Wayne Delport

Datamonkey 2010

Datamonkey is a popular web-based suite of phylogenetic analysis tools for use in evolutionary biology. Since the original release in 2005, we have expanded the analysis options to include recently developed algorithmic methods for recombination detection, evolutionary fingerprinting of genes, codon model selection, co-evolution between sites, identification of sites, which rapidly escape host-immune pressure and HIV-1 subtype assignment. The traditional selection tools have also been augmented to include recent developments in the field. Here, we summarize the analyses options currently available on Datamonkey, and provide guidelines for their use in evolutionary biology. Availability and documentation: http://www.datamonkey.org.

Genome-wide analysis of the structure of the South African Coloured Population in the Western Cape.

Admixed populations present unique opportunities to discover the genetic factors underlying many multifactorial diseases. The geographical position and complex history of South Africa has led to the establishment of the unique admixed population known as the South African Coloured. Not much is known about the genetic make-up of this population, and the historical record is patchy. We genotyped 959 individuals from the Western Cape area, self-identified as belonging to this population, using the Affymetrix 500k genotyping platform. This resulted in nearly 75,000 autosomal SNPs that could be compared with populations represented in the International HapMap Project and the Human Genome Diversity Project. Analysis by means of both the admixture and linkage models in STRUCTURE revealed that the major ancestral components of this population are predominantly Khoesan (32–43%), Bantu-speaking Africans (20–36%), European (21–28%) and a smaller Asian contribution (9–11%), depending on the model used. This is consistent with historical data. While of great historical and genealogical interest, this information is also essential for future admixture mapping of disease genes in this population.

Investigating the Origin and Spread of Hepatitis C Virus Genotype 5a

ABSTRACT Epidemiological and phylogenetic studies of hepatitis C virus (HCV) have identified six major HCV genotypes and have attempted to characterize their origin and spread worldwide. Putative regions of endemic infection have been identified for all HCV genotypes except HCV genotype 5a. Although HCV genotype 5a was previously thought to be largely restricted to the northern part of South Africa, this study reports an unexpected cluster of the genotype in West Flanders Province in Belgium. To investigate the molecular epidemiology of this cluster and of HCV genotype 5a in general, a rigorous phylogenetic analysis of Belgian and South African HCV genotype 5a samples was performed. Remarkably, the Belgian and South African strains form two distinct clusters of similar diversity. We used a Bayesian coalescent method to estimate the rate of virus spread through time for HCV genotype 5a in both regions. Our results indicate that HCV genotype 5a strains have been spreading independently in Belgium and South Africa for more than 100 years, with a rate of spread characteristic of an epidemic genotype. These findings have major implications for tracing the origin of HCV genotype 5a. Here, we speculate about the possible origins of these clusters.

Detection of Minority Resistance during Early HIV-1 Infection: Natural Variation and Spurious Detection rather than Transmission and Evolution of Multiple Viral Variants†

ABSTRACT Reports of a high frequency of the transmission of minority viral populations with drug-resistant mutations (DRM) are inconsistent with evidence that HIV-1 infections usually arise from mono- or oligoclonal transmission. We performed ultradeep sequencing (UDS) of partial HIV-1 gag, pol, and env genes from 32 recently infected individuals. We then evaluated overall and per-site diversity levels, selective pressure, sequence reproducibility, and presence of DRM and accessory mutations (AM). To differentiate biologically meaningful mutations from those caused by methodological errors, we obtained multinomial confidence intervals (CI) for the proportion of DRM at each site and fitted a binomial mixture model to determine background error rates for each sample. We then examined the association between detected minority DRM and the virologic failure of first-line antiretroviral therapy (ART). Similar to other studies, we observed increased detection of DRM at low frequencies (average, 0.56%; 95% CI, 0.43 to 0.69; expected UDS error, 0.21 ± 0.08% mutations/site). For 8 duplicate runs, there was variability in the proportions of minority DRM. There was no indication of increased diversity or selection at DRM sites compared to other sites and no association between minority DRM and AM. There was no correlation between detected minority DRM and clinical failure of first-line ART. It is unlikely that minority viral variants harboring DRM are transmitted and maintained in the recipient host. The majority of low-frequency DRM detected using UDS are likely errors inherent to UDS methodology or a consequence of error-prone HIV-1 replication.

The Origins of Sexually Transmitted HIV Among Men Who Have Sex with Men

Cell-free virus in seminal plasma is the origin of sexually transmitted HIV among men who have sex with men. Starting at the Source It is no mystery that the majority of HIV infections result from sexual transmission of virus, yet the cellular and molecular mechanisms involved are still poorly understood. The principal cell types in human semen are spermatozoa, immature germ cells, and white blood cells. HIV can potentially be carried in any of these or in the seminal fluid. Some men with acute HIV infection may be particularly contagious as a result of abnormally elevated HIV-associated white blood cells or high levels of HIV RNA in semen. Most quantitative studies of HIV in semen have used commercially available HIV RNA assays to measure copy numbers of cell-free virions in seminal plasma; only a few have used HIV DNA polymerase chain reaction assays to assess the prevalence or number of HIV-infected cells in semen. Even with these technological advances, it remains unclear whether transmitted strains originate as RNA virions or as integrated proviral DNA in infected seminal white blood cells. The HIV-1 species in semen often differ genetically from those in the peripheral blood of the same infected person, and the genetic sequences of cell-free HIV in semen differ from those of cell-associated HIV in semen. It therefore should be possible to determine whether the initial transmission event is mediated by a cell-free virion or an HIV-infected cell. Mounting evidence from clinical, animal, and in vitro studies indicates that HIV-1–infected cells may be the critical vectors of transmission. However, the identification of the true source is complicated by the prevalence of cell-associated over cell-free HIV transmission, the risk factors associated with transmission, and the type and location of infected cells. In a search for the source reservoir of sexually transmitted HIV, Butler and colleagues examined the genetic and structural elements of a conserved genetic region of the viral envelope in blood- and semen-derived HIV RNA and DNA from source and recipient partners in six transmission pairs of men who have sex with men. In all transmission pairs, phylogenetic analyses revealed that viral sequences derived from the recipient partner’s blood plasma clustered with the sequences from his source partner’s seminal plasma rather than those from his seminal cells, providing compelling evidence for extracellular seminal HIV RNA as the source of infection. These seminal non–cell-associated RNA populations may represent a unique and tractable target to block transmission. Although it is known that most HIV-1 infections worldwide result from exposure to virus in semen, it has not yet been established whether transmitted strains originate as RNA virions in seminal plasma or as integrated proviral DNA in infected seminal leukocytes. We present phylogenetic evidence that among six transmitting pairs of men who have sex with men, blood plasma virus in the recipient is consistently more closely related to the seminal plasma virus in the source. All sequences were subtype B, and the env C2V3 of transmitted variants tended to have higher mean isoelectric points, contain potential N-linked glycosylation sites, and favor CCR5 co-receptor usage. A statistically robust phylogenetically corrected analysis did not detect genetic signatures reliably associated with transmission, but further investigation of larger samples of transmitting pairs holds promise for determining which structural and genetic features of viral genomes are associated with transmission.

Models of coding sequence evolution

Probabilistic models of sequence evolution are in widespread use in phylogenetics and molecular sequence evolution. These models have become increasingly sophisticated and combined with statistical model comparison techniques have helped to shed light on how genes and proteins evolve. Models of codon evolution have been particularly useful, because, in addition to providing a significant improvement in model realism for protein-coding sequences, codon models can also be designed to test hypotheses about the selective pressures that shape the evolution of the sequences. Such models typically assume a phylogeny and can be used to identify sites or lineages that have evolved adaptively. Recently some of the key assumptions that underlie phylogenetic tests of selection have been questioned, such as the assumption that the rate of synonymous changes is constant across sites or that a single phylogenetic tree can be assumed at all sites for recombining sequences. While some of these issues have been addressed through the development of novel methods, others remain as caveats that need to be considered on a case-by-case basis. Here, we outline the theory of codon models and their application to the detection of positive selection. We review some of the more recent developments that have improved their power and utility, laying a foundation for further advances in the modeling of coding sequence evolution.

CodonTest: Modeling Amino Acid Substitution Preferences in Coding Sequences

Codon models of evolution have facilitated the interpretation of selective forces operating on genomes. These models, however, assume a single rate of non-synonymous substitution irrespective of the nature of amino acids being exchanged. Recent developments have shown that models which allow for amino acid pairs to have independent rates of substitution offer improved fit over single rate models. However, these approaches have been limited by the necessity for large alignments in their estimation. An alternative approach is to assume that substitution rates between amino acid pairs can be subdivided into rate classes, dependent on the information content of the alignment. However, given the combinatorially large number of such models, an efficient model search strategy is needed. Here we develop a Genetic Algorithm (GA) method for the estimation of such models. A GA is used to assign amino acid substitution pairs to a series of rate classes, where is estimated from the alignment. Other parameters of the phylogenetic Markov model, including substitution rates, character frequencies and branch lengths are estimated using standard maximum likelihood optimization procedures. We apply the GA to empirical alignments and show improved model fit over existing models of codon evolution. Our results suggest that current models are poor approximations of protein evolution and thus gene and organism specific multi-rate models that incorporate amino acid substitution biases are preferred. We further anticipate that the clustering of amino acid substitution rates into classes will be biologically informative, such that genes with similar functions exhibit similar clustering, and hence this clustering will be useful for the evolutionary fingerprinting of genes.

Frequent toggling between alternative amino acids is driven by selection in HIV-1.

Host immune responses against infectious pathogens exert strong selective pressures favouring the emergence of escape mutations that prevent immune recognition. Escape mutations within or flanking functionally conserved epitopes can occur at a significant cost to the pathogen in terms of its ability to replicate effectively. Such mutations come under selective pressure to revert to the wild type in hosts that do not mount an immune response against the epitope. Amino acid positions exhibiting this pattern of escape and reversion are of interest because they tend to coincide with immune responses that control pathogen replication effectively. We have used a probabilistic model of protein coding sequence evolution to detect sites in HIV-1 exhibiting a pattern of rapid escape and reversion. Our model is designed to detect sites that toggle between a wild type amino acid, which is susceptible to a specific immune response, and amino acids with lower replicative fitness that evade immune recognition. Through simulation, we show that this model has significantly greater power to detect selection involving immune escape and reversion than standard models of diversifying selection, which are sensitive to an overall increased rate of non-synonymous substitution. Applied to alignments of HIV-1 protein coding sequences, the model of immune escape and reversion detects a significantly greater number of adaptively evolving sites in env and nef. In all genes tested, the model provides a significantly better description of adaptively evolving sites than standard models of diversifying selection. Several of the sites detected are corroborated by association between Human Leukocyte Antigen (HLA) and viral sequence polymorphisms. Overall, there is evidence for a large number of sites in HIV-1 evolving under strong selective pressure, but exhibiting low sequence diversity. A phylogenetic model designed to detect rapid toggling between wild type and escape amino acids identifies a larger number of adaptively evolving sites in HIV-1, and can in some cases correctly identify the amino acid that is susceptible to the immune response.

A Population Genetics Pedigree Perspective on the Transmission of Helicobacter pylori

The inference of transmission pathways for medicinally important bacteria is important to our understanding of pathogens. Here we report analyses of transmission in Helicobacter pylori, a major carcinogen. Our study is novel in that the focal community comprises detailed family pedigrees and has a high prevalence of H. pylori. To infer transmission, we performed high-resolution analyses of nucleotide sequences for three genes and accounted for the occurrence of mutation and recombination through the use of simulation modeling. Our results demonstrate that transmission has a strong nonfamilial component potentially the result of a large proportion of infections derived from the community. These results are interesting from both a medical and an evolutionary standpoint. First, efficient control measures and beliefs about the sources of H. pylori infection should be reevaluated. Evolutionarily, our results contradict the hypothesis of strict vertical transmission, presented as an explanation for the strong correlation between human population history and H. pylori diversity. Thus the paradox of persistent phylogenetic structure, despite a permissive mode of transmission and high recombination rates, must be solved elsewhere. Here we consider the potential for recombination events to maintain genetic structure in light of horizontal transmission.

Incidence of Helicobacter felis and the Effect of Coinfection with Helicobacter pylori on the Gastric Mucosa in the African Population

ABSTRACT Helicobacter pylori and Helicobacter felis are two of the Helicobacter spp. that infect humans. H. pylori has been linked to significant gastric pathology. Coinfection with Helicobacter spp. may influence infectious burden, pathogenesis, and antibiotic resistance; however, this has not been studied. The aims of this study were to identify the incidence of H. felis and to analyze the effects of coinfection with both organisms on gastric pathology in a well-characterized South African population. Biopsy samples from the gastric corpora and antra of volunteers (n = 90) were subjected to histological examination and PCR for the identification of H. pylori and H. felis. We further investigated the effect of global strain type on the occurrence of precursor lesions by assigning nucleotide sequences derived from PCR amplification of three genes to global groupings (ancestral Africa1, ancestral Africa2, ancestral Europe, ancestral Asia, and mixed). H. pylori was detected in 75 (83.3%), H. felis in 23 (25.6%), and coinfection in 21 (23.3%) of the volunteers by PCR. H. felis was randomly distributed among adults and children but clustered within families, suggesting intrafamilial transmission. Analysis of histopathology scores revealed no differences in atrophy, activity, and helicobacter density between H. felis-positive and H. felis-negative volunteers. H. pylori substrains common to southern Africa showed no differences in inflammation or atrophy scores. The incidences of H. felis and coinfection with H. pylori in the African population are high. H. felis infection, however, does not influence specific gastric pathology in this population.