Elevated HIV Viral Load is Associated with Higher Recombination Rate In Vivo.

HIV's exceptionally high recombination rate drives its intrahost diversification, enabling immune escape and multidrug resistance within people living with HIV. While we know that HIV's recombination rate varies by genomic position, we have little understanding of how recombination varies throughout infection or between individuals as a function of the rate of cellular coinfection. We hypothesize that denser intrahost populations may have higher rates of coinfection and therefore recombination. To test this hypothesis, we develop a new approach (recombination analysis via time series linkage decay or RATS-LD) to quantify recombination using autocorrelation of linkage between mutations across time points. We validate RATS-LD on simulated data under short read sequencing conditions and then apply it to longitudinal, high-throughput intrahost viral sequencing data, stratifying populations by viral load (a proxy for density). Among sampled viral populations with the lowest viral loads (<26,800 copies/mL), we estimate a recombination rate of 1.5×10-5 events/bp/generation (95% CI: 7×10-6 to 2.9×10-5), similar to existing estimates. However, among samples with the highest viral loads (>82,000 copies/mL), our median estimate is approximately 6 times higher. In addition to co-varying across individuals, we also find that recombination rate and viral load are associated within single individuals across different time points. Our findings suggest that rather than acting as a constant, uniform force, recombination can vary dynamically and drastically across intrahost viral populations and within them over time. More broadly, we hypothesize that this phenomenon may affect other facultatively asexual populations where spatial co-localization varies.

Brain tropism acquisition: The spatial dynamics and evolution of a measles virus collective infectious unit that drove lethal subacute sclerosing panencephalitis.

It is increasingly appreciated that pathogens can spread as infectious units constituted by multiple, genetically diverse genomes, also called collective infectious units or genome collectives. However, genetic characterization of the spatial dynamics of collective infectious units in animal hosts is demanding, and it is rarely feasible in humans. Measles virus (MeV), whose spread in lymphatic tissues and airway epithelia relies on collective infectious units, can, in rare cases, cause subacute sclerosing panencephalitis (SSPE), a lethal human brain disease. In different SSPE cases, MeV acquisition of brain tropism has been attributed to mutations affecting either the fusion or the matrix protein, or both, but the overarching mechanism driving brain adaptation is not understood. Here we analyzed MeV RNA from several spatially distinct brain regions of an individual who succumbed to SSPE. Surprisingly, we identified two major MeV genome subpopulations present at variable frequencies in all 15 brain specimens examined. Both genome types accumulated mutations like those shown to favor receptor-independent cell-cell spread in other SSPE cases. Most infected cells carried both genome types, suggesting the possibility of genetic complementation. We cannot definitively chart the history of the spread of this virus in the brain, but several observations suggest that mutant genomes generated in the frontal cortex moved outwards as a collective and diversified. During diversification, mutations affecting the cytoplasmic tails of both viral envelope proteins emerged and fluctuated in frequency across genetic backgrounds, suggesting convergent and potentially frequency-dependent evolution for modulation of fusogenicity. We propose that a collective infectious unit drove MeV pathogenesis in this brain. Re-examination of published data suggests that similar processes may have occurred in other SSPE cases. Our studies provide a primer for analyses of the evolution of collective infectious units of other pathogens that cause lethal disease in humans.

The clarifying role of time series data in the population genetics of HIV.

HIV can evolve remarkably quickly in response to antiretroviral therapies and the immune system. This evolution stymies treatment effectiveness and prevents the development of an HIV vaccine. Consequently, there has been a great interest in using population genetics to disentangle the forces that govern the HIV adaptive landscape (selection, drift, mutation, and recombination). Traditional population genetics approaches look at the current state of genetic variation and infer the processes that can generate it. However, because HIV evolves rapidly, we can also sample populations repeatedly over time and watch evolution in action. In this paper, we demonstrate how time series data can bound evolutionary parameters in a way that complements and informs traditional population genetic approaches. Specifically, we focus on our recent paper (Feder et al., 2016, eLife), in which we show that, as improved HIV drugs have led to fewer patients failing therapy due to resistance evolution, less genetic diversity has been maintained following the fixation of drug resistance mutations. Because soft sweeps of multiple drug resistance mutations spreading simultaneously have been previously documented in response to the less effective HIV therapies used early in the epidemic, we interpret the maintenance of post-sweep diversity in response to poor therapies as further evidence of soft sweeps and therefore a high population mutation rate (θ) in these intra-patient HIV populations. Because improved drugs resulted in rarer resistance evolution accompanied by lower post-sweep diversity, we suggest that both observations can be explained by decreased population mutation rates and a resultant transition to hard selective sweeps. A recent paper (Harris et al., 2018, PLOS Genetics) proposed an alternative interpretation: Diversity maintenance following drug resistance evolution in response to poor therapies may have been driven by recombination during slow, hard selective sweeps of single mutations. Then, if better drugs have led to faster hard selective sweeps of resistance, recombination will have less time to rescue diversity during the sweep, recapitulating the decrease in post-sweep diversity as drugs have improved. In this paper, we use time series data to show that drug resistance evolution during ineffective treatment is very fast, providing new evidence that soft sweeps drove early HIV treatment failure.

Correction: Within-patient mutation frequencies reveal fitness costs of CpG dinucleotides and drastic amino acid changes in HIV.

[This corrects the article DOI: 10.1371/journal.pgen.1007420.].

Within-patient mutation frequencies reveal fitness costs of CpG dinucleotides and drastic amino acid changes in HIV.

HIV has a high mutation rate, which contributes to its ability to evolve quickly. However, we know little about the fitness costs of individual HIV mutations in vivo, their distribution and the different factors shaping the viral fitness landscape. We calculated the mean frequency of transition mutations at 870 sites of the pol gene in 160 patients, allowing us to determine the cost of these mutations. As expected, we found high costs for non-synonymous and nonsense mutations as compared to synonymous mutations. In addition, we found that non-synonymous mutations that lead to drastic amino acid changes are twice as costly as those that do not and mutations that create new CpG dinucleotides are also twice as costly as those that do not. We also found that G→A and C→T mutations are more costly than A→G mutations. We anticipate that our new in vivo frequency-based approach will provide insights into the fitness landscape and evolvability of not only HIV, but a variety of microbes.

A spatio-temporal assessment of simian/human immunodeficiency virus (SHIV) evolution reveals a highly dynamic process within the host.

The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIVmne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1.

The population genetics of drug resistance evolution in natural populations of viral, bacterial and eukaryotic pathogens.

Drug resistance is a costly consequence of pathogen evolution and a major concern in public health. In this review, we show how population genetics can be used to study the evolution of drug resistance and also how drug resistance evolution is informative as an evolutionary model system. We highlight five examples from diverse organisms with particular focus on: (i) identifying drug resistance loci in the malaria parasite Plasmodium falciparum using the genomic signatures of selective sweeps, (ii) determining the role of epistasis in drug resistance evolution in influenza, (iii) quantifying the role of standing genetic variation in the evolution of drug resistance in HIV, (iv) using drug resistance mutations to study clonal interference dynamics in tuberculosis and (v) analysing the population structure of the core and accessory genome of Staphylococcus aureus to understand the spread of methicillin resistance. Throughout this review, we discuss the uses of sequence data and population genetic theory in studying the evolution of drug resistance.

Natural selection affects multiple aspects of genetic variation at putatively neutral sites across the human genome.

A major question in evolutionary biology is how natural selection has shaped patterns of genetic variation across the human genome. Previous work has documented a reduction in genetic diversity in regions of the genome with low recombination rates. However, it is unclear whether other summaries of genetic variation, like allele frequencies, are also correlated with recombination rate and whether these correlations can be explained solely by negative selection against deleterious mutations or whether positive selection acting on favorable alleles is also required. Here we attempt to address these questions by analyzing three different genome-wide resequencing datasets from European individuals. We document several significant correlations between different genomic features. In particular, we find that average minor allele frequency and diversity are reduced in regions of low recombination and that human diversity, human-chimp divergence, and average minor allele frequency are reduced near genes. Population genetic simulations show that either positive natural selection acting on favorable mutations or negative natural selection acting against deleterious mutations can explain these correlations. However, models with strong positive selection on nonsynonymous mutations and little negative selection predict a stronger negative correlation between neutral diversity and nonsynonymous divergence than observed in the actual data, supporting the importance of negative, rather than positive, selection throughout the genome. Further, we show that the widespread presence of weakly deleterious alleles, rather than a small number of strongly positively selected mutations, is responsible for the correlation between neutral genetic diversity and recombination rate. This work suggests that natural selection has affected multiple aspects of linked neutral variation throughout the human genome and that positive selection is not required to explain these observations.

Elevated HIV viral load is associated with higher recombination rate in vivo.

HIV's exceptionally high recombination rate drives its intra-host diversification, enabling immune escape and multi-drug resistance within people living with HIV. While we know that HIV's recombination rate varies by genomic position, we have little understanding of how recombination varies throughout infection or between individuals as a function of the rate of cellular coinfection. We hypothesize that denser intra-host populations may have higher rates of coinfection and therefore recombination. To test this hypothesis, we develop a new approach (Recombination Analysis via Time Series Linkage Decay, or RATS-LD) to quantify recombination using autocorrelation of linkage between mutations across time points. We validate RATS-LD on simulated data under short read sequencing conditions and then apply it to longitudinal, high-throughput intra-host viral sequencing data, stratifying populations by viral load (a proxy for density). Among sampled viral populations with the lowest viral loads (< 26,800 copies/mL), we estimate a recombination rate of 1.5 × 10-5 events/bp/generation (95% CI: 7 × 10-6 - 2.9 × 10-5), similar to existing estimates. However, among samples with the highest viral loads (> 82,000 copies/mL), our median estimate is approximately 6 times higher. In addition to co-varying across individuals, we also find that recombination rate and viral load are associated within single individuals across different time points. Our findings suggest that rather than acting as a constant, uniform force, recombination can vary dynamically and drastically across intra-host viral populations and within them over time. More broadly, we hypothesize that this phenomenon may affect other facultatively asexual populations where spatial co-localization varies.

State-dependent evolutionary models reveal modes of solid tumour growth.

Spatial properties of tumour growth have profound implications for cancer progression, therapeutic resistance and metastasis. Yet, how spatial position governs tumour cell division remains difficult to evaluate in clinical tumours. Here, we demonstrate that faster division on the tumour periphery leaves characteristic genetic patterns, which become evident when a phylogenetic tree is reconstructed from spatially sampled cells. Namely, rapidly dividing peripheral lineages branch more extensively and acquire more mutations than slower-dividing centre lineages. We develop a Bayesian state-dependent evolutionary phylodynamic model (SDevo) that quantifies these patterns to infer the differential division rates between peripheral and central cells. We demonstrate that this approach accurately infers spatially varying birth rates of simulated tumours across a range of growth conditions and sampling strategies. We then show that SDevo outperforms state-of-the-art, non-cancer multi-state phylodynamic methods that ignore differential sequence evolution. Finally, we apply SDevo to single-time-point, multi-region sequencing data from clinical hepatocellular carcinomas and find evidence of a three- to six-times-higher division rate on the tumour edge. With the increasing availability of high-resolution, multi-region sequencing, we anticipate that SDevo will be useful in interrogating spatial growth restrictions and could be extended to model non-spatial factors that influence tumour progression.

Understanding patterns of HIV multi-drug resistance through models of temporal and spatial drug heterogeneity.

Triple-drug therapies have transformed HIV from a fatal condition to a chronic one. These therapies should prevent HIV drug resistance evolution, because one or more drugs suppress any partially resistant viruses. In practice, such therapies drastically reduced, but did not eliminate, resistance evolution. In this article, we reanalyze published data from an evolutionary perspective and demonstrate several intriguing patterns about HIV resistance evolution - resistance evolves (1) even after years on successful therapy, (2) sequentially, often via one mutation at a time and (3) in a partially predictable order. We describe how these observations might emerge under two models of HIV drugs varying in space or time. Despite decades of work in this area, much opportunity remains to create models with realistic parameters for three drugs, and to match model outcomes to resistance rates and genetic patterns from individuals on triple-drug therapy. Further, lessons from HIV may inform other systems.

Evolutionary Dynamics in Structured Populations Under Strong Population Genetic Forces.

In the long-term neutral equilibrium, high rates of migration between subpopulations result in little population differentiation. However, in the short-term, even very abundant migration may not be enough for subpopulations to equilibrate immediately. In this study, we investigate dynamical patterns of short-term population differentiation in adapting populations via stochastic and analytical modeling through time. We characterize a regime in which selection and migration interact to create non-monotonic patterns of population differentiation over time when migration is weaker than selection, but stronger than drift. We demonstrate how these patterns can be leveraged to estimate high migration rates using approximate Bayesian computation. We apply this approach to estimate fast migration in a rapidly adapting intra-host Simian-HIV population sampled from different anatomical locations. We find differences in estimated migration rates between different compartments, even though all are above [Formula: see text] = 1. This work demonstrates how studying demographic processes on the timescale of selective sweeps illuminates processes too fast to leave signatures on neutral timescales.

The Relationship Between Haplotype-Based FST and Haplotype Length.

The population-genetic statistic [Formula: see text] is used widely to describe allele frequency distributions in subdivided populations. The increasing availability of DNA sequence data has recently enabled computations of [Formula: see text] from sequence-based "haplotype loci." At the same time, theoretical work has revealed that [Formula: see text] has a strong dependence on the underlying genetic diversity of a locus from which it is computed, with high diversity constraining values of [Formula: see text] to be low. In the case of haplotype loci, for which two haplotypes that are distinct over a specified length along a chromosome are treated as distinct alleles, genetic diversity is influenced by haplotype length: longer haplotype loci have the potential for greater genetic diversity. Here, we study the dependence of [Formula: see text] on haplotype length. Using a model in which a haplotype locus is sequentially incremented by one biallelic locus at a time, we show that increasing the length of the haplotype locus can either increase or decrease the value of [Formula: see text], and usually decreases it. We compute [Formula: see text] on haplotype loci in human populations, finding a close correspondence between the observed values and our theoretical predictions. We conclude that effects of haplotype length are valuable to consider when interpreting [Formula: see text] calculated on haplotypic data.

More effective drugs lead to harder selective sweeps in the evolution of drug resistance in HIV-1.

In the early days of HIV treatment, drug resistance occurred rapidly and predictably in all patients, but under modern treatments, resistance arises slowly, if at all. The probability of resistance should be controlled by the rate of generation of resistance mutations. If many adaptive mutations arise simultaneously, then adaptation proceeds by soft selective sweeps in which multiple adaptive mutations spread concomitantly, but if adaptive mutations occur rarely in the population, then a single adaptive mutation should spread alone in a hard selective sweep. Here, we use 6717 HIV-1 consensus sequences from patients treated with first-line therapies between 1989 and 2013 to confirm that the transition from fast to slow evolution of drug resistance was indeed accompanied with the expected transition from soft to hard selective sweeps. This suggests more generally that evolution proceeds via hard sweeps if resistance is unlikely and via soft sweeps if it is likely.

Identifying signatures of selection in genetic time series.

Both genetic drift and natural selection cause the frequencies of alleles in a population to vary over time. Discriminating between these two evolutionary forces, based on a time series of samples from a population, remains an outstanding problem with increasing relevance to modern data sets. Even in the idealized situation when the sampled locus is independent of all other loci, this problem is difficult to solve, especially when the size of the population from which the samples are drawn is unknown. A standard χ(2)-based likelihood-ratio test was previously proposed to address this problem. Here we show that the χ(2)-test of selection substantially underestimates the probability of type I error, leading to more false positives than indicated by its P-value, especially at stringent P-values. We introduce two methods to correct this bias. The empirical likelihood-ratio test (ELRT) rejects neutrality when the likelihood-ratio statistic falls in the tail of the empirical distribution obtained under the most likely neutral population size. The frequency increment test (FIT) rejects neutrality if the distribution of normalized allele-frequency increments exhibits a mean that deviates significantly from zero. We characterize the statistical power of these two tests for selection, and we apply them to three experimental data sets. We demonstrate that both ELRT and FIT have power to detect selection in practical parameter regimes, such as those encountered in microbial evolution experiments. Our analysis applies to a single diallelic locus, assumed independent of all other loci, which is most relevant to full-genome selection scans in sexual organisms, and also to evolution experiments in asexual organisms as long as clonal interference is weak. Different techniques will be required to detect selection in time series of cosegregating linked loci.

LDx: estimation of linkage disequilibrium from high-throughput pooled resequencing data.

High-throughput pooled resequencing offers significant potential for whole genome population sequencing. However, its main drawback is the loss of haplotype information. In order to regain some of this information, we present LDx, a computational tool for estimating linkage disequilibrium (LD) from pooled resequencing data. LDx uses an approximate maximum likelihood approach to estimate LD (r(2)) between pairs of SNPs that can be observed within and among single reads. LDx also reports r(2) estimates derived solely from observed genotype counts. We demonstrate that the LDx estimates are highly correlated with r(2) estimated from individually resequenced strains. We discuss the performance of LDx using more stringent quality conditions and infer via simulation the degree to which performance can improve based on read depth. Finally we demonstrate two possible uses of LDx with real and simulated pooled resequencing data. First, we use LDx to infer genomewide patterns of decay of LD with physical distance in D. melanogaster population resequencing data. Second, we demonstrate that r(2) estimates from LDx are capable of distinguishing alternative demographic models representing plausible demographic histories of D. melanogaster.