Linking influenza virus evolution within and between human hosts.
ABSTRACT: Influenza viruses rapidly diversify within individual human infections. Several recent studies have deep-sequenced clinical influenza infections to identify viral variation within hosts, but it remains unclear how within-host mutations fare at the between-host scale. Here, we compare the genetic variation of H3N2 influenza within and between hosts to link viral evolutionary dynamics across scales. Synonymous sites evolve at similar rates at both scales, indicating that global evolution at these putatively neutral sites results from the accumulation of within-host variation. However, nonsynonymous mutations are depleted between hosts compared to within hosts, suggesting that selection purges many of the protein-altering changes that arise within hosts. The exception is at antigenic sites, where selection detectably favors nonsynonymous mutations at the global scale, but not within hosts. These results suggest that selection against deleterious mutations and selection for antigenic change are the main forces that act on within-host variants of influenza virus as they transmit and circulate between hosts.
Project description:Influenza vaccines must be frequently reformulated to account for antigenic changes in the viral envelope protein, hemagglutinin (HA). The rapid evolution of influenza virus under immune pressure is likely enhanced by the virus's genetic diversity within a host, although antigenic change has rarely been investigated on the level of individual infected humans. We used deep sequencing to characterize the between- and within-host genetic diversity of influenza viruses in a cohort of patients that included individuals who were vaccinated and then infected in the same season. We characterized influenza HA segments from the predominant circulating influenza A subtypes during the 2012-2013 (H3N2) and 2013-2014 (pandemic H1N1; H1N1pdm) flu seasons. We found that HA consensus sequences were similar in nonvaccinated and vaccinated subjects. In both groups, purifying selection was the dominant force shaping HA genetic diversity. Interestingly, viruses from multiple individuals harbored low-frequency mutations encoding amino acid substitutions in HA antigenic sites at or near the receptor-binding domain. These mutations included two substitutions in H1N1pdm viruses, G158K and N159K, which were recently found to confer escape from virus-specific antibodies. These findings raise the possibility that influenza antigenic diversity can be generated within individual human hosts but may not become fixed in the viral population even when they would be expected to have a strong fitness advantage. Understanding constraints on influenza antigenic evolution within individual hosts may elucidate potential future pathways of antigenic evolution at the population level.Influenza vaccines must be frequently reformulated due to the virus's rapid evolution rate. We know that influenza viruses exist within each infected host as a "swarm" of genetically distinct viruses, but the role of this within-host diversity in the antigenic evolution of influenza has been unclear. We characterized here the genetic and potential antigenic diversity of influenza viruses infecting humans, some of whom became infected despite recent vaccination. Influenza virus between- and within-host genetic diversity was not significantly different in nonvaccinated and vaccinated humans, suggesting that vaccine-induced immunity does not exert strong selective pressure on viruses replicating in individual people. We found low-frequency mutations, below the detection threshold of traditional surveillance methods, in nonvaccinated and vaccinated humans that were recently associated with antibody escape. Interestingly, these potential antigenic variants did not reach fixation in infected people, suggesting that other evolutionary factors may be hindering their emergence in individual humans.
Project description:The evolutionary dynamics of influenza virus ultimately derive from processes that take place within and between infected individuals. Here we define influenza virus dynamics in human hosts through sequencing of 249 specimens from 200 individuals collected over 6290 person-seasons of observation. Because these viruses were collected from individuals in a prospective community-based cohort, they are broadly representative of natural infections with seasonal viruses. Consistent with a neutral model of evolution, sequence data from 49 serially sampled individuals illustrated the dynamic turnover of synonymous and nonsynonymous single nucleotide variants and provided little evidence for positive selection of antigenic variants. We also identified 43 genetically-validated transmission pairs in this cohort. Maximum likelihood optimization of multiple transmission models estimated an effective transmission bottleneck of 1-2 genomes. Our data suggest that positive selection is inefficient at the level of the individual host and that stochastic processes dominate the host-level evolution of influenza viruses.
Project description:The surface glycoprotein hemagglutinin (HA) helps the influenza A virus to evade the host immune system by antigenic variation and is a major driving force for viral evolution. In this study, the selection pressure on HA of H5N1 influenza A virus was analyzed using bioinformatics algorithms. Most of the identified positive selection (PS) sites were found to be within or adjacent to epitope sites. Some of the identified PS sites are consistent with previous experimental studies, providing further support to the biological significance of our findings. The highest frequency of PS sites was observed in recent strains isolated during 2005-2007. Phylogenetic analysis was also conducted on HA sequences from various hosts. Viral drift is almost similar in both avian and human species with a progressive trend over the years. Our study reports new mutations in functional regions of HA that might provide markers for vaccine design or can be used to predict isolates of pandemic potential.
Project description:In recent years a number of studies have brought attention to the role of positive selection during the evolution of antigenic escape by influenza strains. Particularly, the identification of positively selected sites within antigenic domains of viral surface proteins has been used to suggest that the evolution of viral-host receptor binding specificity is driven by selection. Here we show that, following the 1918 outbreak, the antigenic sites of the hemagglutinin (HA) viral surface protein and the stalk region of neuraminidase became substitution hotspots. The hotspots show similar patterns of nucleotide substitution bias at synonymous and nonsynonymous sites. Such bias imposes directionality in amino acid replacements that can influence signals of selection at antigenic sites. Our results suggest that the high accumulation of substitutions within the antigenic sites of HA can explain not only cases of antigenic escape by antigenic drift but also lead to occasional episodes of viral extinction.
Project description:CD8(+) cytotoxic T-lymphocytes (CTLs) perform a critical role in the immune control of viral infections, including those caused by human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV). As a result, genetic variation at CTL epitopes is strongly influenced by host-specific selection for either escape from the immune response, or reversion due to the replicative costs of escape mutations in the absence of CTL recognition. Under strong CTL-mediated selection, codon positions within epitopes may immediately "toggle" in response to each host, such that genetic variation in the circulating virus population is shaped by rapid adaptation to immune variation in the host population. However, this hypothesis neglects the substantial genetic variation that accumulates in virus populations within hosts. Here, we evaluate this quantity for a large number of HIV-1- (n > or = 3,000) and HCV-infected patients (n > or = 2,600) by screening bulk RT-PCR sequences for sequencing "mixtures" (i.e., ambiguous nucleotides), which act as site-specific markers of genetic variation within each host. We find that nonsynonymous mixtures are abundant and significantly associated with codon positions under host-specific CTL selection, which should deplete within-host variation by driving the fixation of the favored variant. Using a simple model, we demonstrate that this apparently contradictory outcome can be explained by the transmission of unfavorable variants to new hosts before they are removed by selection, which occurs more frequently when selection and transmission occur on similar time scales. Consequently, the circulating virus population is shaped by the transmission rate and the disparity in selection intensities for escape or reversion as much as it is shaped by the immune diversity of the host population, with potentially serious implications for vaccine design.
Project description:The patterns and dynamics of evolution in acutely infecting viruses within individual hosts are largely unknown. To this end, we investigated the intrahost variation of canine influenza virus (CIV) during the course of experimental infections in naïve and partially immune dogs and in naturally infected dogs. Tracing sequence diversity in the gene encoding domain 1 of the hemagglutinin (HA1) protein over the time course of infection provided information on the patterns and processes of intrahost viral evolution and revealed some of the effects of partial host immunity. Viral populations sampled on any given day were generally characterized by mean pairwise genetic diversities between 0.1 and 0.2% and by mutational spectra that changed considerably on different days. Some observed mutations may have affected antigenicity or host range, including reversions of CIV host-associated mutations. Patterns of sequence diversity differed between naïve and vaccinated dogs, with some presumably antigenic mutations transiently reaching high frequency in the latter. CIV populations are therefore characterized by the rapid generation and clearance of genetic diversity. Potentially advantageous mutations arise readily during the course of single infections and may give rise to antigenic escape or host range variants.
Project description:To examine whether positive selection operates on the hemagglutinin 1 (HA1) gene of human influenza A viruses (H1 subtype), 21 nucleotide sequences of the HA1 gene were statistically analyzed. The nucleotide sequences were divided into antigenic and nonantigenic sites. The nucleotide diversities for antigenic and nonantigenic sites of the HA1 gene were computed at synonymous and nonsynonymous sites separately. For nonantigenic sites, the nucleotide diversities were larger at synonymous sites than at nonsynonymous sites. This is consistent with the neutral theory of molecular evolution. For antigenic sites, however, the nucleotide diversities at nonsynonymous sites were larger than those at synonymous sites. These results suggest that positive selection operates on antigenic sites of the HA1 gene of human influenza A viruses (H1 subtype).
Project description:Human influenza viruses are rapidly evolving RNA viruses that cause short-term respiratory infections with substantial morbidity and mortality in annual epidemics. Uncovering the general principles of viral coevolution with human hosts is important for pathogen surveillance and vaccine design. Protein regions are an appropriate model for the interactions between two macromolecules, but the currently used epitope definition for the major antigen of influenza viruses, namely hemagglutinin, is very broad. Here, we combined genetic, evolutionary, antigenic, and structural information to determine the most relevant regions of the hemagglutinin of human influenza A/H3N2 viruses for interaction with human immunoglobulins. We estimated the antigenic weights of amino acid changes at individual sites from hemagglutination inhibition data using antigenic tree inference followed by spatial clustering of antigenicity-altering protein sites on the protein structure. This approach determined six relevant areas (patches) for antigenic variation that had a key role in the past antigenic evolution of the viruses. Previous transitions between successive predominating antigenic types of H3N2 viruses always included amino acid changes in either the first or second antigenic patch. Interestingly, there was only partial overlap between the antigenic patches and the patches under strong positive selection. Therefore, besides alterations of antigenicity, other interactions with the host may shape the evolution of human influenza A/H3N2 viruses.
Project description:Influenza A virus (IAV) infects a remarkably wide variety of avian and mammalian hosts. Evolution finely hones IAV genes to optimally infect and be transmitted in a particular host species. Sporadically, IAV manages to jump between species, introducing novel antigenic strains into the new host population that wreak havoc until herd immunity develops. IAV adaptation to new hosts typically involves reassortment of IAV gene segments from coinfecting virus strains adapted to different hosts in conjunction with multiple adaptive mutations in the various IAV genes. To better understand host adaptation between mammalian species in real time, we passaged mouse-adapted A/PR8/34 (PR8) in guinea pigs. Guinea pigs, unlike mice, support spontaneous and robust IAV transmission. For some IAV strains, including PR8, adaptation is required for a virus to attain transmissibility, providing an opportunity to understand the evolution of transmissibility in guinea pigs. Multiple guinea pig-adapted PR8 mutants generated by serial nasal wash passaging in independent lines replicated more efficiently and were transmitted by cocaging. All transmissible variants possessed one of two nonsynonymous mutations in M1, either alone or in combination with mutations in PB2, HA, NP, or NA. Rapid reassortment between independently selected variants combined beneficial mutations in NP and M1 to form the fittest virus capable of being transmitted. These findings provide further insight into genetic determinants in NP and M1 involved in PR8 IAV adaptation to be transmitted in a new host and clearly show the benefit of a segmented genome in rapidly generating optimal combinations of mutations in IAV evolution.
Project description:Given the frequent mutation of antigenic features, the constancy of genetic and antigenic diversity of influenza within a subtype is surprising. While the emergence of new strains and antigenic features is commonly attributed to selection by the human immune system, the mechanism that ensures the extinction of older strains remains controversial. To replicate this dynamics of replacement current models utilize mechanisms such as short-lived strain-transcending immunity, a direct competition for hosts, stochastic extinction or constrained antigenic evolution. Building on the idea of short-lived immunity we introduce a minimal model that exhibits the aforementioned dynamics of replacement. Our model relies only on competition due to an antigen specific immune-response in an unconstrained antigenic space. Furthermore the model explains the size of typical influenza epidemics as well as the tendency that new epidemics are associated with mutations of old antigens.