site codon model, without positive selection; h <dig> is the alternative model that allows for positive selection at sites in the foreground branches.
the p-values are derived from a χ <dig> distribution .
parameter estimates for ω and its proportion of sites pω are for rate categories ω <  <dig> under h <dig>  and ω ≥  <dig> under h <dig>
notes--na: not applicable; nfd: no fixed differences.
sites in boldface are those for which the lrt rejects h <dig> at the 1% level.
one potential caveat with our analysis is that codon models assume that all nonsynonymous differences observed in the data are fixed  <cit> .
however, when data are sampled at the population level, as is most likely the case here, it is possible that most of the observed differences do in fact represent segregating polymorphisms.
this is known to render the use of non-synonymous to synonymous rate ratios  potentially problematic, as estimated ω ratios can take values <  <dig> within a population even in the presence of very strong positive selection  <cit> .
as some of the nonsynonymous differences in our data are potentially transient polymorphisms, we reanalyzed the same data with two tests based on population genetics principles.
first, we employed the mcdonald-kreitman test , which is a two-population neutrality test that compares the ratio of fixed nonsynonymous to synonymous differences to the ratio of polymorphic nonsynonymous to synonymous differences  <cit> .
here, a first "population" consisted of the sequences from the pandemic clade, while the other "population" contained all the remaining sequences in order to match the specification of the codon-based test of positive selection.
the results suggest that there is no evidence for selection at the 1% level , which supports the results of the likelihood ratio test based on codon models.
second, the results of the tajima test, which compares two different estimates of nucleotide diversity under the infinite-site model, appear more contrasted, with only three genes  for which neutrality cannot be rejected.
this set of genes matches exactly the list of genes with decreasing incidence .
alternatively, the genes with an indication that neutrality could be rejected  are those that underwent a rapid and recent expansion.
the results of the tajima test are therefore potentially compounded by the effect of a recent "population" expansion of these segments, which is known to inflate the type-i error rate of this test  <cit> .
in the absence of a clear rejection of the neutral hypothesis both with population genetics and phylogenetic approaches, the emergence of the  <dig> h1n <dig> pandemic was therefore most likely due to nonadaptive processes such as drift .
test of positive selection for swine-to-human host-switch events
a complementary hypothesis is that the acquisition of the competence to be transmitted between humans requires some adaptive changes in the genome of the h1n <dig> virus of non-human origin.
to evaluate this hypothesis, we first performed the mkt, defining the first "population" as that of viruses found in swines, and the second "population" as that of viruses found in humans.
the results show that all the comparisons of nonsynonymous/synonymous polymorphisms to nonsynonymous/synonymous fixations failed because of the systematic absence of fixed differences .
this result raises some concern about saturation, which is probably not an issue here since the longest branch length is ≤  <dig>  substitutions per site, except for ns <dig> due to the presence in our data of a swine virus sampled in  <dig> .
the results were identical when this sequence was excluded from the data.
the absence of fixed differences, whose deficit usually indicates the action of purifying selection, here might also suggest that the mkt is not the most appropriate test for these data.
this interpretation is supported by a simulation study that shows that the mkt exhibits unduly high type-i error rates at the large mutation rates  typically found in rna viruses such as influenza viruses  <cit> .
on the other hand, the tajima test failed to reject the null hypothesis of neutral evolution for all protein-coding genes.
although all p-values are far from the 1% threshold used here , this test has been shown to be conservative  <cit> .
results of the mcdonald-kreitman test , tajima's d test , as well as log-likelihood values , parameter estimates and p-values for the ten standard protein-coding genes of  <dig> h1n <dig> influenza a genomes.
h <dig> is the null branch-site codon model, without positive selection; h <dig> is the alternative model that allows for positive selection at sites in the foreground branches.
the p-values are derived from a χ <dig> distribution .
parameter estimates for ω and its proportion of sites pω are for rate categories ω <  <dig> under h <dig>  and ω ≥  <dig> under h <dig>
notes--na: not applicable; nfd: no fixed differences.
sites in boldface are those for which the lrt rejects h <dig> at the 1% level.
from a phylogenetic standpoint, the test of positive selection based on codon models, although used conservatively here , detected evidence for positive selection at the 1% level in the ha and the na genes .
the ha gene codes for the principal surface antigen which is responsible for viral binding to host receptors via receptor-binding pockets, permitting entry into the host cell by membrane fusion and endocytosis  <cit> .
as such, the ha gene appears to be a critical factor for efficient transmission from host to host.
the na gene codes for a tetrameric protein that facilitates the release and spread of viral particles to neighboring cells by cleaving sialic acids from infected cell surfaces and newly formed viral particles .
note that no evidence for adaptive evolution during host-switch events was found in the pb <dig> gene, which is often associated with host restriction .
this lack of evidence for adaptive evolution during host switch might in turn be associated with the mild symptoms of the  <dig> h1n <dig> pandemic viruses in the human population .
of the two amino-acid sites in ha that were potentially under positive selection for adaptation to human hosts, only site  <dig>  belongs directly to one of the four epitopes proximal to the receptor-binding pocket, while site  <dig>  is two positions downstream of sites participating in the exact same epitope .
it is therefore very likely that these two amino-acid changes improve viral binding to human hosts.
on the other hand, the interpretation of the results for na is not so clear.
of the three amino-acid sites in na that were identified to play a potential role in the adaptation to human hosts , site  <dig> does not belong to the part of the na protein that is usually crystalized , and therefore its functional role is difficult to predict.
site  <dig> is in proximity of the catalytic pocket, but is not part of the sites that interact directly with the substrate .
site  <dig> is located in a loop that contributes to the binding of two antibodies  <cit> .
it should be emphasized that the identification of sites under positive selection is difficult and not always absolutely reliable  <cit> .
in spite of this, because our aligned sequences are quite conserved, it is unlikely that an unreliable alignment might have caused false positive identification of sites potentially under selection  <cit> .
therefore, we posit that the identified sites might play a role in reshaping the antigenic properties of the na protein during the host-switch event from swine to human h1n <dig> viruses.
characterization of the pandemic n <dig> structure and epitopes
in order to test the hypothesis that the three sites identified above with the codon models do affect the antigenic properties of the na protein, we first set out to predict the structural changes involved between pandemic and non-pandemic molecules.
recent work has focused on the ha protein, and has shown that the  <dig> and  <dig> proteins have very similar tridimensional  structures  <cit> .
unfortunately, to date, the structure of the na protein has not been obtained.
we therefore reconstructed 3d models by homology modeling with swiss-model  <cit> .
the prediction of the pandemic na was based on pdb template 2hty , which is of type n <dig> but derived from an h5n <dig> virus isolated in  <dig>  <cit> , which is consistent with the avian origin of h <dig>  <cit> .
both the non-pandemic and the swine models were derived from pdb template 3b7e , which is the structure of the pandemic  <dig> n <dig>  <cit> .
the three models covered residues 83- <dig> and were quite similar .
the main structural difference between the pandemic and the non-pandemic structure is due to a nonsynonymous substitution in the  <dig> loop, which contains the active site of na, at position  <dig> .
most of the pandemic sequences have an isoleucine at this position, just like the swine sequences isolated in  <dig>  on the other hand, all the non-pandemic sequences and swine sequences isolated before  <dig> have a valine, which is also the amino acid used by the  <dig> h1n <dig> na protein and that was found to distort the structure of the na protein  <cit> .
this v149i substitution was previously reported in the context of studying two antineuraminidase compounds , and was judged to be too far from the drug binding pocket to impact antiviral susceptibility  <cit> .
our results therefore suggest that  the v149i substitution affected the active site of na but  could not be solely responsible for the onset of the pandemic since the  <dig> protein had a valine, unless epistatic interactions exist.
because we have found evidence for linkage between the segments of h1n <dig> , such epistatic interactions cannot be ruled out; but since we found that all segments had the same tmrca, our current approach cannot identify the segment and the site interacting directly with loop  <dig> of na.
however, in spite of this change in 3d structure of the pandemic na protein, site  <dig> which is at the core of this structural change is not a site detected by our analyses of positive selection.
could nonetheless loop  <dig> still be a strong epitope?
to address this question, we predicted the epitopes of the non-pandemic na proteins.
figure  <dig> shows the epitopes predicted for the human and swine sequences, with each peak above the  <dig>  threshold indicating the presence of an epitope.
more specifically, figure 5a shows that a small number of differences exist between the pandemic and the non-pandemic proteins, as a total of five regions differ .
notably, the sites detected to be potentially under positive selection for host-switch events, at positions  <dig>   <dig> and  <dig>  are included or in very close proximity of the regions where epitope differences are detected between pandemic and non-pandemic na proteins.
yet, while no new epitope emerged within loop  <dig>  this region represents an epitope present in both pandemic and non-pandemic proteins, while its conformation changed nonadaptively in the  <dig> sequences .
on the other hand, the comparison of predicted b-cell epitopes of swine sequences vs. human pandemic sequences  shows that the predicted epitopes are almost identical between these two viruses, to the exception of a small region n-terminal region 37- <dig>  importantly, none of the sites potentially under positive selection falls within a predicted epitope difference.
as a consequence, the emergence of novel na epitopes in the  <dig> h1n <dig> pandemic viruses is most likely nonadaptive.
CONCLUSIONS
to summarize the results found in this study, we showed that  the only evidence for positive selection is in the ha and na antigenes during host-switch events to human hosts, while the emergence of the pandemic was nonadaptive ,  loop  <dig>  which contains the active site of na, is an epitope present in all sampled sequences , and a recent substitution in this epitope  spread rapidly but nonadaptively through the viral na sequences in  <dig> irrespective of their host, potentially by means of a demographic bottleneck in human viruses following a reassortment event with a virus of swine origin  <cit> ,  this substitution  caused a structural modification of the  <dig> pandemic na protein, and  although this loop  <dig> is found in all viruses sampled here, the pandemic na proteins are predicted to possess four novel epitopes not found in viruses circulating in swines or non-pandemic humans.
these results are significant on two fronts.
first, in terms of methodology, the use of codon models proved here to return more sensible results than the use of population genetics test of neutrality such as the mkt or the tajima test, which both have limitations when it comes to analyzing viral data as population genetics-based tests can be sensitive to demographic changes and/or high levels of diversity, so that these tests can have low power  <cit> .
however, the use of codon models in the context of a population study is also not fully satisfactory, as not all nonsynonymous substitutions can be assumed to be fixed.
to circumvent this limitation, mutation-selection models that aim at bridging the gap between these two evolutionary scales have been developed  <cit> .
however, mutation-selection models have, so far, not been extended to detecting selection at certain amino acid sites in particular lineages and thus, do not allow us to investigate the complex question as to when  and where  evidence for positive selection can be found.
second, in terms of the biology of influenza a viruses, our study shows that, while host-switch events and pandemics are not tightly coupled, it is likely that the emergence of new epitopes in a population is first nonadaptive for the virus.
then, the frequency of these epitopes increases very quickly due to drift, before plummeting again because they are linked to a highly virulent phenotype that kills its hosts too quickly.
a similar argument is generally derived from modeling the population dynamics of influenza viruses, potentially including very sophisticated immune interactions between hosts and viruses , and was also put forward based on the study of h3n <dig> subtypes  <cit> .
it is also significant to note that, while certain amino acid substitutions may be linked to the emergence of a particular epidemic or pandemic influenza strain, such as v149i, the persistence or re-emergence of this very same substitution is no guarantee of an up-coming threat to public health.
more likely, the emergence of an epidemic or pandemic 'phenotype' is the result of epistatic interactions between sites within  <cit>  or across segments, forming a constellation or network of epistatic interactions that are changing over time in ways that we do not currently fully understand.
it is tempting to associate these changes of epistatic interactions to antigenic shifts, and future studies should address this potential link.
the observation that evidence for positive selection was found only in the ha and na genes, which are two of the genes for which neτ increases, while no evidence for positive selection was found in the other genes showing such an increase may suggest that ha, na, pb <dig> and the m and ns genes might be linked.
yet, this linkage is not constant in space or in time as these five different segments in  <dig> h1n <dig> viruses have different origins, with ha and ns coming from classical swine, while na and m come from an eurasian avian-like swine and pb <dig> comes from a human h3n <dig> virus  <cit> .
a better insight into the timing and the forces at play in the emergence of influenza viruses and into the dynamics of gene constellations in influenza viruses will require a continuous and in-depth surveillance of the viruses circulating around the world in its various hosts  <cit> .

 viruses are the cause of several deadly diseases such as yellow fever, dengue, hepatitis or seasonal influenza. the etiologic agent of the latter, the influenza virus, can cause mild to severe illnesses depending on the influenza type and strain. the case of the h1n outbreak, first detected in humans in early, was caused by a antigenically novel strain that led the world health organization to declare the outbreak as the first influenza pandemic of the 21st century. the emergence of such viruses in the human population has since attracted intense scrutiny, with a particular focus on two of their properties: virulence and interspecies transmission. the h1n virus is an influenza a virus that belongs to the family of orthomyxoviruses, and has a segmented negative single-stranded rna genome made of eight segments that each encode 1- proteins necessary for virus attachment to host cells and spread of viral infection. by approximate order of decreasing sizes, these genes code for polymerase subunits, the hemagglutinin and neuraminidase antigens, a nucleoprotein, a ribonucleoprotein exporter, an interferon antagonist, an ion channel protein and a matrix protein. two other proteins, pb2-f and pb1-n, whose roles are now emerging, have also been characterized. this segmented genome is constantly evolving either by accumulating mutations, which generally lead to small antigenic differences or by exchanging genomic segments, a process termed reassortment, which, when occurring between different subtypes, can lead to dramatic changes in antigenic properties, also called "antigenic shift". the actual changes that may have led to the emergence of past pandemics start to become clearer thanks to a number of studies. for instance, the first pandemic of the 20th century in also known as'spanish flu', was caused by an h1n virus, which was isolated and sequenced from a casualty preserved in the alaskan permafrost. structural and genetic studies have shown that this particular virus lacked a cleavage site in ha, that virulence was determined by several proteins including ha, the replication complex, ns and pb1-f, while ha and pb played an important role in viral transmissibility. the precise origin of this virus is however difficult to trace back in time due to the absence of genetic information on the viruses circulating before the 20th century. the emergence of the h1n pandemic is, on the other hand, not as well understood. structural information revealed that the ha protein had a striking similarity to its counterpart. in a landmark study, smith and collaborators showed that the etiologic agent of the pandemic had three key features: the polymerase genes as well as ha, np and the ns genes emerged from triple reassortant north-american swine viruses while the na and m genes originated from avian-like swine viruses, that the pandemic viruses diversified about a year before the onset of the pandemic and that a long branch separated the diversification of these pandemic viruses from their first emergence. these authors suggested that the long branch leading to the diversification of the pandemic viruses both reflects a long unsampled history and mild evidence for positive selection, but they did not fully characterize the adaptive nature of the pandemic. it is also unclear whether the actual host-switch events from non-human animals to humans have an adaptive nature. here we revisit the adaptive nature of the h1n pandemic with a detailed analysis of the role of selection in the emergence of this virus, and its adaptation to human hosts. on the basis of an extended data set compared to, we show that while the acquisition of efficient human-to-human transmission was driven by positive selection, the emergence of the h1n pandemic was essentially nonadaptive, and resulted from stochastic processes, which in turn are expected to make the prediction of such dramatic events difficult.  phylodynamics of the h1n pandemic we downloaded complete influenza a genomes of the h1n subtype in north america collected between year and and selected only the gene sequences with at most % similarity for each of the ten "canonical" protein-coding genes. this clustering step allowed us to performed all phylogenetic analyses in a reasonable timeframe while conserving most of the sequence diversity present in the original data set. in order to reconstruct rooted phylogenetic trees for each of these genes, we used the'relaxed molecular clock' approach implemented in beast, where tip dates were set to the collection year of each virus. a calibration scheme at a finer time-scale was not used because the information about the collection month was missing from some of the sampled genomes. the substitution models selected by the akaike information criterion were all gtr + Γ + i, except for pb, m m and ns and ns. since tvm-based models are not implemented in beauti, we employed the next best aic model which in all cases was based on gtr for the relaxed clock analyses with beast. the results show that h1n sequences across all ten genes have very similar histories. if we assume that the ancestral h1n genome is of swine or other non-human origin, there were a minimum of three host-switch events to human: two occurred on internal branches, one of which led to the pandemic. this particular host-switch event was placed on the long branch sustaining the clade rather than on the short branch leading to the mexico-swine- genome because the position of this latter genome within the clade is weakly supported. the third host-switch event occurred on a terminal branch of the tree. it is notable that only one of the two internal host-switch events led to a pandemic, which suggests that the two processes of host-switch event and'pandemicity' are not tightly coupled, as already suggested by the h5n viruses. in the rest of the text, we will denote the part of the tree that leads to the human pandemic sequences as the "pandemic clade", while all the other human sequences are part of the "non-pandemic clade". the relaxed clock analyses also allow us to derive three additional results on the population dynamics of the different segments and genes, their rates of evolution and their coalescence times. first, the results of the skyline analyses show that the population dynamics of the different segments and genes exhibit two very contrasted trajectories. while most segments followed similar and downwards dynamics in the past, a decoupling event or a series of such events took place at most five years before ca. when seven of the ten genes underwent a rapid expansion suggestive of a selective sweep. the time resolution of our analyses is too low for us to derive more accurate dates, but the suddenness of this expansion suggests that it would have been difficult to forecast as it represents a dramatic departure from the previously decreasing trend. these "expanding genes" include one of the polymerase genes, the two antigenic determinants, and the genes on the last two segments, m and ns. on the other hand, two of the polymerase genes as well as the nucleoprotein underwent a steady decrease in terms of scaled effective population size. note first that these estimates are relative to the viral population, not to the host's dynamics, and therefore represent the incidence of the virus rather than its prevalence. second, segment dynamics are not linked to the origin of the segments or genes, as pa and np, which come form a north american avian and a classical avian source, respectively, still exhibit similar dynamics. yet, such a decoupling of segment dynamics is not atypical in influenza a viruses. one notable difference with the latter study however is that our reconstruction goes years back in time before the outbreak without encountering any of the oscillations reconstructed over a year period for h3n viruses. a potential explanation is that the pattern observed here is due to our smaller effective sample size, and/or to the lower temporal resolution of our analysis. in spite of these potential confounding factors, the lack of oscillations detected in our results might also reflect the lack of evidence for seasonality in h1n dynamics, which is consistent with the dominant incidence of h3n viruses in the human population between and. while in the face of the pandemic it makes sense that neτ for both the ha and na antigens increased, it is unclear why neτ decreased for some segments and why a decoupling is inferred within the polymerase genes, setting pb which has a role in host restriction, apart. this decoupling of segments cannot be due to our sequence clustering that eliminated highly similar sequences, but under-sampling of genomes cannot be ruled out. second, the posterior distributions of the absolute rates of evolution are summarized in figure these rates are similar to those estimated in previous studies, and our results suggest that there is extensive rate heterogeneity between the different segments of the influenza a genomes of h1n viruses, and even within segments as demonstrated in particular by the posterior estimates for m and m. post-hoc comparisons of rates sampled from their posterior distributions, either by means of tukey hsd or pairwise t tests, show significant differences at the α = level, even under the very conservative bonferroni correction. therefore, influenza a viruses of different subtypes evolve at different rates as reviewed before, and each of their protein-coding genes, even on the same segment, exhibit significant rate heterogeneity. summarizing rates of evolution of influenza a viruses and possibly other segmented rna viruses by a single number might therefore not give a realistic picture of the extensive rate variation found in these viruses. third, table shows that the pandemic and non-pandemic h1n protein-coding genes analyzed here coalesced on average years before that is, around. ns and in particular pb have both been circulating for much longer periods of time. keeping in mind that the accuracy of the estimated dates depends on the density of sampled genomes, three points can be made here: these dates are much deeper than in, where time to the most recent common ancestor of the sampled sequences goes back to ~ due to the lower breadth of their sampling strategy. the inclusion of the brevig genome a/brevig mission/1/ for instance would only pull this root age further back in time; we also detected variation of coalescence times within segments: the protein-coding genes on segment and m2-m on the one hand and ns2-ns on the other hand, coalesced at slightly different dates, although their 95% hpds overlap - which might be due to a combination of short sequences and small numbers of variable sites in the overlapping genes on segments and 8; the observation that different segments share the same coalescence times has already been documented and interpreted as evidence for the correlated evolution and co-transmission of segments, so that there would be genetic linkage between segments. however, found that coalescence times were shared by the pb2/pb1/pa/np/m segments, while our table suggests that pb2/pa/ha/na/ns have similar root age and therefore could be linked. the different linkage groups or constellations might be a characteristic of the different viruses studied. however, it is also possible that such gene constellations are highly labile both in time and across subtypes. all dates are in years before notes--hpd: highest posterior density; mrca: most recent common ancestor. a way to test this lability hypothesis is to estimate the tmrca of the pandemic sequences. unlike the tmrca of the sampled h1n sequences, the emergence of the pandemic sequences shows a very consistent date across all segments and genes at years before that is during the last semester of. this date is slightly older than previous estimates that put the tmrca of pandemic sequences sometime between mid- to early january; this difference can be due to relaxation of selective constraints that are not directly accounted for here, slight differences in model specifications and to our generally broader sampling of genomes. based on the synchrony argument used above and in, this result suggests the formation of a new gene constellation in the late the emergence of this constellation could be the consequence of a selective sweep, as suggested in the case of avian influenza viruses, but it could also be due to a demographic bottleneck in the viral population or other nonadaptive processes. the lability hypothesis has a corollary that is easily testable: although the reassortment events that led to the emergence of the pandemic strain have a history that goes back to the early 1990's, consistently with the tmrca estimated here, the coalescence times of the genomes analyzed here occurred only shortly before the pandemic. the most recent common ancestor of the pandemic clade has a mean age of years before, which corresponds to the end of the years gap separating this mrcapandemic from the pandemic clade represents a long period of time when sequences leading to the pandemic were not sampled. but the long branch leading to the pandemic clade could also be due to the simultaneous action of positive selection. test of positive selection for the h1n pandemic to test the hypothesis that the long branch leading to the pandemic might represent the action of positive selection, we performed a branch-site test of positive selection along this branch in all ten protein-coding genes of the h1n influenza a genome. the results, presented in table demonstrate quite dramatically that none of the ten protein-coding genes shows any evidence for positive selection, hereby suggesting that this long branch reflects exclusively a period of years of unsampled history, and hence a dramatic failure of the current surveillance system of circulating influenza viruses. results of the mcdonald-kreitman test, tajima's d test, as well as log-likelihood values, parameter estimates and p-values for the ten standard protein-coding genes of h1n influenza a genomes. h is the null branch-site codon model, without positive selection; h is the alternative model that allows for positive selection at sites in the foreground branches. the p-values are derived from a χ distribution. parameter estimates for ω and its proportion of sites pω are for rate categories ω < under h and ω ≥ under h notes--na: not applicable; nfd: no fixed differences. sites in boldface are those for which the lrt rejects h at the 1% level. one potential caveat with our analysis is that codon models assume that all nonsynonymous differences observed in the data are fixed. however, when data are sampled at the population level, as is most likely the case here, it is possible that most of the observed differences do in fact represent segregating polymorphisms. this is known to render the use of non-synonymous to synonymous rate ratios potentially problematic, as estimated ω ratios can take values < within a population even in the presence of very strong positive selection. as some of the nonsynonymous differences in our data are potentially transient polymorphisms, we reanalyzed the same data with two tests based on population genetics principles. first, we employed the mcdonald-kreitman test, which is a two-population neutrality test that compares the ratio of fixed nonsynonymous to synonymous differences to the ratio of polymorphic nonsynonymous to synonymous differences. here, a first "population" consisted of the sequences from the pandemic clade, while the other "population" contained all the remaining sequences in order to match the specification of the codon-based test of positive selection. the results suggest that there is no evidence for selection at the 1% level, which supports the results of the likelihood ratio test based on codon models. second, the results of the tajima test, which compares two different estimates of nucleotide diversity under the infinite-site model, appear more contrasted, with only three genes for which neutrality cannot be rejected. this set of genes matches exactly the list of genes with decreasing incidence. alternatively, the genes with an indication that neutrality could be rejected are those that underwent a rapid and recent expansion. the results of the tajima test are therefore potentially compounded by the effect of a recent "population" expansion of these segments, which is known to inflate the type-i error rate of this test. in the absence of a clear rejection of the neutral hypothesis both with population genetics and phylogenetic approaches, the emergence of the h1n pandemic was therefore most likely due to nonadaptive processes such as drift. test of positive selection for swine-to-human host-switch events a complementary hypothesis is that the acquisition of the competence to be transmitted between humans requires some adaptive changes in the genome of the h1n virus of non-human origin. to evaluate this hypothesis, we first performed the mkt, defining the first "population" as that of viruses found in swines, and the second "population" as that of viruses found in humans. the results show that all the comparisons of nonsynonymous/synonymous polymorphisms to nonsynonymous/synonymous fixations failed because of the systematic absence of fixed differences. this result raises some concern about saturation, which is probably not an issue here since the longest branch length is ≤ substitutions per site, except for ns due to the presence in our data of a swine virus sampled in. the results were identical when this sequence was excluded from the data. the absence of fixed differences, whose deficit usually indicates the action of purifying selection, here might also suggest that the mkt is not the most appropriate test for these data. this interpretation is supported by a simulation study that shows that the mkt exhibits unduly high type-i error rates at the large mutation rates typically found in rna viruses such as influenza viruses. on the other hand, the tajima test failed to reject the null hypothesis of neutral evolution for all protein-coding genes. although all p-values are far from the 1% threshold used here, this test has been shown to be conservative. results of the mcdonald-kreitman test, tajima's d test, as well as log-likelihood values, parameter estimates and p-values for the ten standard protein-coding genes of h1n influenza a genomes. h is the null branch-site codon model, without positive selection; h is the alternative model that allows for positive selection at sites in the foreground branches. the p-values are derived from a χ distribution. parameter estimates for ω and its proportion of sites pω are for rate categories ω < under h and ω ≥ under h notes--na: not applicable; nfd: no fixed differences. sites in boldface are those for which the lrt rejects h at the 1% level. from a phylogenetic standpoint, the test of positive selection based on codon models, although used conservatively here, detected evidence for positive selection at the 1% level in the ha and the na genes. the ha gene codes for the principal surface antigen which is responsible for viral binding to host receptors via receptor-binding pockets, permitting entry into the host cell by membrane fusion and endocytosis. as such, the ha gene appears to be a critical factor for efficient transmission from host to host. the na gene codes for a tetrameric protein that facilitates the release and spread of viral particles to neighboring cells by cleaving sialic acids from infected cell surfaces and newly formed viral particles. note that no evidence for adaptive evolution during host-switch events was found in the pb gene, which is often associated with host restriction. this lack of evidence for adaptive evolution during host switch might in turn be associated with the mild symptoms of the h1n pandemic viruses in the human population. of the two amino-acid sites in ha that were potentially under positive selection for adaptation to human hosts, only site belongs directly to one of the four epitopes proximal to the receptor-binding pocket, while site is two positions downstream of sites participating in the exact same epitope. it is therefore very likely that these two amino-acid changes improve viral binding to human hosts. on the other hand, the interpretation of the results for na is not so clear. of the three amino-acid sites in na that were identified to play a potential role in the adaptation to human hosts, site does not belong to the part of the na protein that is usually crystalized, and therefore its functional role is difficult to predict. site is in proximity of the catalytic pocket, but is not part of the sites that interact directly with the substrate. site is located in a loop that contributes to the binding of two antibodies. it should be emphasized that the identification of sites under positive selection is difficult and not always absolutely reliable. in spite of this, because our aligned sequences are quite conserved, it is unlikely that an unreliable alignment might have caused false positive identification of sites potentially under selection. therefore, we posit that the identified sites might play a role in reshaping the antigenic properties of the na protein during the host-switch event from swine to human h1n viruses. characterization of the pandemic n structure and epitopes in order to test the hypothesis that the three sites identified above with the codon models do affect the antigenic properties of the na protein, we first set out to predict the structural changes involved between pandemic and non-pandemic molecules. recent work has focused on the ha protein, and has shown that the and proteins have very similar tridimensional structures. unfortunately, to date, the structure of the na protein has not been obtained. we therefore reconstructed 3d models by homology modeling with swiss-model. the prediction of the pandemic na was based on pdb template 2hty, which is of type n but derived from an h5n virus isolated in, which is consistent with the avian origin of h. both the non-pandemic and the swine models were derived from pdb template 3b7e, which is the structure of the pandemic n. the three models covered residues 83- and were quite similar. the main structural difference between the pandemic and the non-pandemic structure is due to a nonsynonymous substitution in the loop, which contains the active site of na, at position. most of the pandemic sequences have an isoleucine at this position, just like the swine sequences isolated in on the other hand, all the non-pandemic sequences and swine sequences isolated before have a valine, which is also the amino acid used by the h1n na protein and that was found to distort the structure of the na protein. this v149i substitution was previously reported in the context of studying two antineuraminidase compounds, and was judged to be too far from the drug binding pocket to impact antiviral susceptibility. our results therefore suggest that the v149i substitution affected the active site of na but could not be solely responsible for the onset of the pandemic since the protein had a valine, unless epistatic interactions exist. because we have found evidence for linkage between the segments of h1n, such epistatic interactions cannot be ruled out; but since we found that all segments had the same tmrca, our current approach cannot identify the segment and the site interacting directly with loop of na. however, in spite of this change in 3d structure of the pandemic na protein, site which is at the core of this structural change is not a site detected by our analyses of positive selection. could nonetheless loop still be a strong epitope? to address this question, we predicted the epitopes of the non-pandemic na proteins. figure shows the epitopes predicted for the human and swine sequences, with each peak above the threshold indicating the presence of an epitope. more specifically, figure 5a shows that a small number of differences exist between the pandemic and the non-pandemic proteins, as a total of five regions differ. notably, the sites detected to be potentially under positive selection for host-switch events, at positions and are included or in very close proximity of the regions where epitope differences are detected between pandemic and non-pandemic na proteins. yet, while no new epitope emerged within loop this region represents an epitope present in both pandemic and non-pandemic proteins, while its conformation changed nonadaptively in the sequences. on the other hand, the comparison of predicted b-cell epitopes of swine sequences vs. human pandemic sequences shows that the predicted epitopes are almost identical between these two viruses, to the exception of a small region n-terminal region 37- importantly, none of the sites potentially under positive selection falls within a predicted epitope difference. as a consequence, the emergence of novel na epitopes in the h1n pandemic viruses is most likely nonadaptive.  to summarize the results found in this study, we showed that the only evidence for positive selection is in the ha and na antigenes during host-switch events to human hosts, while the emergence of the pandemic was nonadaptive, loop which contains the active site of na, is an epitope present in all sampled sequences, and a recent substitution in this epitope spread rapidly but nonadaptively through the viral na sequences in irrespective of their host, potentially by means of a demographic bottleneck in human viruses following a reassortment event with a virus of swine origin, this substitution caused a structural modification of the pandemic na protein, and although this loop is found in all viruses sampled here, the pandemic na proteins are predicted to possess four novel epitopes not found in viruses circulating in swines or non-pandemic humans. these results are significant on two fronts. first, in terms of methodology, the use of codon models proved here to return more sensible results than the use of population genetics test of neutrality such as the mkt or the tajima test, which both have limitations when it comes to analyzing viral data as population genetics-based tests can be sensitive to demographic changes and/or high levels of diversity, so that these tests can have low power. however, the use of codon models in the context of a population study is also not fully satisfactory, as not all nonsynonymous substitutions can be assumed to be fixed. to circumvent this limitation, mutation-selection models that aim at bridging the gap between these two evolutionary scales have been developed. however, mutation-selection models have, so far, not been extended to detecting selection at certain amino acid sites in particular lineages and thus, do not allow us to investigate the complex question as to when and where evidence for positive selection can be found. second, in terms of the biology of influenza a viruses, our study shows that, while host-switch events and pandemics are not tightly coupled, it is likely that the emergence of new epitopes in a population is first nonadaptive for the virus. then, the frequency of these epitopes increases very quickly due to drift, before plummeting again because they are linked to a highly virulent phenotype that kills its hosts too quickly. a similar argument is generally derived from modeling the population dynamics of influenza viruses, potentially including very sophisticated immune interactions between hosts and viruses, and was also put forward based on the study of h3n subtypes. it is also significant to note that, while certain amino acid substitutions may be linked to the emergence of a particular epidemic or pandemic influenza strain, such as v149i, the persistence or re-emergence of this very same substitution is no guarantee of an up-coming threat to public health. more likely, the emergence of an epidemic or pandemic'phenotype' is the result of epistatic interactions between sites within or across segments, forming a constellation or network of epistatic interactions that are changing over time in ways that we do not currently fully understand. it is tempting to associate these changes of epistatic interactions to antigenic shifts, and future studies should address this potential link. the observation that evidence for positive selection was found only in the ha and na genes, which are two of the genes for which neτ increases, while no evidence for positive selection was found in the other genes showing such an increase may suggest that ha, na, pb and the m and ns genes might be linked. yet, this linkage is not constant in space or in time as these five different segments in h1n viruses have different origins, with ha and ns coming from classical swine, while na and m come from an eurasian avian-like swine and pb comes from a human h3n virus. a better insight into the timing and the forces at play in the emergence of influenza viruses and into the dynamics of gene constellations in influenza viruses will require a continuous and in-depth surveillance of the viruses circulating around the world in its various hosts. 
because this substitution was not present in the h1n pandemic virus, we posit that the emergence of pandemics is due to epistatic interactions between sites distributed over different segments. altogether, our results are consistent with population dynamics models that highlight the epistatic and nonadaptive rise of novel epitopes in viral populations, followed by their demise when the resulting virus is too virulent. the emergence of the h1n influenza pandemic followed a multiple reassortment event from viruses originally circulating in swines and humans, but the adaptive nature of this emergence is poorly understood. a more detailed analysis of the na protein shows that the pandemic sequence is characterized by novel epitopes and by a particular substitution in loop which is responsible for a nonadaptive structural change tightly associated with the emergence of the pandemic. we show that while transmission to a human host might require an adaptive phase in the ha and na antigens, the emergence of the pandemic was essentially nonadaptive. here we base our analysis on complete genomes of h1n viruses sampled in north america between and in swine and human hosts.
 the emergence of the h1n influenza pandemic followed a multiple reassortment event from viruses originally circulating in swines and humans, but the adaptive nature of this emergence is poorly understood.  here we base our analysis on complete genomes of h1n viruses sampled in north america between and in swine and human hosts. we show that while transmission to a human host might require an adaptive phase in the ha and na antigens, the emergence of the pandemic was essentially nonadaptive. a more detailed analysis of the na protein shows that the pandemic sequence is characterized by novel epitopes and by a particular substitution in loop which is responsible for a nonadaptive structural change tightly associated with the emergence of the pandemic.  because this substitution was not present in the h1n pandemic virus, we posit that the emergence of pandemics is due to epistatic interactions between sites distributed over different segments. altogether, our results are consistent with population dynamics models that highlight the epistatic and nonadaptive rise of novel epitopes in viral populations, followed by their demise when the resulting virus is too virulent. 