A key priority in infectious disease research is to understand the ecological and evolutionary drivers of viral diseases from data on disease incidence as well as viral genetic and antigenic variation. We propose using a simulation-based, Bayesian method known as Approximate Bayesian Computation (ABC) to fit and assess phylodynamic models that simulate pathogen evolution and ecology against summaries of these data. We illustrate the versatility of the method by analyzing two spatial models describing the phylodynamics of interpandemic human influenza virus subtype A(H3N2). The first model captures antigenic drift phenomenologically with continuously waning immunity, and the second epochal evolution model describes the replacement of major, relatively long-lived antigenic clusters. Combining features of long-term surveillance data from the Netherlands with features of influenza A (H3N2) hemagglutinin gene sequences sampled in northern Europe, key phylodynamic parameters can be estimated with ABC. Goodness-of-fit analyses reveal that the irregularity in interannual incidence and H3N2's ladder-like hemagglutinin phylogeny are quantitatively only reproduced under the epochal evolution model within a spatial context. However, the concomitant incidence dynamics result in a very large reproductive number and are not consistent with empirical estimates of H3N2's population level attack rate. These results demonstrate that the interactions between the evolutionary and ecological processes impose multiple quantitative constraints on the phylodynamic trajectories of influenza A(H3N2), so that sequence and surveillance data can be used synergistically. ABC, one of several data synthesis approaches, can easily interface a broad class of phylodynamic models with various types of data but requires careful calibration of the summaries and tolerance parameters.
The infectious disease dynamics of many viral pathogens like influenza, norovirus and coronavirus are inextricably tied to their evolution. This interaction between evolutionary and ecological processes complicates our ability to understand the infectious disease behavior of rapidly evolving pathogens. Most statistical methods for the analysis of these “phylodynamics” require that the likelihood of the data can be explicitly calculated. Currently, this is not possible for many phylodynamic models, so that questions on the interaction between viral variants cannot be well-addressed within this framework. Simulation-based statistical methods circumvent likelihood calculations. Considering interpandemic human influenza A virus subtype H3N2, we here illustrate the effectiveness of these methods to fit and assess complex phylodynamic models against both sequence and surveillance data. We find that combining molecular genetic and epidemiological data is key to estimate phylodynamic parameters reliably. Moreover, the information in the available data taken together is enough to expose quantitative model inconsistencies. Methods such as ABC which can combine sequence and surveillance data appear to be well-suited to fit and assess mechanistic hypotheses on the phylodynamics of RNA viruses.
Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.
Rapid antigenic evolution in the influenza A virus hemagglutinin precludes effective vaccination with existing vaccines. To understand this phenomenon, we passaged virus in mice immunized with influenza. Neutralizing antibodies selected mutants with single amino acid hemagglutinin substitutions that increased virus binding to cell surface glycan receptors. Passaging these high avidity-binding mutants in naïve mice, but not immune mice, selected for additional hemagglutinin substitutions that decreased cellular receptor binding avidity. Analyzing a panel of monoclonal antibody hemagglutinin escape mutants revealed a positive correlation between receptor binding avidity and escape from polyclonal antibodies. We propose that in response to variation in neutralizing antibody pressure between individuals, influenza A virus evolves by adjusting receptor binding avidity via amino acid substitutions throughout the hemagglutinin globular domain, many of which simultaneously alter antigenicity.
The evolution of haemagglutinin (HA), an important influenza virus antigen, has been the subject of intensive research for more than two decades. Many characteristics of HA's sequence evolution are captured by standard Markov chain substitution models. Such models assign equal fitness to all accessible amino acids at a site. We show, however, that such models strongly underestimate the number of homoplastic amino acid substitutions during the course of HA's evolution, i.e. substitutions that repeatedly give rise to the same amino acid at a site. We develop statistics to detect individual homoplastic events and find that they preferentially occur at positively selected epitopic sites. Our results suggest that the evolution of the influenza A HA, including evolution by positive selection, is strongly affected by the long-term site-specific preferences for individual amino acids.
directional selection; dN/dS; haemagglutinin; homoplasy; influenza A
Human Influenza A virus undergoes recurrent changes in the hemagglutinin (HA) surface protein, primarily involved in the human antibody recognition. Relevant antigenic changes, enabling the virus to evade host immune response, have been recognized to occur in parallel to multiple mutations at antigenic sites in HA. Yet, the role of correlated mutations (epistasis) in driving the molecular evolution of the virus still represents a challenging puzzle. Further, though circulation at a global geographic level is key for the survival of Influenza A, its role in shaping the viral phylodynamics remains largely unexplored. Here we show, through a sequence based epidemiological model, that epistatic effects between amino acids substitutions, coupled with a reservoir that mimics worldwide circulating viruses, are key determinants that drive human Influenza A evolution. Our approach explains all the up-to-date observations characterizing the evolution of H3N2 subtype, including phylogenetic properties, nucleotide fixation patterns, and composition of antigenic clusters.
The rapid evolution of influenza viruses presents difficulties in maintaining the optimal efficiency of vaccines. Amino acid substitutions result in antigenic drift, a process whereby antisera raised in response to one virus have reduced effectiveness against future viruses. Interestingly, while amino acid substitutions occur at a relatively constant rate, the antigenic properties of H3 move in a discontinuous, step-wise manner. It is not clear why this punctuated evolution occurs, whether this represents simply the fact that some substitutions affect these properties more than others, or if this is indicative of a changing relationship between the virus and the host. In addition, the role of changing glycosylation of the haemagglutinin in these shifts in antigenic properties is unknown. We analysed the antigenic drift of HA1 from human influenza H3 using a model of sequence change that allows for variation in selective pressure at different locations in the sequence, as well as at different parts of the phylogenetic tree. We detect significant changes in selective pressure that occur preferentially during major changes in antigenic properties. Despite the large increase in glycosylation during the past 40 years, changes in glycosylation did not correlate either with changes in antigenic properties or with significantly more rapid changes in selective pressure. The locations that undergo changes in selective pressure are largely in places undergoing adaptive evolution, in antigenic locations, and in locations or near locations undergoing substitutions that characterise the change in antigenicity of the virus. Our results suggest that the relationship of the virus to the host changes with time, with the shifts in antigenic properties representing changes in this relationship. This suggests that the virus and host immune system are evolving different methods to counter each other. While we are able to characterise the rapid increase in glycosylation of the haemagglutinin during time in human influenza H3, an increase not present in influenza in birds, this increase seems unrelated to the observed changes in antigenic properties.
H3N2-type influenza is responsible for widespread disease and significant mortality. The virus evolves rapidly, changing its antigenic properties, allowing it to escape clearance by the immune response as well as complicating the maintenance of vaccine effectiveness. Part of this evolution has been the rapid increase in glycosylation, an increase not observed either in H9 evolution in birds or in H1 evolution in humans. It has been observed that the antigenic properties change in a punctuated, discontinuous manner. This could be either because some mutations are more significant than others, or it could mean that the antigenic changes correspond to adjustments in the antagonistic relationship between virus and host. By studying the sequence evolution of the H3 haemagglutinin, we can demonstrate that the selective pressure acting on the virus protein changes with time, and that these changes are especially rapid during changes in antigenic properties. This indicates that the antigenic changes correspond to modifications in the virus–host relationship. Surprisingly, neither the changes in selective pressure nor the changes in antigenic properties correspond to changes in glycosylation.
Influenza A virus is one of the best-studied viruses and a model organism for the study of molecular evolution; in particular, much research has focused on detecting natural selection on influenza virus proteins. Here, we study the dynamics of the synonymous and nonsynonymous nucleotide composition of influenza A virus genes. In several genes, the nucleotide frequencies at synonymous positions drift away from the equilibria predicted from the synonymous substitution matrices. We investigate possible reasons for this unexpected behavior by fitting several regression models. Relaxation toward a mutation-selection equilibrium following a host jump fails to explain the dynamics of the synonymous nucleotide composition, even if we allow for slow temporal changes in the substitution matrix. Instead, we find that deep internal branches of the phylogeny show distinct patterns of nucleotide substitution and that these branches strongly influence the dynamics of nucleotide composition, suggesting that the observed trends are at least in part a result of natural selection acting on synonymous sites. Moreover, we find that the dynamics of the nucleotide composition at synonymous and nonsynonymous sites are highly correlated, providing evidence that even nonsynonymous sites can be influenced by selection pressure for nucleotide composition.
Whole-genome scans for positive Darwinian selection are widely used to detect evolution of genome novelty. Most approaches are based on evaluation of nonsynonymous to synonymous substitution rate ratio across evolutionary lineages. These methods are sensitive to saturation of synonymous sites and thus cannot be used to study evolution of distantly related organisms. In contrast, indels occur less frequently than amino acid replacements, accumulate more slowly, and can be employed to characterize evolution of diverged organisms. As indels are also subject to the forces of natural selection, they can generate functional changes through positive selection. Here, we present a new computational approach to detect selective constraints on indel substitutions at the whole-genome level for distantly related organisms. Our method is based on ancestral sequence reconstruction, takes into account the varying susceptibility of different types of secondary structure to indels, and according to simulation studies is conservative. We applied this newly developed framework to characterize the evolution of organisms of the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) bacterial superphylum. The superphylum contains organisms with unique cell biology, physiology, and diverse lifestyles. It includes bacteria with simple cell organization and more complex eukaryote-like compartmentalization. Lifestyles range from free-living organisms to obligate pathogens. In this study, we conduct a whole-genome level analysis of indel substitutions specific to evolutionary lineages of the PVC superphylum and found that indels evolved under positive selection on up to 12% of gene tree branches. We also analyzed possible functional consequences for several case studies of predicted indel events.
selection; indel substitutions; PVC superphylum
Influenza viruses undergo continual antigenic evolution allowing mutant viruses to evade host immunity acquired to previous virus strains. Antigenic phenotype is often assessed through pairwise measurement of cross-reactivity between influenza strains using the hemagglutination inhibition (HI) assay. Here, we extend previous approaches to antigenic cartography, and simultaneously characterize antigenic and genetic evolution by modeling the diffusion of antigenic phenotype over a shared virus phylogeny. Using HI data from influenza lineages A/H3N2, A/H1N1, B/Victoria and B/Yamagata, we determine patterns of antigenic drift across viral lineages, showing that A/H3N2 evolves faster and in a more punctuated fashion than other influenza lineages. We also show that year-to-year antigenic drift appears to drive incidence patterns within each influenza lineage. This work makes possible substantial future advances in investigating the dynamics of influenza and other antigenically-variable pathogens by providing a model that intimately combines molecular and antigenic evolution.
Every year, seasonal influenza, commonly called flu, infects up to one in five people around the world, and causes up to half a million deaths. Even though the human immune system can detect and destroy the virus that causes influenza, people can catch flu many times throughout their lifetimes because the virus keeps evolving in an effort to avoid the immune system. This antigenic drift—so-called because the antigens displayed by the virus keep changing—also explains why influenza vaccines become less effective over time and need to be reformulated every year.
It is possible to determine which antigens are displayed by a new strain of the virus by observing how blood samples that respond to known strains respond to the new strain. This information about the “antigenic phenotype” of the virus can be plotted on an antigenic map in which strains with similar antigens cluster together. Gene sequencing has shown that there are four subtypes of the flu virus that commonly infect people; but the relationship between changes in antigenic phenotype and changes in gene sequences of the influenza virus is poorly understood.
Bedford et al. have now developed an approach to combine antigenic maps with genetic information about the four subtypes of the human flu virus. This revealed that the antigenic phenotype of H3N2—a subtype that is becoming increasingly common—evolved faster than the other three subtypes. Further, a correlation was observed between antigenic drift and the number of new influenza cases per year for each flu strain. This suggests that knowing which antigenic phenotypes are present at the start of flu season could help predict which strains of the virus will predominate later on.
The work of Bedford et al. provides a useful framework to study influenza, and could help to pinpoint which changes in viral genes cause the changes in antigens. This information could potentially speed up the development of new flu vaccines for each flu season.
influenza; evolution; antigenic cartography; phylogenetics; Bayesian inference; multidimensional scaling; viruses
The seasonal influenza A virus undergoes rapid evolution to escape human immune response. Adaptive changes occur primarily in antigenic epitopes, the antibody-binding domains of the viral hemagglutinin. This process involves recurrent selective sweeps, in which clusters of simultaneous nucleotide fixations in the hemagglutinin coding sequence are observed about every 4 years. Here, we show that influenza A (H3N2) evolves by strong clonal interference. This mode of evolution is a red queen race between viral strains with different beneficial mutations. Clonal interference explains and quantifies the observed sweep pattern: we find an average of at least one strongly beneficial amino acid substitution per year, and a given selective sweep has three to four driving mutations on average. The inference of selection and clonal interference is based on frequency time series of single-nucleotide polymorphisms, which are obtained from a sample of influenza genome sequences over 39 years. Our results imply that mode and speed of influenza evolution are governed not only by positive selection within, but also by background selection outside antigenic epitopes: immune adaptation and conservation of other viral functions interfere with each other. Hence, adapting viral proteins are predicted to be particularly brittle. We conclude that a quantitative understanding of influenza’s evolutionary and epidemiological dynamics must be based on all genomic domains and functions coupled by clonal interference.
adaptive evolution; inference of selection; mutation rate; seasonal influenza
Influenza B virus hemagglutinin (HA) is a major surface glycoprotein with frequent amino acid substitutions. However, the roles of antibody selection in the amino acid substitutions of HA were still poorly understood. In order to gain insights into this important issue, an analysis was conducted on a total of 271 HA1. sequences of influenza B virus strains isolated during 1940–2007. In this analysis, phylogenetic analysis by maximum likelihood (PAML) package was used to detect the existence of positive selection and to identify positively selected sites on HA1. Strikingly, all the positively selected sites were located in the four major epitopes (120-loop, 150-loop, 160-loop, and 190-helix) of HA identified in previous studies, thus supporting a predominant role of antibody selection in HA evolution. Of particular significance is the involvement of the 120-loop in positive selection, which may become increasingly important in future field isolates. Despite the absence of different subtypes, influenza B virus HA continued to evolve into new sublineages, within which the four major epitopes were targeted selectively in positive selection. Thus, any newly emerging strains need to be placed in the context of their evolutionary history in order to understand and predict their epidemic potential.
positive selection; antigenic drift; molecular evolution; antibody selection
After the emergence of influenza A viruses in the human population, the number of N-glycosylation sites (NGS) in the globular head region of hemagglutinin (HA) has increased continuously for several decades. It has been speculated that the addition of NGS to the globular head region of HA has conferred selective advantages to the virus by preventing the binding of antibodies (Ab) to antigenic sites (AS). Here, the effect of N-glycosylation on the binding of Ab to AS in human influenza A virus subtype H3N2 (A/H3N2) was examined by inferring natural selection at AS and other sites (NAS) that are located close to and distantly from the NGS in the three-dimensional structure of HA through a comparison of the rates of synonymous (dS) and nonsynonymous (dN) substitutions. When positions 63, 122, 126, 133, 144, and 246 in the globular head region of HA were non-NGS, the dN/dS was >1 and positive selection was detected at the AS located near these positions. However, the dN/dS value decreased and the evidence of positive selection disappeared when these positions became NGS. In contrast, dN/dS at the AS distantly located from the positions mentioned above and at the NAS of any location were generally <1 and did not decrease when these positions changed from non-NGS to NGS. These results suggest that the attachment of N-glycans to the NGS in the globular head region of HA prevented the binding of Ab to AS in the evolutionary history of human A/H3N2 virus.
Despite their close phylogenetic relationship, type A and B influenza viruses exhibit major epidemiological differences in humans, with the latter both less common and less often associated with severe disease. However, it is unclear what processes determine the evolutionary dynamics of influenza B virus, and how influenza viruses A and B interact at the evolutionary scale. To address these questions we inferred the phylogenetic history of human influenza B virus using complete genome sequences for which the date (day) of isolation was available. By comparing the phylogenetic patterns of all eight viral segments we determined the occurrence of segment reassortment over a 30-year sampling period. An analysis of rates of nucleotide substitution and selection pressures revealed sporadic occurrences of adaptive evolution, most notably in the viral hemagglutinin and compatible with the action of antigenic drift, yet lower rates of overall and nonsynonymous nucleotide substitution compared to influenza A virus. Overall, these results led us to propose a model in which evolutionary changes within and between the antigenically distinct ‘Yam88’ and ‘Vic87’ lineages of influenza B virus are the result of changes in herd immunity, with reassortment continuously generating novel genetic variation. Additionally, we suggest that the interaction with influenza A virus may be central in shaping the evolutionary dynamics of influenza B virus, facilitating the shift of dominance between the Vic87 and the Yam88 lineages.
Influenza B virus; Phylogeny; Reassortment; Coalescent; Antigenic drift; Epidemiology
To investigate the process of human immunodeficiency virus type 1 (HIV-1) evolution in vivo, a total of 179 HIV-1 V3 sequences derived from cell-free plasma were determined from serial samples in three epidemiologically linked individuals (one infected blood donor and two transfusion recipients) over a maximum period of 8 years. A systematic analysis of pairwise comparisons of intrapatient sequences, both within and between each sample time point, revealed a preponderance and accumulation of nonsynonymous rather than synonymous substitutions in the V3 loop and flanking regions as they diverged over time. This strongly argues for the dominant role that positive selection for amino acid change plays in governing the pattern and process of HIV-1 env V3 evolution in vivo and nullifies hypotheses of purely neutral or mutation-driven evolution or completely chance events. In addition, different rates of evolution of HIV-1 were observed in these three different individuals infected with the same viral strain, suggesting that the degree of positive pressure for HIV-1 amino acid change is host dependent. Finally, the observed similar rate of accumulation in divergence within and between infected individuals suggests that the process of genetic divergence in the HIV epidemic proceeds regardless of host-to-host transmission events, i.e., that transmission does not reset the evolutionary clock.
Influenza virus undergoes rapid evolution by both antigenic shift and antigenic drift. Antibodies, particularly those binding near the receptor-binding site of hemagglutinin (HA) or the neuraminidase (NA) active site, are thought to be the primary defense against influenza infection, and mutations in antibody binding sites can reduce or eliminate antibody binding. The binding of antibodies to their cognate antigens is governed by such biophysical properties of the interacting surfaces as shape, non-polar and polar surface area, and charge.
To understand forces shaping evolution of influenza virus, we have examined HA sequences of human influenza A and B viruses, assigning each amino acid values reflecting total accessible surface area, non-polar and polar surface area, and net charge due to the side chain. Changes in each of these values between neighboring sequences were calculated for each residue and mapped onto the crystal structures.
Areas of HA showing the highest frequency of pairwise changes agreed well with previously identified antigenic sites in H3 and H1 HAs, and allowed us to propose more detailed antigenic maps and novel antigenic sites for H1 and influenza B HA. Changes in biophysical properties differed between HAs of different subtypes, and between different antigenic sites of the same HA. For H1, statistically significant differences in several biophysical quantities compared to residues lying outside antigenic sites were seen for some antigenic sites but not others. Influenza B antigenic sites all show statistically significant differences in biophysical quantities for all antigenic sites, whereas no statistically significant differences in biophysical quantities were seen for any antigenic site is seen for H3. In many cases, residues previously shown to be under positive selection at the genetic level also undergo rapid change in biophysical properties.
The biophysical consequences of amino acid changes introduced by antigenic drift vary from subtype to subtype, and between different antigenic sites. This suggests that the significance of antibody binding in selecting new variants may also be variable for different antigenic sites and influenza subtypes.
Adaptive evolution is characterized by positive and parallel, or repeated selection of mutations. Mouse adaptation of influenza A virus (IAV) produces virulent mutants that demonstrate positive and parallel evolution of mutations in the hemagglutinin (HA) receptor and non-structural protein 1 (NS1) interferon antagonist genes. We now present a genomic analysis of all 11 genes of 39 mouse adapted IAV variants from 10 replicate adaptation experiments. Mutations were mapped on the primary and structural maps of each protein and specific mutations were validated with respect to virulence, replication, and RNA polymerase activity. Mouse adapted (MA) variants obtained after 12 or 20–21 serial infections acquired on average 5.8 and 7.9 nonsynonymous mutations per genome of 11 genes, respectively. Among a total of 115 nonsynonymous mutations, 51 demonstrated properties of natural selection including 27 parallel mutations. The greatest degree of parallel evolution occurred in the HA receptor and ribonucleocapsid components, polymerase subunits (PB1, PB2, PA) and NP. Mutations occurred in host nuclear trafficking factor binding sites as well as sites of virus-virus protein subunit interaction for NP, NS1, HA and NA proteins. Adaptive regions included cap binding and endonuclease domains in the PB2 and PA polymerase subunits. Four mutations in NS1 resulted in loss of binding to the host cleavage and polyadenylation specificity factor (CPSF30) suggesting that a reduction in inhibition of host gene expression was being selected. The most prevalent mutations in PB2 and NP were shown to increase virulence but differed in their ability to enhance replication and demonstrated epistatic effects. Several positively selected RNA polymerase mutations demonstrated increased virulence associated with >300% enhanced polymerase activity. Adaptive mutations that control host range and virulence were identified by their repeated selection to comprise a defined model for studying IAV evolution to increased virulence in the mouse.
Tat-specific cytotoxic T cells have previously been shown to exert positive Darwinian selection favoring amino acid replacements of an epitope of simian immunodeficiency virus (SIV). The region of the tat gene encoding this epitope falls within a region of overlap between the tat and vpr reading frames, and nonsynonymous nucleotide substitutions in the tat reading frame were found to occur disproportionately in such a way as to cause synonymous changes in the vpr reading frame. Comparison of published complete SIV genomes showed Tat to be the least conserved at the amino acid level of nine proteins encoded by the virus, while Vpr was one of the most conserved. Numerous parallel amino acid changes occurred within the Tat epitope independently in different monkeys, and purifying selection on the vpr reading frame, by limiting acceptable nonsynonymous substitutions in the tat reading frame, evidently has enhanced the probability of parallel evolution.
The gene encoding the progesterone receptor (PGR) acts as a transcription factor, and participates in the regulation of reproductive processes including menstruation, implantation, pregnancy maintenance, parturition, mammary development, and lactation. Unlike other mammals, primates do not exhibit progesterone withdrawal at the time of parturition. Because progesterone-mediated reproductive features vary among mammals, PGR is an attractive candidate gene for studies of adaptive evolution. Thus, we sequenced the progesterone receptor coding regions in a diverse range of species including apes, Old World monkeys, New World monkeys, prosimian primates and other mammals. Adaptive evolution occurred on the human and chimpanzee lineages as evidenced by statistically significant increases in nonsynonymous substitution rates compared to synonymous substitution rates. Positive selection was rarely observed in other lineages. In humans, amino acid replacements occurred mostly in a region of the gene that has been shown to have an inhibitory function (IF) on the ability of the progesterone receptor to act as a transcription factor. Moreover, many of the nonsynonymous substitutions in primates occurred in the N-terminus. This suggests that cofactor interaction surfaces might have been altered, resulting in altered progesterone-regulated gene transcriptional effects. Further evidence that the changes conferred an adaptive advantage comes from SNP analysis indicating only one of the IF changes is polymorphic in humans. In chimpanzees, amino acid changes occurred in both the inhibitory and transactivation domains. Positive selection provides the basis for the hypothesis that changes in structure and function of the progesterone receptor during evolution contributes to the diversity of primate reproductive biology, especially in parturition.
The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8+ T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8+ T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8+ T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.
We used a panel of monoclonal antibodies to H9 hemagglutinin to select 18 escape mutants of mouse-adapted influenza A/Swine/Hong Kong/9/98 (H9N2) virus. Cross-reactions of the mutants with the antibodies and the sequencing of hemagglutinin genes revealed two minimally overlapping epitopes. We mapped the amino acid changes to two areas of the recently reported three-dimensional structure of A/Swine/Hong Kong/9/98 hemagglutinin. The grouping of the antigenically relevant amino acid positions in H9 hemagglutinin differs from the pattern observed in H3 and H5 hemagglutinins. Several positions in site B of H3 hemagglutinin are distributed in two sites of H9 hemagglutinin. Unlike any subtype analyzed so far, H9 hemagglutinin does not contain an antigenic site corresponding to site A in H3 hemagglutinin. Positions 145 and 193 (H3 numbering), which in H3 hemagglutinin belong to sites A and B, respectively, are within one site in H9 hemagglutinin. This finding is consistent with the peculiarity of the three-dimensional structure of the H9 molecule, that is, the absence from H9 hemagglutinin of the lateral loop that forms site A in H3 and the equivalent site in H5 hemagglutinins. The escape mutants analyzed displayed phenotypic variations, including decreased virulence for mice and changes in affinity for sialyl substrates. Our results demonstrate a correlation between intersubtype differences in three-dimensional structure and variations among subtypes in the distribution of antigenic areas. Our findings also suggest that covariation and pleiotropic effects of antibody-selected mutations may be important in the evolution of H9 influenza virus, a possible causative agent of a future pandemic.
Models based on amino acid changes in influenza hemagglutinin protein were compared to predict antigenic variants of influenza A/H3N2 viruses.
Current inactivated influenza vaccines provide protection when vaccine antigens and circulating viruses share a high degree of similarity in hemagglutinin protein. Five antigenic sites in the hemagglutinin protein have been proposed, and 131 amino acid positions have been identified in the five antigenic sites. In addition, 20, 18, and 32 amino acid positions in the hemagglutinin protein have been identified as mouse monoclonal antibody–binding sites, positively selected codons, and substantially diverse codons, respectively. We investigated these amino acid positions for predicting antigenic variants of influenza A/H3N2 viruses in ferrets. Results indicate that the model based on the number of amino acid changes in the five antigenic sites is best for predicting antigenic variants (agreement = 83%). The methods described in this study could be applied to predict vaccine-induced cross-reactive antibody responses in humans, which may further improve the selection of vaccine strains.
influenza; antigenicity; vaccine strain; hemagglutinin; prediction model; antigenic variants; bioinformatics; research
Understanding the evolutionary dynamics of influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. Here we show with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype. These clades cocirculated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.
A comparative analysis of 413 complete genomes of the H3N2 subtype of human influenza A virus sampled from New York State, United States, the largest and first population-based study of its kind, reveals that viral evolution within epidemic seasons is dominated by the random importation of genetically different viral strains from other geographic areas, rather than by natural selection favoring local strains able to escape the human immune response. Multiple clades of genetically distinct viral strains cocirculate across the entire state within any season and occasionally exchange genes through reassortment. Both genetic diversity and geographic viral “traffic” are extensive within epidemics. Therefore, the evolution of influenza A virus is strongly shaped by random migration and reassortment and is far harder to predict than previously realized. Consequently, intensive sampling and whole-genome sequencing are essential for selecting viral strains for future vaccine production.
Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.
Genome Stability; Hemagglutinin; Neuraminidase; Pandemic Influenza H1N1 2009; Respiratory Disease; Swine Flu.
Distinguishing mutations that determine an organism's phenotype from (near-) neutral ‘hitchhikers’ is a fundamental challenge in genome research, and is relevant for numerous medical and biotechnological applications. For human influenza viruses, recognizing changes in the antigenic phenotype and a strains' capability to evade pre-existing host immunity is important for the production of efficient vaccines. We have developed a method for inferring ‘antigenic trees’ for the major viral surface protein hemagglutinin. In the antigenic tree, antigenic weights are assigned to all tree branches, which allows us to resolve the antigenic impact of the associated amino acid changes. Our technique predicted antigenic distances with comparable accuracy to antigenic cartography. Additionally, it identified both known and novel sites, and amino acid changes with antigenic impact in the evolution of influenza A (H3N2) viruses from 1968 to 2003. The technique can also be applied for inference of ‘phenotype trees’ and genotype–phenotype relationships from other types of pairwise phenotype distances.
The molecular evolution of any organism is described by changes in the genotype resulting from genetic drift or selection to maintain or establish fitness under the given environmental conditions. Identification of phenotype-defining changes and their distinction from (near-) neutral (‘hitchhikers’) ones is a fundamental challenge in genome research. The standard approach involves time- and cost-intensive mutation experiments, which are typically low throughput, due to their experimental nature. We have developed a computational method for the inference of phenotypic impact of genotypic changes that is applicable to any system, within or across species, where homologous genetic sequences and associated pairwise phenotype distances are available. We demonstrate the accuracy of our method by application to the human influenza A (H3N2) virus. This exemplary system is of particular interest, as recognizing changes in the antigenic phenotype and a viral strains' capability to evade pre-existing host immunity is important for the production of efficient vaccines. We accurately identified known sites and amino acid changes with antigenic impact over 35 years of evolution, and provide further details on individual antigenically relevant changes in the evolution of influenza A (H3N2) viruses.
Non-long terminal repeat retroelements continue to impact the human genome through
cis-activity of long interspersed element-1 (LINE-1 or L1) and trans-mobilization of Alu.
Current activity is dominated by modern subfamilies of these elements, leaving behind an
evolutionary graveyard of extinct Alu and L1 subfamilies. Because Alu is a nonautonomous
element that relies on L1 to retrotranspose, there is the possibility that competition
between these elements has driven selection and antagonistic coevolution between Alu and
L1. Through analysis of synonymous versus nonsynonymous codon evolution across L1
subfamilies, we find that the C-terminal ORF2 cys domain experienced a dramatic increase
in amino acid substitution rate in the transition from L1PA5 to L1PA4 subfamilies. This
observation coincides with the previously reported rapid evolution of ORF1 during the same
transition period. Ancestral Alu sequences have been previously reconstructed, as their
short size and ubiquity have made it relatively easy to retrieve consensus sequences from
the human genome. In contrast, creating constructs of extinct L1 copies is a more
laborious task. Here, we report our efforts to recreate and evaluate the
retrotransposition capabilities of two ancestral L1 elements, L1PA4 and L1PA8 that were
active ∼18 and ∼40 Ma, respectively. Relative to the modern L1PA1 subfamily, we
find that both elements are similarly active in a cell culture retrotransposition assay in
HeLa, and both are able to efficiently trans-mobilize Alu elements from several
subfamilies. Although we observe some variation in Alu subfamily retrotransposition
efficiency, any coevolution that may have occurred between LINEs and SINEs is not evident
from these data. Population dynamics and stochastic variation in the number of active
source elements likely play an important role in individual LINE or SINE subfamily
amplification. If coevolution also contributes to changing retrotransposition rates and
the progression of subfamilies, cell factors are likely to play an important mediating
role in changing LINE-SINE interactions over evolutionary time.
Alu; extinction; LINE-1; L1; ORF1 protein; ORF2 protein; retroelement; SINE amplification