Scalable relaxed clock phylogenetic dating

Molecular clock models relate observed genetic diversity to calendar time, enabling estimation of times of common ancestry. Many large datasets of fast-evolving viruses are not well fitted by molecular clock models that assume a constant substitution rate through time, and more flexible relaxed clock models are required for robust inference of rates and dates. Estimation of relaxed molecular clocks using Bayesian Markov chain Monte Carlo is computationally expensive and may not scale well to large datasets. We build on recent advances in maximum likelihood and least-squares phylogenetic and molecular clock dating methods to develop a fast relaxed-clock method based on a Gamma-Poisson mixture model of substitution rates. This method estimates a distinct substitution rate for every lineage in the phylogeny while being scalable to large phylogenies. Unknown lineage sample dates can be estimated as well as unknown root position. We estimate confidence intervals for rates, dates, and tip dates using parametric and non-parametric bootstrap approaches. This method is implemented as an open-source R package, treedater

Viral dynamics during structured treatment interruptions of chronic human immunodeficiency virus type 1 infection

Although antiviral agents which block human immunodeficiency virus (HIV) replication can result in long-term suppression of viral loads to undetectable levels in plasma, long-term therapy fails to eradicate virus, which generally rebounds after a single treatment interruption. Multiple structured treatment interruptions (STIs) have been suggested as a possible strategy that may boost HIV-specific immune responses and control viral replication. We analyze viral dynamics during four consecutive STI cycles in 12 chronically infected patients with a history (>2 years) of viral suppression under highly active antiretroviral therapy. We fitted a simple model of viral rebound to the viral load data from each patient by using a novel statistical approach that allows us to overcome problems of estimating viral dynamics parameters when there are many viral load measurements below the limit of detection. There is an approximate halving of the average viral growth rate between the first and fourth STI cycles, yet the average time between treatment interruption and detection of viral loads in the plasma is approximately the same in the first and fourth interruptions. We hypothesize that reseeding of viral reservoirs during treatment interruptions can account for this discrepancy, although factors such as stochastic effects and the strength of HIV-specific immune responses may also affect the time to viral rebound. We also demonstrate spontaneous drops in viral load in later STIs, which reflect fluctuations in the rates of viral production and/or clearance that may be caused by a complex interaction between virus and target cells and/or immune responses

Phenotypic hypersusceptibility to multiple protease inhibitors and low replicative capacity in patients who are chronically infected with human immunodeficiency virus type 1

Increased susceptibility to the protease inhibitors saquinavir and amprenavir has been observed in human immunodeficiency virus type 1 (HIV-1) with specific mutations in protease (V82T and N88S). Increased susceptibility to ritonavir has also been described in some viruses from antiretroviral agent-naïve patients with primary HIV-1 infection in association with combinations of amino acid changes at polymorphic sites in the protease. Many of the viruses displaying increased susceptibility to protease inhibitors also had low replication capacity. In this retrospective study, we analyze the drug susceptibility phenotype and the replication capacity of virus isolates obtained at the peaks of viremia during five consecutive structured treatment interruptions in 12 chronically HIV-1-infected patients. Ten out of 12 patients had at least one sample with protease inhibitor hypersusceptibility (change ≤0.4-fold) to one or more protease inhibitor. Hypersusceptibility to different protease inhibitors was observed at variable frequency, ranging from 38% to amprenavir to 11% to nelfinavir. Pairwise comparisons between susceptibilities for the protease inhibitors showed a consistent correlation among all pairs. There was also a significant relationship between susceptibility to protease inhibitors and replication capacity in all patients. Replication capacity remained stable over the course of repetitive cycles of structured treatment interruptions. We could find no association between in vitro replication capacity and in vivo plasma viral load doubling time and CD4(+) and CD8(+) T-cell counts at each treatment interruption. Several mutations were associated with hypersusceptibility to each protease inhibitor in a univariate analysis. This study extends the association between hypersusceptibility to protease inhibitors and low replication capacity to virus isolated from chronically infected patients and highlights the complexity of determining the genetic basis of this phenomenon. The potential clinical relevance of protease inhibitor hypersusceptibility and low replication capacity to virologic response to protease inhibitor-based therapies deserves to be investigated further

Identification of hidden population structure in time-scaled phylogenies

Abstract Population structure influences genealogical patterns, however data pertaining to how populations are structured are often unavailable or not directly observable. Inference of population structure is highly important in molecular epidemiology where pathogen phylogenetics is increasingly used to infer transmission patterns and detect outbreaks. Discrepancies between observed and idealised genealogies, such as those generated by the coalescent process, can be quantified, and where significant differences occur, may reveal the action of natural selection, host population structure, or other demographic and epidemiological heterogeneities. We have developed a fast non-parametric statistical test for detection of cryptic population structure in time-scaled phylogenetic trees. The test is based on contrasting estimated phylogenies with the theoretically expected phylodynamic ordering of common ancestors in two clades within a coalescent framework. These statistical tests have also motivated the development of algorithms which can be used to quickly screen a phylogenetic tree for clades which are likely to share a distinct demographic or epidemiological history. Epidemiological applications include identification of outbreaks in vulnerable host populations or rapid expansion of genotypes with a fitness advantage. To demonstrate the utility of these methods for outbreak detection, we applied the new methods to large phylogenies reconstructed from thousands of HIV-1 partial pol sequences. This revealed the presence of clades which had grown rapidly in the recent past, and was significantly concentrated in young men, suggesting recent and rapid transmission in that group. Furthermore, to demonstrate the utility of these methods for the study of antimicrobial resistance, we applied the new methods to a large phylogeny reconstructed from whole genome Neisseria gonorrhoeae sequences. We find that population structure detected using these methods closely overlaps with the appearance and expansion of mutations conferring antimicrobial resistance

Genotype-Specific Evolution of Hepatitis E Virus

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis globally. HEV comprises four genotypes with different geographic distributions and host ranges. We utilize this natural case-control study for investigating the evolution of zoonotic viruses compared to single-host viruses, using 244 near-full-length HEV genomes. Genome-wide estimates of the ratio of nonsynonymous to synonymous evolutionary changes (dN/dS ratio) located a region of overlapping reading frames, which is subject to positive selection in genotypes 3 and 4. The open reading frames (ORFs) involved have functions related to host-pathogen interaction, so genotype-specific evolution of these regions may reflect their fitness. Bayesian inference of evolutionary rates shows that genotypes 3 and 4 have significantly higher rates than genotype 1 across all ORFs. Reconstruction of the phylogenies of zoonotic genotypes demonstrates significant intermingling of isolates between hosts. We speculate that the genotype-specific differences may result from cyclical adaptation to different hosts in genotypes 3 and 4.IMPORTANCE Hepatitis E virus (HEV) is increasingly recognized as a pathogen that affects both the developing and the developed world. While most often clinically mild, HEV can be severe or fatal in certain demographics, such as expectant mothers. Like many other viral pathogens, HEV has been classified into several distinct genotypes. We show that most of the HEV genome is evolutionarily constrained. One locus of positive selection is unusual in that it encodes two distinct protein products. We are the first to detect positive selection in this overlap region. Genotype 1, which infects humans only, appears to be evolving differently from genotypes 3 and 4, which infect multiple species, possibly because genotypes 3 and 4 are unable to achieve the same fitness due to repeated host jumps.This work was funded in part by a Medical Research Council Methodology Research Programme grant (MR/J013862/1) to SDWF, as well as by the Department of Pathology at the Unive rsity of Cambridge. SLKP and HyPhy development was supported by the US National Institutes of Health (grants GM093 939 and GM110749)

Adaptation of avian influenza virus to a swine host

The emergence of pathogenic RNA viruses into new hosts can have dramatic consequences for both livestock and public health. Here we characterize the viral genetic changes that were observed in a previous study which experimentally adapted a field isolate of duck influenza virus to swine respiratory cells. Both pre-existing and $\textit{de novo}$ mutations were selected during this adaptation. We compare the $\textit{in vitro}$ growth dynamics of the adapted virus with those of the original strain as well as all possible reassortants using reverse genetics. This full factorial design showed that viral gene segments are involved in complex epistatic interactions on virus fitness, including negative and sign epistasis. We also identify two point mutations at positions 67 and 113 of the HA2 subunit of the hemagglutinin protein conferring a fast growth phenotype on the naïve avian virus in swine cells. These HA2 mutations enhance the pH dependent, HA-mediated membrane fusion. A global H1 maximum-likelihood phylogenetic analysis, combined with comprehensive ancestry reconstruction and tests for directional selection, confirmed the field relevance of the mutation at position 113 of HA2. Most notably, this mutation was associated with the establishment of the H1 ‘avian-like’ swine influenza lineage, regarded as the most likely to cause the next influenza pandemic in humans. This multidisciplinary approach to study the genetics of viral adaptation provides unique insights on the underlying processes leading to influenza emergence in a new host species, and identifies specific targets for future surveillance and functional studies.This work was supported by a grant from DEFRA and HEFCE under the Veterinary Training and Research Initiative to the Cambridge Infectious Diseases Consortium (VB, LT), the French Ministry of Agriculture and INRA (VB, AT, J-LG), BBSRC grants BB/H014306/1 (LT) and BB/G00479X/1 (LT, JL), and the Medical Research Council Methodology Research Programme grant MR/J013862/1 (SDWF)

The role of venues in structuring HIV, sexually transmitted infections, and risk networks among men who have sex with men.

Background Venues form part of the sampling frame for time-location sampling, an approach often used for HIV surveillance. While sampling location is often regarded as a nuisance factor, venues may play a central role in structuring risk networks. We investigated individual reports of risk behaviors and infections among men who have sex with men (MSM) attending different venues to examine structuring of HIV risk behaviors. However, teasing apart ‘risky people’ from ‘risky places’ is difficult, as individuals cannot be randomized to attend different venues. However, we can emulate this statistically using marginal structural models, which inversely weight individuals according to their estimated probability of attending the venue. Methods We conducted a cross-sectional survey of 609 MSM patrons of 14 bars in San Diego, California, recruited using the Priorities for Local AIDS Control Efforts (PLACE) methodology, which consists of a multi-level identification and assessment of venues for HIV risk through population surveys. Results and Discussion Venues differed by many factors, including participants’ reported age, ethnicity, number of lifetime male partners, past sexually transmitted infection (STI), and HIV status. In multivariable marginal structural models, venues demonstrated structuring of HIV+ status, past STI, and methamphetamine use, independently of individual-level characteristics. Conclusions Studies using time-location sampling should consider venue as an important covariate, and the use of marginal structural models may help to identify risky venues. This may assist in widespread, economically feasible and sustainable targeted surveillance and prevention. A more mechanistic understanding of how 'risky venues' emerge and structure risk is needed

The nonadaptive nature of the H1N1 2009 Swine Flu pandemic contrasts with the adaptive facilitation of transmission to a new host

<p>Abstract</p> <p>Background</p> <p>The emergence of the 2009 H1N1 Influenza pandemic followed a multiple reassortment event from viruses originally circulating in swines and humans, but the adaptive nature of this emergence is poorly understood.</p> <p>Results</p> <p>Here we base our analysis on 1180 complete genomes of H1N1 viruses sampled in North America between 2000 and 2010 in swine and human hosts. We show that while transmission to a human host might require an adaptive phase in the HA and NA antigens, the emergence of the 2009 pandemic was essentially nonadaptive. A more detailed analysis of the NA protein shows that the 2009 pandemic sequence is characterized by novel epitopes and by a particular substitution in loop 150, which is responsible for a nonadaptive structural change tightly associated with the emergence of the pandemic.</p> <p>Conclusions</p> <p>Because this substitution was not present in the 1918 H1N1 pandemic virus, we posit that the emergence of pandemics is due to epistatic interactions between sites distributed over different segments. Altogether, our results are consistent with population dynamics models that highlight the epistatic and nonadaptive rise of novel epitopes in viral populations, followed by their demise when the resulting virus is too virulent.</p