Dominant role of host selective pressure in driving hepatitis C virus evolution in perinatal infection

The dynamics of the genetic diversification of hepatitis C virus (HCV) populations was addressed in perinatal infection. Clonal sequences of hypervariable region 1 of the putative E2 envelope protein of HCV were obtained from four HCV-infected newborns (sequential samples spanning a period of 6 to 13 months after birth) and from their mothers (all samples collected at delivery). The data show that the variants detected between birth and the third month of life in samples from the four newborns were present in the HCV populations of their mothers at delivery. In the newborns, a unique viral variant (or a small group of closely related variants) remained stable for weeks despite active viral replication. Diversification of the intrahost HCV population was observed 6 to 13 months after birth and was substantially higher in two of the four subjects, as documented by the intersample genetic distance (GD) (P = 0.007). Importantly, a significant correlation between increasing GD and high values for the intersample K(a)/K(s) ratio (the ratio between anoffymous and synonymous substitutions; an index of the action of selective forces) was observed, as documented by the increase of both parameters over time (P = 0.01). These data argue for a dominant role of positive selection for amino acid changes in driving the pattern of genetic diversification of HCV populations, indicate that the intrahost evolution of HCV populations is compatible with a Darwinian model system, and may have implications in the designing of future antiviral strategies.     

Daily sampling of an HIV-1 patient with slowly progressing disease displays persistence of multiple env subpopulations consistent with neutrality

The molecular evolution of HIV-1 is characterized by frequent substitutions, indels and recombination events. In addition, a HIV-1 population may adapt through frequency changes of its variants. To reveal such population dynamics we analyzed HIV-1 subpopulation frequencies in an untreated patient with stable, low plasma HIV-1 RNA levels and close to normal CD4+ T-cell levels. The patient was intensively sampled during a 32-day period as well as approximately 1.5 years before and after this period (days -664, 1, 2, 3, 11, 18, 25, 32 and 522). 77 sequences of HIV-1 env (approximately 3100 nucleotides) were obtained from plasma by limiting dilution with 7-11 sequences per time point, except day -664. Phylogenetic analysis using maximum likelihood methods showed that the sequences clustered in six distinct subpopulations. We devised a method that took into account the relatively coarse sampling of the population. Data from days 1 through 32 were consistent with constant within-patient subpopulation frequencies. However, over longer time periods, i.e. between days 1...32 and 522, there were significant changes in subpopulation frequencies, which were consistent with evolutionarily neutral fluctuations. We found no clear signal of natural selection within the subpopulations over the study period, but positive selection was evident on the long branches that connected the subpopulations, which corresponds to >3 years as the subpopulations already were established when we started the study. Thus, selective forces may have been involved when the subpopulations were established. Genetic drift within subpopulations caused by de novo substitutions could be resolved after approximately one month. Overall, we conclude that subpopulation frequencies within this patient changed significantly over a time period of 1.5 years, but that this does not imply directional or balancing selection. We show that the short-term evolution we study here is likely representative for many patients of slow and normal disease progression.     

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified viral load as a critical factor in variant identification. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.     

Analysis of a stochastic predator–prey model with applications to intrahost HIV genetic diversity

During an infection, HIV experiences strong selection by immune system T cells. Recent experimental work has shown that MHC escape mutations form an important pathway for HIV to avoid such selection. In this paper, we study a model of MHC escape mutation. The model is a predator–prey model with two prey, composed of two HIV variants, and one predator, the immune system CD8 cells. We assume that one HIV variant is visible to CD8 cells and one is not. The model takes the form of a system of stochastic differential equations. Motivated by well-known results concerning the short life-cycle of HIV intrahost, we assume that HIV population dynamics occur on a faster time scale then CD8 population dynamics. This separation of time scales allows us to analyze our model using an asymptotic approach. Using this model we study the impact of an MHC escape mutation on the population dynamics and genetic evolution of the intrahost HIV population. From the perspective of population dynamics, we show that the competition between the visible and invisible HIV variants can reach steady states in which either a single variant exists or in which coexistence occurs depending on the parameter regime. We show that in some parameter regimes the end state of the system is stochastic. From a genetics perspective, we study the impact of the population dynamics on the lineages of an HIV sample taken after an escape mutation occurs. We show that the lineages go through severe bottlenecks and that in certain parameter regimes the lineage distribution can be characterized by a Kingman coalescent. Our results depend on methods from diffusion theory and coalescent theory.     

Subpopulations of equine infectious anemia virus Rev coexist in vivo and differ in phenotype

Lentiviruses exist in vivo as a population of related, nonidentical genotypes, commonly referred to as quasispecies. The quasispecies structure is characteristic of complex adaptive systems and contributes to the high rate of evolution in lentiviruses that confounds efforts to develop effective vaccines and antiviral therapies. Here, we describe analyses of genetic data from longitudinal studies of genetic variation in a lentivirus regulatory protein, Rev, over the course of disease in ponies experimentally infected with equine infectious anemia virus. As observed with other lentivirus data, the Rev variants exhibited a quasispecies character. Phylogenetic and partition analyses suggested that the Rev quasispecies comprised two distinct subpopulations that coexisted during infection. One subpopulation appeared to accumulate changes in a linear, time-dependent manner, while the other evolved radially from a common variant. Over time, the two subpopulations cycled in predominance coincident with changes in the disease state, suggesting that the two groups differed in selective advantage. Transient expression assays indicated the two populations differed significantly in Rev nuclear export activity. Chimeric proviral clones containing Rev genotypes representative of each population differed in rate and overall level of virus replication in vitro. The coexistence of genetically distinct viral subpopulations that differ in phenotype provides great adaptability to environmental changes within the infected host. A quasispecies model with multiple subpopulations may provide additional insight into the nature of lentivirus reservoirs and the evolution of antigenic and drug-resistant variants.     

Dynamic Correlation between Intrahost HIV-1 Quasispecies Evolution and Disease Progression

Quantifying the dynamics of intrahost HIV-1 sequence evolution is one means of uncovering information about the interaction between HIV-1 and the host immune system. In the chronic phase of infection, common dynamics of sequence divergence and diversity have been reported. We developed an HIV-1 sequence evolution model that simulated the effects of mutation and fitness of sequence variants. The amount of evolution was described by the distance from the founder strain, and fitness was described by the number of offspring a parent sequence produces. Analysis of the model suggested that the previously observed saturation of divergence and decrease of diversity in later stages of infection can be explained by a decrease in the proportion of offspring that are mutants as the distance from the founder strain increases rather than due to an increase of viral fitness. The prediction of the model was examined by performing phylogenetic analysis to estimate the change in the rate of evolution during infection. In agreement with our modeling, in 13 out of 15 patients (followed for 3–12 years) we found that the rate of intrahost HIV-1 evolution was not constant but rather slowed down at a rate correlated with the rate of CD4+ T-cell decline. The correlation between the dynamics of the evolutionary rate and the rate of CD4+ T-cell decline, coupled with our HIV-1 sequence evolution model, explains previously conflicting observations of the relationships between the rate of HIV-1 quasispecies evolution and disease progression.     

Genome-wide patterns of intrahuman dengue virus diversity reveal associations with viral phylogenetic clade and interhost diversity

Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured ～75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.     

Coevolution of the hepatitis C virus polyprotein sites in patients on combined pegylated interferon and ribavirin therapy

Genotype-specific sensitivity of the hepatitis C virus (HCV) to interferon-ribavirin (IFN-RBV) combination therapy and reduced HCV response to IFN-RBV as infection progresses from acute to chronic infection suggest that HCV genetic factors and intrahost HCV evolution play important roles in therapy outcomes. HCV polyprotein sequences (n = 40) from 10 patients with unsustainable response (UR) (breakthrough and relapse) and 10 patients with no response (NR) following therapy were identified through the Virahep-C study. Bayesian networks (BNs) were constructed to relate interrelationships among HCV polymorphic sites to UR/NR outcomes. All models showed an extensive interdependence of HCV sites and strong connections (P ≤ 0.003) to therapy response. Although all HCV proteins contributed to the networks, the topological properties of sites differed among proteins. E2 and NS5A together contributed ～40% of all sites and ～62% of all links to the polyprotein BN. The NS5A BN and E2 BN predicted UR/NR outcomes with 85% and 97.5% accuracy, respectively, in 10-fold cross-validation experiments. The NS5A model constructed using physicochemical properties of only five sites was shown to predict the UR/NR outcomes with 83.3% accuracy for 6 UR and 12 NR cases of the HALT-C study. Thus, HCV adaptation to IFN-RBV is a complex trait encoded in the interrelationships among many sites along the entire HCV polyprotein. E2 and NS5A generate broad epistatic connectivity across the HCV polyprotein and essentially shape intrahost HCV evolution toward the IFN-RBV resistance. Both proteins can be used to accurately predict the outcomes of IFN-RBV therapy.     

Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies.Hepatitis C virus (HCV) infection is a major global health issue leading to persistent viral infection in the majority of those infected and is associated with progressive liver disease, cirrhosis, and hepatocellular carcinoma. Six major genotypes of HCV have been described that have evolved in geographically distinct regions and that share approximately. 80% nucleotide homology with one another. HCV viral genotypes have been further classified into subtypes (25). HCV subtype 3a infection is now the most common subtype in the United Kingdom (11), although it is globally distributed and frequently associated with intravenous drug use.The classification of HCV viral strains by genotype and subtype has proven informative not only in terms of the epidemic and evolutionary history of the virus but also in terms of clinical outcomes. In particular, the response rates to current gold standard therapy (9) and the prevalence of hepatic steatosis (20) are significantly higher for subtype 3a than for genotype 1 infections. The reasons for this are not understood but must relate to viral genetic and phenotypic differences between strains, or to differences in the ability of hosts to exert an effective immune response against particular viral sequences, or to a combination of both factors.To date, detailed assessment of the HCV genome has largely focused on HCV genotype 1. Indeed, only a few full-length HCV subtype 3a viral sequences are currently published and available within the major HCV databases (Los Alamos; http://hcv.lanl.gov/components/hcv-db/combined_search/searchi.html and euHCVdb; http://euhcvdb.ibcp.fr/euHCVdb/) (16).To characterize HCV subtype 3a in detail, we performed whole-genome analysis of a cohort of patients with persistent HCV subtype 3a infection. We subsequently focus on the highly variable regions observed in the envelope protein E2 in both acute and chronic infection, since it was apparent that these regions were not restricted to the well-documented hypervariable region 1 (HVR1) that is found at the 5′ end of E2 in all HCV genotypes.Viral genomic variability can be assessed at a number of different levels; first, intergenotypic variability may arise in genomic regions that are conserved within the same subtype but are distinct between subtypes. Second, there is intragenotypic variability, which may be defined as regions of viral variability within the same genotype or subtype. Finally, intrahost variability is where viral genomic variability occurs within the same viral subtype and also the same host when individual clonal sequences are assessed. Although intergenotypic variability may simply be a feature of the existence of geographically distinct HCV subtypes, intragenotypic and intrahost variability may reflect viral regions subject to specific selection pressures, with important functional implications.We observed two distinct regions of intrahost and intragenotypic hypervariability within genotype 3a envelope 2 (E2)—in addition to the previously described HVR1—that we have named HVR495 and HVR575. We show that these regions are subject to positive selection pressure, sometimes very early in acute infection. Although HVR575 has been previously recognized as a site of intergenotypic variation (18), the identification of this region as a hypervariable site within genotype 3a and as a site under early selection pressure leading to variability within the same host has not been previously described.     

Exceptional Heterogeneity in Viral Evolutionary Dynamics Characterises Chronic Hepatitis C Virus Infection

The treatment of HCV infection has seen significant progress, particularly since the approval of new direct-acting antiviral drugs. However these clinical achievements have been made despite an incomplete understanding of HCV replication and within-host evolution, especially compared with HIV-1. Here, we undertake a comprehensive analysis of HCV within-host evolution during chronic infection by investigating over 4000 viral sequences sampled longitudinally from 15 HCV-infected patients. We compare our HCV results to those from a well-studied HIV-1 cohort, revealing key differences in the evolutionary behaviour of these two chronic-infecting pathogens. Notably, we find an exceptional level of heterogeneity in the molecular evolution of HCV, both within and among infected individuals. Furthermore, these patterns are associated with the long-term maintenance of viral lineages within patients, which fluctuate in relative frequency in peripheral blood. Together, our findings demonstrate that HCV replication behavior is complex and likely comprises multiple viral subpopulations with distinct evolutionary dynamics. The presence of a structured viral population can explain apparent paradoxes in chronic HCV infection, such as rapid fluctuations in viral diversity and the reappearance of viral strains years after their initial detection.     

Genetic and epigenetic divergence between disturbed and undisturbed subpopulations of a Mediterranean shrub: a 20‐year field experiment

Little is known on the potential of ecological disturbance to cause genetic and epigenetic changes in plant populations. We take advantage of a long‐term field experiment initiated in 1986 to study the demography of the shrub Lavandula latifolia, and compare genetic and epigenetic characteristics of plants in two adjacent subplots, one experimentally disturbed and one left undisturbed, 20 years after disturbance. Experimental setup was comparable to an unreplicated ‘Before‐After‐Control‐Impact’ (BACI) design where a single pair of perturbed and control areas were compared. When sampled in 2005, plants in the two subplots had roughly similar ages, but they had established in contrasting environments: dense conspecific population (‘Undisturbed’ subpopulation) versus open area with all conspecifics removed (‘Disturbed’ subpopulation). Plants were characterized genetically and epigenetically using amplified fragment length polymorphism (AFLP) and two classes of methylation‐sensitive AFLP (MSAP) markers. Subpopulations were similar in genetic diversity but differed in epigenetic diversity and multilocus genetic and epigenetic characteristics. Epigenetic divergence between subpopulations was statistically unrelated to genetic divergence. Bayesian clustering revealed an abrupt linear boundary between subpopulations closely coincident with the arbitrary demarcation line between subplots drawn 20 years back, which supports that genetic and epigenetic divergence between subpopulations was caused by artificial disturbance. There was significant fine‐scale spatial structuring of MSAP markers in both subpopulations, which in the Undisturbed one was indistinguishable from that of AFLP markers. Genetic differences between subpopulations could be explained by divergent selection alone, while the concerted action of divergent selection and disturbance‐driven appearance of new methylation variants in the Disturbed subpopulation is proposed to explain epigenetic differences. This study provides the first empirical evidence to date suggesting that relatively mild disturbances could leave genetic and epigenetic signatures on the next adult generation of long‐lived plants.     

Impact of CCR5delta32 host genetic background and disease progression on HIV-1 intrahost evolutionary processes: efficient hypothesis testing through hierarchical phylogenetic models

The interplay between C-C chemokine receptor type 5 (CCR5) host genetic background, disease progression, and intrahost HIV-1 evolutionary dynamics remains unclear because differences in viral evolution between hosts limit the ability to draw conclusions across hosts stratified into clinically relevant populations. Similar inference problems are proliferating across many measurably evolving pathogens for which intrahost sequence samples are readily available. To this end, we propose novel hierarchical phylogenetic models (HPMs) that incorporate fixed effects to test for differences in dynamics across host populations in a formal statistical framework employing stochastic search variable selection and model averaging. To clarify the role of CCR5 host genetic background and disease progression on viral evolutionary patterns, we obtain gp120 envelope sequences from clonal HIV-1 variants isolated at multiple time points in the course of infection from populations of HIV-1-infected individuals who only harbored CCR5-using HIV-1 variants at all time points. Presence or absence of a CCR5 wt/Δ32 genotype and progressive or long-term nonprogressive course of infection stratify the clinical populations in a two-way design. As compared with the standard approach of analyzing sequences from each patient independently, the HPM provides more efficient estimation of evolutionary parameters such as nucleotide substitution rates and d(N)/d(S) rate ratios, as shown by significant shrinkage of the estimator variance. The fixed effects also correct for nonindependence of data between populations and results in even further shrinkage of individual patient estimates. Model selection suggests an association between nucleotide substitution rate and disease progression, but a role for CCR5 genotype remains elusive. Given the absence of clear d(N)/d(S) differences between patient groups, delayed onset of AIDS symptoms appears to be solely associated with lower viral replication rates rather than with differences in selection on amino acid fixation.     

Transmission of clonal hepatitis C virus genomes reveals the dominant but transitory role of CD8⁺ T cells in early viral evolution

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.     

Evolution of the Hepatitis C Virus Second Envelope Protein Hypervariable Region in Chronically Infected Patients Receiving Alpha Interferon Therapy

Sustained hepatitis C virus (HCV) RNA clearance is achieved in 8 to 12% of patients with chronic HCV infection treated with alpha interferon (IFN-alpha) at the approved dose of 3 MU three times a week for 6 months and in about 25% of those receiving this treatment for 12 months. We used single-strand conformation polymorphism analysis combined with cloning and sequencing strategies to characterize the genetic evolution of HCV second envelope gene hypervariable region 1 (HVR1) quasispecies during and after IFN therapy in patients who failed to clear HCV RNA. Sustained HCV RNA clearance was achieved in 6% of patients. Profound changes in HVR1 quasispecies major variants were estimated to occur in 70% of the patients during and after therapy. These changes were evolutionary and were characterized by shifts in the virus population, related to selection and subsequent diversification of minor pretreatment variants. The quasispecies changes appeared to be induced by changes in the host environment likely resulting from the IFN-induced enhancement and post-IFN attenuation of neutralizing and possibly cytotoxic responses against HVR1. The remaining patients had no apparent changes in HVR1 quasispecies major variants, suggesting selection of major pretreatment variants, but some changes were observed in other genomic regions. We conclude that IFN-alpha administration and withdrawal profoundly alters the nature of circulating HCV quasispecies, owing to profound changes in virus-host interactions, in patients in whom sustained HCV RNA clearance fails to occur. These changes are associated with profound alterations of the natural outcome of HCV-related liver disease, raising the hypothesis of a causal relationship.     

Sequential bottlenecks drive viral evolution in early acute hepatitis C virus infection

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection.     

Opportunity to assimilate and pressure to discriminate can generate cultural divergence in the laboratory

Formal models of cultural evolution have illustrated circumstances under which behavioral traits that have no inherent advantage over others can undergo positive selection pressure. One situation in which this may occur is when the behavior functions as a social marker, and there is pressure to identify oneself as a member of a particular group. Our aim in the current study was to determine whether participants organized into subpopulations could effectively exploit variation in a completely novel behavior to advertise themselves as belonging to a particular subpopulation, such that discrimination between in-group and out-group members was possible and subpopulations exhibited increasing distinctiveness. Eighty participants took part, organized into four subpopulations, each composed of five four-member generations. They each completed a tower-building task, used in previous experimental studies of cultural evolution. An incentive payment structure was imposed with the aim of motivating participants to advertise themselves as belonging to a particular subpopulation and to distinguish in-group members from members of other subpopulations. The first generation were exposed to photographs of randomly assigned “seed” towers, and later generations were exposed to photographs of the towers built by the members of the previous generation of their own subpopulation. Participants were able to discriminate towers built by in-group members of the same generation, from towers built by out-group members. Over generations, tower designs evolved such that they were increasingly identifiable as belonging to a particular subpopulation. Arbitrary traits which had no prior advantage became associated with group membership, providing empirical support for theoretical models.     

Sequence Variation in the Gene Encoding the Nonstructural 3 Protein of Hepatitis C Virus: Evidence for Immune Selection

To determine whether the persistent nature of hepatitis C infection is related to the emergence of antigenic variants driven by immune selection, we examined the sequence heterogeneity in a portion of the hepatitis C virus (HCV) nonstructural 3 (NS3) gene of a patient infected over the course of more than 2 years. By PCR amplification, cloning, and sequencing, we observed several variable and conserved regions in the NS3 segment of the HCV genome. All variable regions had higher ratios of nonsynonymous/synonymous mutations and encompassed immunodominant epitopes, and their locations were not essential to maintain the known function of HCV RNA helicase. In contrast, the regions that are critical for HCV RNA helicase activity were found to be conserved with lower heterogeneity or lower ratios of nonsynonymous/synonymous mutations, and none except one of these regions was encoded within immunodominant epitopes. Our results are consistent with immune selection of viral variants at the epitope and molecular levels that may enable HCV to evade host defenses over time. Plotting the relatedness of sequence variants revealed a star topology suggesting that a wild-type HCV sequence is maintained, unlike HIV. Received: 2 November 2000 / Accepted: 1 October 2001     

Evolution of Syncytium-Inducing and Non-Syncytium-Inducing Biological Virus Clones in Relation to Replication Kinetics during the Course of Human Immunodeficiency Virus Type 1 Infection

To investigate the temporal relationship between human immunodeficiency virus type 1 (HIV-1) replicative capacity and syncytium-inducing (SI) phenotype, biological and genetic characteristics of longitudinally obtained virus clones from two HIV-1-infected individuals who developed SI variants were studied. In one individual, the emergence of rapidly replicating SI and non-syncytium-inducing (NSI) variants was accompanied by a loss of the slowly replicating NSI variants. In the other subject, NSI variants were always slowly replicating, while the coexisting SI variants showed an increase in the rate of replication. Irrespective their replicative capacity, the NSI variants remained present throughout the infection in both individuals. Phylogenetic analysis of the V3 region showed early branching of the SI variants from the NSI tree. Successful SI conversion seemed a unique event since no SI variants were found among later-stage NSI variants. This was also confirmed by the increasing evolutionary distance between the two subpopulations. At any time point during the course of the infection, the variation within the coexisting SI and NSI populations did not exceed 2%, indicating continuous competition within each viral subpopulation.     

Population-Enrichment Adaptive Design Strategy for an Event-Driven Vaccine Efficacy Trial

A population-enrichment adaptive design allows a prospective use for study population selection. It has the flexibility allowing pre-specified modifications to an ongoing trial to mitigate the potential risk associated with the assumptions made at design stage. In this way, the trial can potentially encompass a broader target patient population, and move forward only with the subpopulations that appear to be benefiting from the treatment. Our work is motivated by a Phase III event-driven vaccine efficacy trial. Two target patient subpopulations were enrolled with the assumption that vaccine efficacy can be demonstrated based on the combined population. It is recognized due to the nature of patients’ underlying conditions, one subpopulation might respond to the treatment better than the other. To maximize the probability of demonstrating vaccine efficacy in at least one patient population while taking advantage of combining two subpopulations in one single trial, an adaptive design strategy with potential population enrichment is developed. Specifically, if the observed vaccine efficacy at interim for one subpopulation is not promising to warrant carrying forward, the population may be enriched with the other subpopulation with better performance. Simulations were conducted to evaluate the operational characteristics from a selection of interim analysis plans. This population-enrichment design provides a more efficient way as compared to the conventional approaches when targeting multiple subpopulations. If executed and planned with caution, this strategy can provide a greater chance of success of the trial and help maintain scientific and regulatory rigors.