Determining the immune response in human immunodefficiency virus infection : HIV -1 diversity as tool for epidemic monitoring

The Human Immunodeficiency Virus type 1 (HIV-1) is characterized by extensive genetic diversity at the population level but also within a single infected individual. The swift capacity of the virus to generate extensive diversity within the human host played a central role in the origin of the disease and is also key for the current global proportions of the HIV-1 pandemic. The epidemic started in Africa with multiple zoonotic transmissions of simian immunodeficiency virus (SIV) to humans. This was followed by a period of diversification and adaptation to the human population that, enhanced by the high rates of mutation and recombination of the virus, allowed the emergence of a virus capable of efficient sexual transmission among humans. The spread of the human adapted virus is estimated to have initiated from late 1950s to the early 1960s from Africa to the rest of the world. The predominance of the subtype B HIV-1 virus in Western Europe suggests that this was the first subtype to be introduced in this region. The subtype diversity pattern of HIV-1 in Portugal resembles the ones found in Central Africa being far more complex than the viral diversity patterns observed in the rest of Western Europe highlighting the relevance of in detailed studies of the Portuguese HIV-1 epidemics. In this work we have characterized the local HIV-1 epidemic of the Portuguese city of Braga in the years from 2000 to 2012. We found that the most frequent HIV-1 subtypes were G and B and by combining epidemiological and phylogenetic analysis we were able to uncover local transmission clusters of non-B and non-G subtypes among locals in association with sexual transmission networks that initiated transmission in the early 2000s. This corroborates Portugal as an early point of introduction of non-B HIV-1 subtypes in Western Europe. Having performed this characterization at the level of this local population we then focused on analyzing the duration of infection at the level of the infected patient. For this purpose we have optimized a methodology to differentiate recent from chronic infections. It was based on the study of ambiguous nucleotide calls obtained from routine HIV-1 genotyping. We found that the analysis of these ambiguities, as an expression of intra-host HIV-1 diversity, allowed the inference of the duration of infection in this study population. Subsequently, we questioned if high HIV-1 subtype diversity found in this region correlated with higher rates of transmission of drug resistance mutations. We found that the level of transmitted drug resistance in this population was similar to other European regions and independent predictors of transmitted drug resistance (TDR) could not be identified supporting the recommendation of universal viral sequencing at patient admission. This study performed in a country that is unique in Western Europe in what regards to HIV-1 diversity supported Portugal as one of the early entry-points of non-B HIV-1 subtypes in Western Europe and also reinforced the need for more efficacious local control measures targeting sexual transmission routes. We believe this study is of general importance especially in a time when several reports suggest that the prevalence of non-B subtypes in Western Europe is increasing. The knowledge herein generated also contributed for the development of method to discriminate recent from non-recent HIV-1 infections, a step of crucial importance to validate prevention strategies. Importantly, it was also shown that the higher HIV-1 subtype diversity found in this study population does not correlate with an increase in the rate of transmission of drug resistance when compared to the rest of the Western Europe.O Vírus da Imunodeficiência Humana Tipo 1 (VIH-1) é caracterizado por uma extensa diversidade genética não só a nível da população, mas também a nível individual, em cada hospedeiro. A rapidez do vírus para gerar grande diversidade dentro do hospedeiro humano desempenhou um papel central na génese da doença e é também essencial para as proporções globais atuais da pandemia do VIH-1. A epidemia começou em África, com várias transmissões zoonóticas de vírus da imunodeficiência símia (SIV) para seres humanos. Isto foi seguido por um período de diversificação e adaptação na população humana que, amplificada pelas altas taxas de mutação e de recombinação do vírus, permitiu o surgimento de uma nova espécie de vírus capaz de transmissão sexual eficiente entre os seres humanos. O início da propagação deste vírus já adaptado ao ser humano é estimada a partir do final dos anos 1950 ao início dos anos 1960, da África Central para o resto do mundo. A predominância do subtipo B do VIH-1 na Europa Ocidental sugere que este foi o primeiro subtipo a ser introduzido nesta região. O padrão de diversidade dos subtipos do VIH-1 em Portugal assemelha-se ao encontrado na África Central, sendo muito mais complexo do que os padrões de diversidade vírica observados no resto da Europa Ocidental. Este facto justifica o relevo que estudos detalhados sobre o VIH-1 em Portugal possam ter para a compreensão das epidemias de VIH-1. Neste trabalho foi caracterizada a epidemia local por VIH-1 na cidade portuguesa de Braga, entre os anos 2000 e 2012. Descobrimos que os subtipos VIH-1 mais frequentes foram G e B. Pela combinação de análise epidemiológica e filogenética pudemos demonstrar grupos de transmissão locais de subtipos não-B e não-G entre os residentes em associação com redes de transmissão sexual que iniciaram a transmissão no início da década de 2000. Isto indicia o papel de Portugal como um ponto de início da introdução de subtipos não-B do VIH-1 na Europa Ocidental. Tendo realizado esta caracterização a nível da população local, o trabalho concentrou-se na análise da duração da infeção ao nível individual. Para este efeito, aperfeiçoou-se uma metodologia para diferenciar infeção recente de infeção crónica. Baseados no estudo de posições nucleotídicas ambíguas obtidas a partir de genotipagem rotineira doVIH-1,descobrimos que a análise dessas ambiguidades, como uma expressão da diversidade intra-hospedeiro do VIH-1, permite inferir a duração da infeção nesta população em estudo. Posteriormente questionamos se a elevada diversidade do VIH-1 encontrada nesta região se poderia correlacionar com maiores taxas de transmissão de mutações de resistência aos antiretrovíricos. Descobrimos que o nível de resistência à terapêutica transmitido nesta população foi semelhante a outras regiões europeias. Não foram identificados preditores independentes de resistência transmissível aos antiretrovíricos, suportando a recomendação de sequenciamento viral universal no momento do contacto do doente com os serviços de saúde. Este estudo realizado num país que é único na Europa Ocidental no que diz respeito à diversidade do VIH-1,validoua noção de Portugal como um dos pontos de entrada iniciais de subtipos não-B do VIH-1 na Europa Ocidental e também reforçou a necessidade de medidas locais de controlo mais eficazes, que visem modos de transmissão sexual. Acreditamos que este estudo é relevante, especialmente num tempo em que vários artigos sugerem que a prevalência de subtipos não-B na Europa Ocidental está a aumentar. O conhecimento aqui gerado também contribuiu para o desenvolvimento de um método para discriminar infeções recentes pelo HIV-1 das não-recentes, um passo de importância crucial para validar as estratégias de prevenção. Relevantemente, também foi demonstrado que à maior diversidade doVIH-1 encontrada na população em estudo, não correspondeu um aumento na taxa de transmissão de resistência à terapêutica, quando comparada com o resto da Europa Ocidental

The role of visual adaptation in cichlid fish speciation

D. Shane Wright (1) , Ole Seehausen (2), Ton G.G. Groothuis (1), Martine E. Maan (1) (1) University of Groningen; GELIFES; EGDB(2) Department of Fish Ecology & Evolution, EAWAG Centre for Ecology, Evolution and Biogeochemistry, Kastanienbaum AND Institute of Ecology and Evolution, Aquatic Ecology, University of Bern.In less than 15,000 years, Lake Victoria cichlid fishes have radiated into as many as 500 different species. Ecological and sexual sel ection are thought to contribute to this ongoing speciation process, but genetic differentiation remains low. However, recent work in visual pigment genes, opsins, has shown more diversity. Unlike neighboring Lakes Malawi and Tanganyika, Lake Victoria is highly turbid, resulting in a long wavelength shift in the light spectrum with increasing depth, providing an environmental gradient for exploring divergent coevolution in sensory systems and colour signals via sensory drive. Pundamilia pundamila and Pundamilia nyererei are two sympatric species found at rocky islands across southern portions of Lake Victoria, differing in male colouration and the depth they reside. Previous work has shown species differentiation in colour discrimination, corresponding to divergent female preferences for conspecific male colouration. A mechanistic link between colour vision and preference would provide a rapid route to reproductive isolation between divergently adapting populations. This link is tested by experimental manip ulation of colour vision - raising both species and their hybrids under light conditions mimicking shallow and deep habitats. We quantify the expression of retinal opsins and test behaviours important for speciation: mate choice, habitat preference, and fo raging performance

Bayesian inference of transmission chains using timing of symptoms, pathogen genomes and contact data.

There exists significant interest in developing statistical and computational tools for inferring 'who infected whom' in an infectious disease outbreak from densely sampled case data, with most recent studies focusing on the analysis of whole genome sequence data. However, genomic data can be poorly informative of transmission events if mutations accumulate too slowly to resolve individual transmission pairs or if there exist multiple pathogens lineages within-host, and there has been little focus on incorporating other types of outbreak data. We present here a methodology that uses contact data for the inference of transmission trees in a statistically rigorous manner, alongside genomic data and temporal data. Contact data is frequently collected in outbreaks of pathogens spread by close contact, including Ebola virus (EBOV), severe acute respiratory syndrome coronavirus (SARS-CoV) and Mycobacterium tuberculosis (TB), and routinely used to reconstruct transmission chains. As an improvement over previous, ad-hoc approaches, we developed a probabilistic model that relates a set of contact data to an underlying transmission tree and integrated this in the outbreaker2 inference framework. By analyzing simulated outbreaks under various contact tracing scenarios, we demonstrate that contact data significantly improves our ability to reconstruct transmission trees, even under realistic limitations on the coverage of the contact tracing effort and the amount of non-infectious mixing between cases. Indeed, contact data is equally or more informative than fully sampled whole genome sequence data in certain scenarios. We then use our method to analyze the early stages of the 2003 SARS outbreak in Singapore and describe the range of transmission scenarios consistent with contact data and genetic sequence in a probabilistic manner for the first time. This simple yet flexible model can easily be incorporated into existing tools for outbreak reconstruction and should permit a better integration of genomic and epidemiological data for inferring transmission chains

The influence of HIV-1 genomic target region selection and sequence length on the accuracy of inferred phylogenies and clustering outcomes.

Masters Degree. University of KwaZulu-Natal, Durban.To improve the methodology of HIV-1 cluster analysis, we addressed how analysis of HIV-1 clustering is associated with parameters that can affect the outcome of viral clustering. The extent of HIV clustering, tree certainty, subtype diversity ratio (SDR), subtype diversity variance (SDV) and Shimodaira-Hasegawa (SH)-like support values were compared between 2881 HIV-1 full genome sequences and sub-genomic regions of which 2567 were retrieved from the LANL HIV Database and 314 were sequenced from blood samples from a cohort in KwaZulu-Natal. Sliding window analysis was based on 99 windows of 1000 bp, 45 windows of 2000 bp and 27 windows of 3000 bp. Clusters were enumerated for each window sequence length, and the optimal sequence length for cluster identification was probed. Potential associations between the extent of HIV clustering and sequence length were also evaluated. The phylogeny based on the full-genome sequences showed the best tree accuracy; it ranked highest with regards to both tree certainty and SH-like support. Product 4, a region associated with env, had the best tree accuracy among the sub-genomic regions. Among the HIV-1 structural genes, env had the best tree certainty, SH-like support, SDR score and the best SDV score overall. The hierarchy of cluster phylotype enumeration mirrored the tree accuracy analysis, with the full genome phylogeny showing the highest extent of clustering, and the product 4 region being second best. Among the structural genes, the highest number of phylotypes was enumerated from the pol phylogeny, followed by env. The extent of HIV-1 clustering was slightly higher for sliding windows of 3 000 bp than 2000 bp and 1000 bp, thus 3000 bp was found to be the optimal length for phylogenetic cluster analysis. We found a moderate association between the length of sequences used and proportion of HIV sequences in clusters; the influence of viral sequence length may have been diminished by the substantial number of taxa. Full-genome sequences could provide the most informative HIV cluster analysis. Selected sub-genomic regions with the best combination of high extent of HIV clustering and high tree accuracy, such as env, could also be considered as a second choice

Emerging concepts of data integration in pathogen phylodynamics

Phylodynamics has become an increasingly popular statistical framework to extract evolutionary and epidemiological information from pathogen genomes. By harnessing such information, epidemiologists aim to shed light on the spatio-temporal patterns of spread and to test hypotheses about the underlying interaction of evolutionary and ecological dynamics in pathogen populations. Although the field has witnessed a rich development of statistical inference tools with increasing levels of sophistication, these tools initially focused on sequences as their sole primary data source. Integrating various sources of information, however, promises to deliver more precise insights in infectious diseases and to increase opportunities for statistical hypothesis testing. Here, we review how the emerging concept of data integration is stimulating new advances in Bayesian evolutionary inference methodology which formalize a marriage of statistical thinking and evolutionary biology. These approaches include connecting sequence to trait evolution, such as for host, phenotypic and geographic sampling information, but also the incorporation of covariates of evolutionary and epidemic processes in the reconstruction procedures. We highlight how a full Bayesian approach to covariate modelling and testing can generate further insights into sequence evolution, trait evolution and population dynamics in pathogen populations. Specific examples demonstrate how such approaches can be used to test the impact of host on rabies and HIV evolutionary rates, to identify the drivers of influenza dispersal as well as the determinants of rabies cross-species transmissions, and to quantify the evolutionary dynamics of influenza antigenicity. Finally, we briefly discuss how data integration is now also permeating through the inference of transmission dynamics, leading to novel insights into tree-generative processes and detailed reconstructions of transmission trees.status: publishe

Phylodynamic Patterns in Pathogen Ecology and Evolution.

The rapid evolution of viral pathogens requires us to consider epidemiological, ecological and evolutionary processes as coupled together and occurring at the same timescale. Rotavirus and influenza account for high levels of morbidity and mortality worldwide and are two important examples of such dynamics. In this work, I investigate the different evolutionary and ecological processes that shape the antigenic structure and phylogenetic characteristics of these two viruses. In the first part of my work, I use a theoretical model of influenza A/H3N2 to identify the relative importance of antigenic novelty, competition between lineages, and changes in the susceptibility of the host population to circulating strains in determining the evolutionary and epidemiological trajectory of the virus. I develop this model further to correspond with patterns of immunity and infection observed in rotavirus, and investigate how reassortment, the swapping of gene segments between viruses, influences the formation and replacement of rotavirus genotypes through immune mediated processes. In the second part of my work, I use a tool (SeasMig), which I developed, to infer alternative stochastically generated migration and mutation events along phylogenetic trees in a Bayesian manner. Using SeasMig, I first show how the seasonality of A/H3N2 influenza incidence corresponds to rates of immigration and emigration of the virus. Subsequently, I tease out the different evolutionary and ecological processes, which drive changes in the US rotavirus population following onset of routine vaccination. My work has implications for identifying likely evolutionary mechanisms, which may lead to reduced vaccine efficacy, and for vaccine strain selection.PhDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113494/1/dzinder_1.pd

Phylogeography of the Solanaceae-infecting Basidiomycota fungus Rhizoctonia solani AG-3 based on sequence analysis of two nuclear DNA loci

<p>Abstract</p> <p>Background</p> <p>The soil fungus <it>Rhizoctonia solani </it>anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of <it>R. solani </it>AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of <it>R. solani </it>AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of <it>R. solani </it>AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of <it>R. solani </it>AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89).</p> <p>Results</p> <p>Populations of <it>R. solani </it>AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of <it>R. solani </it>AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of <it>R. solani </it>AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (Φ_{<it>ST </it>}= 0.257, significant at P < 0.05) but not for locus pP42F (Φ_{<it>ST </it>}= 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of <it>R. solani </it>AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of <it>R. solani </it>AG-3.</p> <p>Conclusion</p> <p>The two sisters populations of the basidiomycete fungus <it>R. solani </it>AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.</p