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Curing HIV will require eliminating the reservoir of integrated, replication-competent proviruses that persist despite antiretroviral therapy (ART). Understanding the burden, genetic diversity and longevity of persisting proviruses in diverse individuals with HIV is critical to this goal, but these characteristics remain understudied in some groups. Among them are viremic controllers, individuals who naturally suppress HIV to low levels but for whom therapy is nevertheless recommended. We reconstructed within-host HIV evolutionary histories from longitudinal single-genome amplified viral sequences in four viremic controllers who eventually initiated ART, and used this information to characterize the age and diversity of proviruses persisting on therapy. We further leveraged these within-host proviral age distributions to estimate rates of proviral turnover prior to ART. This is an important yet understudied metric, since pre-ART proviral turnover dictates reservoir composition at ART initiation (and thereafter), which is when curative interventions, once developed, would be administered. Despite natural viremic control, all participants displayed significant within-host HIV evolution pre-therapy, where overall on-ART proviral burden and diversity broadly reflected the extent of viral replication and diversity pre-ART. Consistent with recent studies of non-controllers, the proviral pools of two participants were skewed towards sequences that integrated near ART initiation, suggesting dynamic proviral turnover during untreated infection. In contrast, proviruses recovered from the two other participants dated to time-points that were more evenly spread throughout infection, suggesting slow or negligible proviral decay following deposition. HIV cure strategies will need to overcome within-host proviral diversity, even in individuals who naturally controlled HIV replication before therapy.ImportanceHIV therapy is life-long because integrated, replication-competent viral copies persist within long-lived cells. To cure HIV, we need to understand when these viral reservoirs form, how large and genetically diverse they are, and how long they endure. Elite controllers, individuals who naturally suppress HIV to undetectable levels, are being intensely studied as models of HIV remission, but viremic controllers, individuals who naturally suppress HIV to low levels, remain understudied even though they too may hold valuable insights. We combined phylogenetics and mathematical modeling to reconstruct proviral seeding and decay from infection to therapy-mediated suppression in four viremic controllers. We recovered diverse proviruses persisting during therapy that broadly reflected HIV’s within-host evolutionary history, where the estimated half-lives of the persistent proviral pool during untreated infection ranged from

The lung is an understudied site of HIV persistence. We isolated 882 subgenomic proviral sequences by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 9% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harbored at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. No participant exhibited genetic compartmentalization so obvious that it was visually apparent in a phylogeny. When formal tests were applied however, two (22%) participants showed modest yet significant support for compartmentalization when analysis was restricted to distinct proviruses per site. This increased to four participants (44%) when considering all within-host sequences. Thus, while a minority of individuals harbor somewhat distinctive proviral populations in blood and lung, these can simply be due to unequal distributions of clonally-expanded sequences. Importantly, the extent of lung proviral diversity on ART strongly reflected that in blood (Spearman ρ = 0.98, p < 0.0001), confirming blood as a strong indicator of total body proviral diversity. Analysis of on-ART proviral diversity in context of pre-therapy viral diversity in two participants revealed marked differences in proviral longevity and dynamics. Whereas one participant’s proviral pool was rich in ancestral sequences that recapitulated more of HIV’s within-host evolutionary history, the other’s largely comprised more contemporary sequences, including ones that re-seeded the reservoir during a three-year treatment interruption. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individual’s proviral composition. Individualized HIV remission and cure strategies may be needed to overcome these challenges.Author SummaryHIV persists in the body despite long-term suppressive antiretroviral therapy. Much of our knowledge about the HIV reservoir comes from studying proviruses in blood, but a fundamental question is whether these are distinct from those in tissues. The lung could theoretically engender genetically distinctive HIV populations, but this remains understudied. Our analysis of nearly 900 proviral sequences from blood and lung of nine individuals with HIV receiving long-term therapy revealed substantial within-host heterogeneity, yet some common patterns. Identical sequences (consistent with clonal expansion of infected cells) were observed in everyone, though at different (9-86%) frequencies. Recovery of shared sequences across blood and lung was common (77% of participants). Fewer than half of participants exhibited blood-lung genetic compartmentalization, and only modestly so. Moreover, when present, compartmentalization was often attributable to differential distribution of identical sequences, not the presence of genetically distinctive populations, across sites. Results also revealed marked inter-individual differences in proviral dynamics, as evidenced by the proportion of persisting proviruses that represented ancestral versus more recently-circulating viral sequences. Critically, the extent of within-host proviral diversity in blood correlated strongly with that in lung, indicating that despite inter-individual heterogeneity, blood is a strong indicator of proviral diversity elsewhere in the body.

Phylogenetic trees and data are often stored in incompatible and inconsistent formats. The outputs of software tools that contain trees with analysis findings are often not compatible with each other, making it hard to integrate the results of different analyses in a comparative study. The treeio package is designed to connect phylogenetic tree input and output. It supports extracting phylogenetic trees as well as the outputs of commonly used analytical software. It can link external data to phylogenies and merge tree data obtained from different sources, enabling analyses of phylogeny-associated data from different disciplines in an evolutionary context. Treeio also supports export of a phylogenetic tree with heterogeneous-associated data to a single tree file, including BEAST compatible NEXUS and jtree formats; these facilitate data sharing as well as file format conversion for downstream analysis. The treeio package is designed to work with the tidytree and ggtree packages. Tree data can be processed using the tidy interface with tidytree and visualized by ggtree. The treeio package is released within the Bioconductor and rOpenSci projects. It is available at https://www.bioconductor.org/packages/treeio/.

Cophylogeny is the congruence of phylogenetic relationships between two different groups of organisms due to their long‐term interaction. We investigated the use of tree shape distance measures to quantify the degree of cophylogeny. We implemented a reverse‐time simulation model of pathogen phylogenies within a fixed host tree, given cospeciation probability, host switching, and pathogen speciation rates. We used this model to evaluate 18 distance measures between host and pathogen trees including two kernel distances that we developed for labeled and unlabeled trees, which use branch lengths and accommodate different size trees. Finally, we used these measures to revisit published cophylogenetic studies, where authors described the observed associations as representing a high or low degree of cophylogeny. Our simulations demonstrated that some measures are more informative than others with respect to specific coevolution parameters especially when these did not assume extreme values. For real datasets, trees’ associations projection revealed clustering of high concordance studies suggesting that investigators are describing it in a consistent way. Our results support the hypothesis that measures can be useful for quantifying cophylogeny. This motivates their usage in the field of coevolution and supports the development of simulation‐based methods, i.e., approximate Bayesian computation, to estimate the underlying coevolutionary parameters.

The complexities of viral evolution can be difficult to elucidate. Software simulating viral evolution provides powerful tools for exploring hypotheses of viral systems, especially in situations where thorough empirical data are difficult to obtain or parameters of interest are difficult to measure. Human immunodeficiency virus 1 (HIV-1) infection has no durable cure; this is primarily due to the virus’ ability to integrate into the genome of host cells, where it can remain in a transcriptionally latent state. An effective cure strategy must eliminate every copy of HIV-1 in this ‘persistent reservoir’ because proviruses can reactivate, even decades later, to resume an active infection. However, many features of the persistent reservoir remain unclear, including the temporal dynamics of HIV-1 integration frequency and the longevity of the resulting reservoir. Thus, sophisticated analyses are required to measure these features and determine their temporal dynamics. Here, we present software that is an extension of SANTA-SIM to include multiple compartments of viral populations. We used the resulting software to create a model of HIV-1 within host evolution that incorporates the persistent HIV-1 reservoir. This model is composed of two compartments, an active compartment and a latent compartment. With this model, we compared five different date estimation methods (Closest Sequence, Clade, Linear Regression, Least Squares, and Maximum Likelihood) to recover the integration dates of genomes in our model’s HIV-1 reservoir. We found that the Least Squares method performed the best with the highest concordance (0.80) between real and estimated dates and the lowest absolute error (all pairwise t tests: P

The HIV reservoir, which comprises diverse proviruses integrated into the genomes of infected, primarily CD4+ T cells, is the main barrier to developing an effective HIV cure. Our understanding of the genetics and dynamics of proviruses persisting within distinct CD4+ T cell subsets, however, remains incomplete. Using single-genome amplification, we characterized subgenomic proviral sequences (nef region) from naive, central memory, transitional memory, and effector memory CD4+ T cells from five HIV-infected individuals on long-term combination antiretroviral therapy (cART) and compared these to HIV RNA sequences isolated longitudinally from archived plasma collected prior to cART initiation, yielding HIV data sets spanning a median of 19.5 years (range, 10 to 20 years) per participant. We inferred a distribution of within-host phylogenies for each participant, from which we characterized proviral ages, phylogenetic diversity, and genetic compartmentalization between CD4+ T cell subsets. While three of five participants exhibited some degree of proviral compartmentalization between CD4+ T cell subsets, combined analyses revealed no evidence that any particular CD4+ T cell subset harbored the longest persisting, most genetically diverse, and/or most genetically distinctive HIV reservoir. In one participant, diverse proviruses archived within naive T cells were significantly younger than those in memory subsets, while for three other participants we observed no significant differences in proviral ages between subsets. In one participant, "old" proviruses were recovered from all subsets, and included one sequence, estimated to be 21.5 years old, that dominated (>93%) their effector memory subset. HIV eradication strategies will need to overcome within- and between-host genetic complexity of proviral landscapes, possibly via personalized approaches.IMPORTANCE The main barrier to HIV cure is the ability of a genetically diverse pool of proviruses, integrated into the genomes of infected CD4+ T cells, to persist despite long-term suppressive combination antiretroviral therapy (cART). CD4+ T cells, however, constitute a heterogeneous population due to their maturation across a developmental continuum, and the genetic "landscapes" of latent proviruses archived within them remains incompletely understood. We applied phylogenetic techniques, largely novel to HIV persistence research, to reconstruct within-host HIV evolutionary history and characterize proviral diversity in CD4+ T cell subsets in five individuals on long-term cART. Participants varied widely in terms of proviral burden, genetic diversity, and age distribution between CD4+ T cell subsets, revealing that proviral landscapes can differ between individuals and between infected cell types within an individual. Our findings expose each within-host latent reservoir as unique in its genetic complexity and support personalized strategies for HIV eradication.

The HIV accessory protein Nef downregulates the viral entry receptor CD4, the Human Leukocyte Antigen (HLA)-A and -B molecules, the Serine incorporator 5 (SERINC5) protein and other molecules from the infected cell surface, thereby promoting viral infectivity, replication and immune evasion. Theneflocus also represents one of the most genetically variable regions in the HIV genome, andnefsequences undergo substantial evolution within a single individual over the course of infection. Few studies however have simultaneously characterized the impact of within-hostnefsequence evolution on Nef protein function over prolonged timescales. Here, we isolated 50 unique Nef clones by single-genome amplification over an 11-year period from the plasma of an individual who was largely naïve to antiretroviral treatment during this time. Together, these clones harbored nonsynonymous substitutions at 13% ofnef’s codons. We assessed their ability to downregulate cell-surface CD4, HLA and SERINC5 and observed that all three Nef functions declined modestly over time, where the reductions in CD4 and HLA downregulation (an average of 0.6% and 2.0% per year, respectively) achieved statistical significance. The results from this case study support all three Nef activities as being important to maintain throughout untreated HIV infection, but nevertheless suggest that, despitenef’s mutational plasticity, within-host viral evolution can compromise Nef function, albeit modestly, over prolonged periods.

The HIV reservoir, which comprises diverse proviruses integrated into the genomes of infected, primarily CD4(+) T cells, is the main barrier to developing an effective HIV cure. Our understanding of the genetics and dynamics of proviruses persisting within distinct CD4(+) T cell subsets, however, remains incomplete. Using single-genome amplification, we characterized subgenomic proviral sequences (nef region) from naive, central memory, transitional memory, and effector memory CD4(+) T cells from five HIV-infected individuals on long-term combination antiretroviral therapy (cART) and compared these to HIV RNA sequences isolated longitudinally from archived plasma collected prior to cART initiation, yielding HIV data sets spanning a median of 19.5 years (range, 10 to 20 years) per participant. We inferred a distribution of within-host phylogenies for each participant, from which we characterized proviral ages, phylogenetic diversity, and genetic compartmentalization between CD4(+) T cell subsets. While three of five participants exhibited some degree of proviral compartmentalization between CD4(+) T cell subsets, combined analyses revealed no evidence that any particular CD4(+) T cell subset harbored the longest persisting, most genetically diverse, and/or most genetically distinctive HIV reservoir. In one participant, diverse proviruses archived within naive T cells were significantly younger than those in memory subsets, while for three other participants we observed no significant differences in proviral ages between subsets. In one participant, “old” proviruses were recovered from all subsets, and included one sequence, estimated to be 21.5 years old, that dominated (>93%) their effector memory subset. HIV eradication strategies will need to overcome within- and between-host genetic complexity of proviral landscapes, possibly via personalized approaches. IMPORTANCE The main barrier to HIV cure is the ability of a genetically diverse pool of proviruses, integrated into the genomes of infected CD4(+) T cells, to persist despite long-term suppressive combination antiretroviral therapy (cART). CD4(+) T cells, however, constitute a heterogeneous population due to their maturation across a developmental continuum, and the genetic “landscapes” of latent proviruses archived within them remains incompletely understood. We applied phylogenetic techniques, largely novel to HIV persistence research, to reconstruct within-host HIV evolutionary history and characterize proviral diversity in CD4(+) T cell subsets in five individuals on long-term cART. Participants varied widely in terms of proviral burden, genetic diversity, and age distribution between CD4(+) T cell subsets, revealing that proviral landscapes can differ between individuals and between infected cell types within an individual. Our findings expose each within-host latent reservoir as unique in its genetic complexity and support personalized strategies for HIV eradication.

The HIV-1 reservoir consists of latently infected cells that persist despite antiretroviral therapy (ART). Elucidating the proviral genetic composition of the reservoir, particularly in the context of pre-therapy viral diversity, is therefore important to understanding reservoir formation and the persistence of latently infected cells. Here we investigate reservoir proviral variants from 13 Zambian acutely-infected individuals with additional pre-therapy sampling for a unique comparison to the ART-naïve quasispecies. We identified complete transmitted/founder (TF) viruses from seroconversion plasma samples, and additionally amplified and sequenced HIV-1 from plasma obtained one year post-infection and just prior to ART initiation. While the majority of proviral variants in the reservoir were most closely related to viral variants from the latest pre-therapy time point, we also identified reservoir proviral variants dating to or near the time of infection, and to intermediate time points between infection and treatment initiation. Reservoir proviral variants differing by five or fewer nucleotide changes from the TF virus persisted during treatment in five individuals, including proviral variants that exactly matched the TF in two individuals, one of whom had remained ART-naïve for more than six years. Proviral variants during treatment were significantly less divergent from the TF virus than plasma variants present at the last ART-naïve time point. These findings indicate that reservoir proviral variants are archived throughout infection, recapitulating much of the viral diversity that arises throughout untreated HIV-1 infection, and strategies to target and reduce the reservoir must therefore permit for the clearance of proviruses encompassing this extensive diversity., Author summary Despite reducing viremia to levels below the limit of detection in standard assays, effective antiretroviral therapy (ART) does not eradicate cells latently infected with HIV-1. These cells serve as a reservoir for viral rebound if therapy is interrupted; thus, understanding the composition of the reservoir may yield further targets for HIV-1 cure strategies. We have taken a genetic approach to elucidating the reservoir in 13 Zambian subtype C seroconvertors who were followed longitudinally through ART initiation and virologic suppression. In five of the 13 individuals, provirus sequences identical to or differing by five or fewer nucleotides from the transmitted/founder virus were detected, indicating archiving and persistence of early infection variants for more than six years following infection. While the majority of proviral variants in latently infected cells were most closely related to plasma virus circulating immediately prior to treatment initiation, additional variants dating to intermediate time points in the infection were also observed. These findings demonstrate that virus is archived during all stages of ART-naïve infection, and these variants persist throughout ART. HIV-1 cure strategies to eliminate the reservoir must address the broad genetic diversity of a within-host proviral quasispecies including variants archived from acute through chronic infection.

HIV's ability to persist during suppressive antiretroviral therapy is the main barrier to cure. Immune-privileged tissues, such as the testes, may constitute distinctive sites of HIV persistence, but this has been challenging to study in humans. We analyzed the proviral burden and genetics in the blood and testes of 10 individuals on suppressive therapy who underwent elective gender-affirming surgery. HIV DNA levels in matched blood and testes were quantified by quantitative PCR, and subgenomic proviral sequences (nef region) were characterized from single templates. HIV diversity, compartmentalization, and immune escape burden were assessed using genetic and phylogenetic approaches. Diverse proviruses were recovered from the blood (396 sequences; 354 nef-intact sequences) and testes (326 sequences; 309 nef-intact sequences) of all participants. Notably, the frequency of identical HIV sequences varied markedly between and within individuals. Nevertheless, proviral loads, within-host unique HIV sequence diversity, and the immune escape burden correlated positively between blood and testes. When all intact nef sequences were evaluated, 60% of participants exhibited significant blood-testis genetic compartmentalization, but none did so when the evaluation was restricted to unique sequences per site, suggesting that compartmentalization, when present, is attributable to the clonal expansion of HIV-infected cells. Our observations confirm the testes as a site of HIV persistence and suggest that individuals with larger and more diverse blood reservoirs will have larger and more diverse testis reservoirs. Furthermore, while the testis microenvironment may not be sufficiently unique to facilitate the seeding of unique viral populations therein, differential clonal expansion dynamics may be at play, which may complicate HIV eradication. IMPORTANCE Two key questions in HIV reservoir biology are whether immune-privileged tissues, such as the testes, harbor distinctive proviral populations during suppressive therapy and, if so, by what mechanism. While our results indicated that blood-testis HIV genetic compartmentalization was reasonably common (60%), it was always attributable to differential frequencies of identical HIV sequences between sites. No blood-tissue data set retained evidence of compartmentalization when only unique HIV sequences per site were considered; moreover, HIV immune escape mutation burdens were highly concordant between sites. We conclude that the principal mechanism by which blood and testis reservoirs differ is not via seeding of divergent HIV sequences therein but, rather, via differential clonal expansion of latently infected cells. Thus, while viral diversity and escape-related barriers to HIV eradication are of a broadly similar magnitude across the blood and testes, clonal expansion represents a challenge. The results support individualized analysis of within-host reservoir diversity to inform curative approaches.

OBJECTIVE: Timely initiation of combination antiretroviral therapy (ART) limits latent HIV reservoir size and should also limit reservoir genetic complexity. However, the relationship between these two factors remains unclear, particularly among HIV-infected youth. DESIGN: Retrospective analysis of replication-competent latent HIV clones serially isolated by limiting-dilution culture from resting CD4+ T-cell reservoirs from ART-suppressed, young adult participants of a historic phase I therapeutic vaccine trial (PACTG/IMPAACT-P1059). METHODS: Replication-competent latent HIV clones isolated from resting CD4+ T-cells of 4 perinatally- and 10 non-perinatally-infected young adults (average 22 versus 6 years uncontrolled infection, respectively) were sequenced in Pol and Nef. Within-host HIV sequence datasets were characterized with respect to their genetic diversity and inferred immune escape mutation burden. RESULTS: While participants were comparable in terms of sociodemographic and HIV sampling characteristics (e.g. on average, a mean 17 Pol sequences were recovered at 5 timepoints over up to 70 weeks) and the length of ART suppression at study entry (average 3 years), replication-competent HIV reservoir size, genetic diversity, immune escape mutation burden and variant complexity were significantly higher among the perinatally-infected participants who experienced longer durations of uncontrolled viremia. Nevertheless, viral sequences inferred to retain susceptibility to host cellular immune responses were detected in all participants, irrespective of uncontrolled viremia duration. CONCLUSIONS: HIV elimination in late-suppressed youth may be doubly challenged by larger and more genetically complex reservoirs. Strategies that integrate host and viral genetic complexity to achieve HIV remission or cure may merit consideration in such cases.

Given that HIV evolution and latent reservoir establishment occur continually within-host, and that latently infected cells can persist long-term, the HIV reservoir should comprise a genetically heterogeneous archive recapitulating within-host HIV evolution. However, this has yet to be conclusively demonstrated, in part due to the challenges of reconstructing within-host reservoir establishment dynamics over long timescales. We developed a phylogenetic framework to reconstruct the integration dates of individual latent HIV lineages. The framework first involves inference and rooting of a maximum-likelihood phylogeny relating plasma HIV RNA sequences serially sampled before the initiation of suppressive antiretroviral therapy, along with putative latent sequences sampled thereafter. A linear model relating root-to-tip distances of plasma HIV RNA sequences to their sampling dates is used to convert root-to-tip distances of putative latent lineages to their establishment (integration) dates. Reconstruction of the ages of putative latent sequences sampled from chronically HIV-infected individuals up to 10 y following initiation of suppressive therapy revealed a genetically heterogeneous reservoir that recapitulated HIV’s within-host evolutionary history. Reservoir sequences were interspersed throughout multiple within-host lineages, with the oldest dating to >20 y before sampling; historic genetic bottleneck events were also recorded therein. Notably, plasma HIV RNA sequences isolated from a viremia blip in an individual receiving otherwise suppressive therapy were highly genetically diverse and spanned a 20-y age range, suggestive of spontaneous in vivo HIV reactivation from a large latently infected cell pool. Our framework for reservoir dating provides a potentially powerful addition to the HIV persistence research toolkit.

Cophylogeny is the congruence of phylogenetic relationships between two different groups of organisms due to their long-term interaction, such as between host and pathogen species. Discordance between host and pathogen phylogenies may occur due to pathogen host-switch events, pathogen speciation within a host species, and extinction. Here, we investigated the use of tree shape distance measures to quantify the degree of cophylogeny for the comparative analysis of host-pathogen interactions across taxonomic groups.We firstly implemented a coalescent model to simulate pathogen phylogenies within a fixed host tree, given the cospeciation probability, migration rate between hosts, and pathogen speciation rate within hosts.Next, we used simulations from this model to evaluate 13 distance metrics between these trees and the host tree, including Robinson-Foulds distance and two kernel distances that we developed for labeled and unlabeled trees, which use branch lengths and can accommodate trees of different sizes. Finally, we used these distance metrics to revisit actual datasets from published cophylogenetic studies across all taxonomic groups, where authors described the observed associations as representing a high or low degree of cophylogeny.Our simulation analyses demonstrated that some metrics are more informative than others with respect to specific coevolution parameters. For example, the Sim metric was the most responsive to variation in coalescence rates, whereas the unlabeled kernel metric was the most responsive to cospeciation probabilities. We also determined that distance metrics were more informative about the model parameters when the underlying parameter values did not assume extreme values,e.g.,rapid host switching. When applied to real datasets, projection of these trees’ associations into a parameter space defined by the 13 distance metrics revealed some clustering of studies reporting low concordance. This suggested that different investigators are describing concordance in a consistent way across biological systems, and that these expert subjective assessments can be at least partly quantified using distance metrics.Our results support the hypothesis that tree distance measures can be useful for quantifying host and pathogen cophylogeny. This motivates the usage of distance metrics in the field of coevolution and supports the development of simulation-based methods,i.e.,approximate Bayesian computation, to estimate coevolutionary parameters from the discordant shapes of host and pathogen trees. [tree shape; cophylogeny; codivergence; coevolution; host switching; tree metrics; kernel]

New, costly, fast acting, therapies targeting the non-structural proteins 5A and 5B (NS5A and NS5B) regions of the hepatitis C virus (HCV) genome are curative in the majority of cases. Variants with certain mutations in the NS5A and NS5B regions of HCV have been shown to reduce susceptibility to direct-acting NS5A and NS5B therapy and are found in treatment naïve patients. Despite this, the ease with which these variants evolve is poorly known, as are their evolutionary and geographic origins. To address this crucial gap we inferred the evolutionary and geographic origins of resistance-associated variants (RAVs) in the HCV NS5A and NS5B regions of subtypes 1a, 1b, and 3a sequences available from global databases. We found that RAVs in the NS5A region of HCV, when prevalent, were widely dispersed throughout the phylogenetic tree of HCV with multiple independent origins and that these variants are globally distributed. In contrast, most of the NS5B C316N variants came from one of two clades in the phylogenetic tree of HCV subtype 1b. The presence of serine (S) at codon 218 of HCV NS5B appears to facilitate the evolution of the C316N RAV. Other NS5B RAVs did not arise very frequently in our data set, except for S556G in subtype 1b and with respect to geography NS5B RAVs were also globally distributed. The inferred distribution of RAVs in the NS5A region and frequency of their origin suggest a low fitness barrier without the need for co-evolution of compensatory mutations. A low fitness barrier may allow rapid selection of de novo resistance to NS5A inhibitors during therapy.

Summary Phylogenetic trees encode the evolutionary distances between species or populations. With sufficient information, these evolutionary distances can be rescaled over time to provide estimates of the dates of the most recent ancestors of the species. Here we present the R program node.dating, divergence-time analysis software, which uses a maximum-likelihood method to estimate the dates of the internal nodes of a phylogenetic tree. Availability and Implementation node.dating is available as a part of the R v3.30 package ape v4.0 (cran.r-project.org). node.dating is also available in the GitHub repository: https://github.com/brj1/node.dating, along with supplementary software and tests. Supplementary information Supplementary data are available at Bioinformatics online.

[The universe] cannot be read until we have learnt the language and become familiar with the characters in which it is written. It is written in mathematical language, and the letters are triangles, circles and other geometrical figures, without which means it is humanly impossible to comprehend a single word.

Summary: ANGES is a suite of Python programs that allows reconstructing ancestral genome maps from the comparison of the organization of extant-related genomes. ANGES can reconstruct ancestral genome maps for multichromosomal linear genomes and unichromosomal circular genomes. It implements methods inspired from techniques developed to compute physical maps of extant genomes. Examples of cereal, amniote, yeast or bacteria ancestral genomes are provided, computed with ANGES. Availability: ANGES is freely available for download at http://paleogenomics.irmacs.sfu.ca/ANGES/. Documentation and examples are available together with the package. Contact: cedric.chauve@sfu.ca

We consider weighted generating functions of trees where the weights are products of functions of the sizes of the subtrees. This work begins with the observation that three different communities, largely independently, found substantially the same result concerning these series. We unify these results with a common generalization. Next we use the insights of one community on the problems of another in two different ways. Namely, we use the differential equation perspective to find a number of new interesting hook length formulae for trees, and we use the body of examples developed by the combinatorial community to give quantum field theory toy examples with nice properties., Comment: 13 pages

Postinhibitory rebound (PIR) is an intrinsic property of many neurons but the underlying mechanism is not well understood. We studied PIR and its relationship to spike adaptation in B-cells isolated from the buccal ganglia of Aplysia. These neurons exhibit PIR following inhibitory synaptic input and following direct membrane hyperpolarization. Hyperpolarizing and depolarizing voltage clamp pulses from the resting potential evoke slow changes in membrane current that persist in the form of tail currents following the pulses. A subtraction method was used to isolate slow tail currents for study. Current-voltage measurements indicate that slow outward tail currents following depolarizing pulses result from increases in membrane conductance, while inward tail currents following hyperpolarizations to −50 and −60 mV result from conductance decreases. The reversal potential of both outward and inward tail current is between −60 and −70 mV. Tail currents activated by pulses more positive than −60 mV are ...

The membrane potentials of the plasmalemma and tonoplast in Griffithsia pacifica Kyl were measured and shown to be significantly different. No consistent cell wall potential was discovered. Adjacent cells in the filament were demonstrated to be electrolonically coupled with a mean coupling coefficient of 0.13. Cobaltions did not move between cells. No dye coupling was seen using the fluorescent dye Lucifer yellow CH. These observations provide new information on the electrophysiology of red algae and indicate that there is intercellular continuity between the cells of a filament of Griffithsia pacifica.