Non-disclosed men who have sex with men in UK HIV transmission networks: phylogenetic analysis of surveillance data.

BACKGROUND: Patients who do not disclose their sexuality, including men who do not disclose same-sex behaviour, are difficult to characterise through traditional epidemiological approaches such as interviews. Using a recently developed method to detect large networks of viral sequences from time-resolved trees, we localised non-disclosed men who have sex with men (MSM) in UK transmission networks, gaining crucial insight into the behaviour of this group. METHODS: For this phylogenetic analysis, we obtained HIV pol sequences from the UK HIV Drug Resistance Database (UKRDB), a central repository for resistance tests done as part of routine clinical care throughout the UK. Sequence data are linked to demographic and clinical data held by the UK Collaborative HIV Cohort study and the national HIV/AIDS reporting system database. Initially, we reconstructed maximum likelihood phylogenies from these sequences, then sequences were selected for time-resolved analysis in BEAST if they were clustered with at least one other sequence at a genetic distance of 4·5% or less with support of at least 90%. We used time-resolved phylogenies to create networks by linking together nodes if sequences shared a common ancestor within the previous 5 years. We identified potential non-disclosed MSM (pnMSM), defined as self-reported heterosexual men who clustered only with men. We measured the network position of pnMSM, including betweenness (a measure of connectedness and importance) and assortativity (the propensity for nodes sharing attributes to link). FINDINGS: 14 405 individuals were in the network, including 8452 MSM, 1743 heterosexual women and 1341 heterosexual men. 249 pnMSM were identified (18·6% of all clustered heterosexual men) in the network. pnMSM were more likely to be black African (p<0·0001), less likely to be infected with subtype B (p=0·006), and were slightly older (p=0·002) than the MSM they clustered with. Mean betweenness centrality was lower for pnMSM than for MSM (1·31, 95% CI 0·48-2·15 in pnMSM vs 2·24, 0·98-3·51 in MSM; p=0·002), indicating that pnMSM were in peripheral positions in MSM clusters. Assortativity by risk group was higher than expected (0·037 vs -0·037, p=0·01) signifying that pnMSM were linked to each other. We found that self-reported heterosexual men were more likely to link MSM and heterosexual women than heterosexual women were to link MSM and heterosexual men (Fisher's exact test p=0·0004; OR 2·24) but the number of such transmission chains was small (only 54 in total vs 32 in women). INTERPRETATION: pnMSM are a subgroup distinct from both MSM and from heterosexual men. They are more likely to choose sexual partners who are also pnMSM and might exhibit lower-risk sexual behaviour than MSM (eg, choosing low-risk partners or consistently using condoms). Heterosexual men are the group most likely to be diagnosed with late-stage disease (ie, low CD4 counts) and non-disclosed MSM might put female partners at higher risk than heterosexual men because non-disclosed MSM have male partners. Hence, pnMSM require specific consideration to ensure they are included in public health interventions. FUNDING: National Institutes of Health.

Using nearly full-genome HIV sequence data improves phylogeny reconstruction in a simulated epidemic.

HIV molecular epidemiology studies analyse viral pol gene sequences due to their availability, but whole genome sequencing allows to use other genes. We aimed to determine what gene(s) provide(s) the best approximation to the real phylogeny by analysing a simulated epidemic (created as part of the PANGEA_HIV project) with a known transmission tree. We sub-sampled a simulated dataset of 4662 sequences into different combinations of genes (gag-pol-env, gag-pol, gag, pol, env and partial pol) and sampling depths (100%, 60%, 20% and 5%), generating 100 replicates for each case. We built maximum-likelihood trees for each combination using RAxML (GTR + Γ), and compared their topologies to the corresponding true tree's using CompareTree. The accuracy of the trees was significantly proportional to the length of the sequences used, with the gag-pol-env datasets showing the best performance and gag and partial pol sequences showing the worst. The lowest sampling depths (20% and 5%) greatly reduced the accuracy of tree reconstruction and showed high variability among replicates, especially when using the shortest gene datasets. In conclusion, using longer sequences derived from nearly whole genomes will improve the reliability of phylogenetic reconstruction. With low sample coverage, results can be highly variable, particularly when based on short sequences.

A Direct Comparison of Two Densely Sampled HIV Epidemics: The UK and Switzerland.

Phylogenetic clustering approaches can elucidate HIV transmission dynamics. Comparisons across countries are essential for evaluating public health policies. Here, we used a standardised approach to compare the UK HIV Drug Resistance Database and the Swiss HIV Cohort Study while maintaining data-protection requirements. Clusters were identified in subtype A1, B and C pol phylogenies. We generated degree distributions for each risk group and compared distributions between countries using Kolmogorov-Smirnov (KS) tests, Degree Distribution Quantification and Comparison (DDQC) and bootstrapping. We used logistic regression to predict cluster membership based on country, sampling date, risk group, ethnicity and sex. We analysed >8,000 Swiss and >30,000 UK subtype B sequences. At 4.5% genetic distance, the UK was more clustered and MSM and heterosexual degree distributions differed significantly by the KS test. The KS test is sensitive to variation in network scale, and jackknifing the UK MSM dataset to the size of the Swiss dataset removed the difference. Only heterosexuals varied based on the DDQC, due to UK male heterosexuals who clustered exclusively with MSM. Their removal eliminated this difference. In conclusion, the UK and Swiss HIV epidemics have similar underlying dynamics and observed differences in clustering are mainly due to different population sizes.

Analysis of the history and spread of HIV-1 in Uganda using phylodynamics.

HIV prevalence has decreased in Uganda since the 1990s, but remains substantial within high-risk groups. Here, we reconstruct the history and spread of HIV subtypes A1 and D in Uganda and explore the transmission dynamics in high-risk populations. We analysed HIV pol sequences from female sex workers in Kampala (n = 42), Lake Victoria fisher-folk (n = 46) and a rural clinical cohort (n = 74), together with publicly available sequences from adjacent regions in Uganda (n = 412) and newly generated sequences from samples taken in Kampala in 1986 (n = 12). Of the sequences from the three Ugandan populations, 60 (37.1 %) were classified as subtype D, 54 (33.3 %) as subtype A1, 31 (19.1 %) as A1/D recombinants, six (3.7 %) as subtype C, one (0.6 %) as subtype G and 10 (6.2 %) as other recombinants. Among the A1/D recombinants we identified a new candidate circulating recombinant form. Phylodynamic and phylogeographic analyses using BEAST indicated that the Ugandan epidemics originated in 1960 (1950-1968) for subtype A1 and 1973 (1970-1977) for D, in rural south-western Uganda with subsequent spread to Kampala. They also showed extensive interconnection with adjacent countries. The sequence analysis shows both epidemics grew exponentially during the 1970s-1980s and decreased from 1992, which agrees with HIV prevalence reports in Uganda. Inclusion of sequences from the 1980s indicated the origin of both epidemics was more recent than expected and substantially narrowed the confidence intervals in comparison to previous estimates. We identified three transmission clusters and ten pairs, none of them including patients from different populations, suggesting active transmission within a structured transmission network.

Intercontinental dispersal of HIV-1 subtype B associated with transmission among men who have sex with men in Japan.

UNLABELLED: Transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who have sex with men (MSM) in East Asia have recently been identified. Using the Los Alamos HIV sequence database and the UK HIV drug resistance database, we explored possible links between HIV MSM epidemics in East Asia and the rest of the world by using phylogenetic and molecular clock analyses. We found that JP.MSM.B-1, a subtype B MSM variant that accounts for approximately one-third of the infections among Japanese MSM, was detected worldwide, in the United Kingdom (n=13), mainland China (n=3), the United States, Germany, Canada, and Taiwan (n=1 each). Interestingly, 10 United Kingdom samples plus two from Germany and the United States formed a distinct monophyletic subgroup within JP.MSM.B-1. The estimated divergence times of JP.MSM.B-1 and the latter subgroup were ∼1989 and ∼1999, respectively. These dates suggest that JP.MSM.B-1 was circulating for many years in Japan among MSM before disseminating to other countries, most likely through global MSM networks. A significant number of other Asian MSM HIV lineages were also detected in the UK HIV drug resistance database. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages. Further study of these strains is warranted to elucidate viral migration and the interrelationship of HIV epidemics on a global scale. IMPORTANCE: We previously identified several transmission clusters of HIV-1 subtype B uniquely associated with the epidemic among men who have sex with men (MSM) in East Asia. Using the Los Alamos HIV sequence database and the UK HIV drug resistance database, we explored the possible interplay of HIV MSM epidemics in the different geographic regions and found previously unrecognized interrelationships among the HIV-1 epidemics in East Asia, the United Kingdom, and the rest of the world. Our study provides insight into the regional and global dispersal of Asian MSM HIV lineages and highlights the importance of strengthening HIV monitoring efforts and the need for implementing effective control measures to reduce HIV transmission on a global scale.

Automated analysis of phylogenetic clusters.

BACKGROUND: As sequence data sets used for the investigation of pathogen transmission patterns increase in size, automated tools and standardized methods for cluster analysis have become necessary. We have developed an automated Cluster Picker which identifies monophyletic clades meeting user-input criteria for bootstrap support and maximum genetic distance within large phylogenetic trees. A second tool, the Cluster Matcher, automates the process of linking genetic data to epidemiological or clinical data, and matches clusters between runs of the Cluster Picker. RESULTS: We explore the effect of different bootstrap and genetic distance thresholds on clusters identified in a data set of publicly available HIV sequences, and compare these results to those of a previously published tool for cluster identification. To demonstrate their utility, we then use the Cluster Picker and Cluster Matcher together to investigate how clusters in the data set changed over time. We find that clusters containing sequences from more than one UK location at the first time point (multiple origin) were significantly more likely to grow than those representing only a single location. CONCLUSIONS: The Cluster Picker and Cluster Matcher can rapidly process phylogenetic trees containing tens of thousands of sequences. Together these tools will facilitate comparisons of pathogen transmission dynamics between studies and countries.

Phylogeography of Japanese encephalitis virus: genotype is associated with climate.

The circulation of vector-borne zoonotic viruses is largely determined by the overlap in the geographical distributions of virus-competent vectors and reservoir hosts. What is less clear are the factors influencing the distribution of virus-specific lineages. Japanese encephalitis virus (JEV) is the most important etiologic agent of epidemic encephalitis worldwide, and is primarily maintained between vertebrate reservoir hosts (avian and swine) and culicine mosquitoes. There are five genotypes of JEV: GI-V. In recent years, GI has displaced GIII as the dominant JEV genotype and GV has re-emerged after almost 60 years of undetected virus circulation. JEV is found throughout most of Asia, extending from maritime Siberia in the north to Australia in the south, and as far as Pakistan to the west and Saipan to the east. Transmission of JEV in temperate zones is epidemic with the majority of cases occurring in summer months, while transmission in tropical zones is endemic and occurs year-round at lower rates. To test the hypothesis that viruses circulating in these two geographical zones are genetically distinct, we applied Bayesian phylogeographic, categorical data analysis and phylogeny-trait association test techniques to the largest JEV dataset compiled to date, representing the envelope (E) gene of 487 isolates collected from 12 countries over 75 years. We demonstrated that GIII and the recently emerged GI-b are temperate genotypes likely maintained year-round in northern latitudes, while GI-a and GII are tropical genotypes likely maintained primarily through mosquito-avian and mosquito-swine transmission cycles. This study represents a new paradigm directly linking viral molecular evolution and climate.

Genetic basis of variation in tenofovir drug susceptibility in HIV-1.

OBJECTIVE: To develop an improved model for the genetic basis of reduced susceptibility to tenofovir in vitro. METHODS: A dataset of 532 HIV-1 subtype B reverse transcriptase genotypes for which matched phenotypic susceptibility data were available was assembled, both as a continuous (transformed) dataset and a categorical dataset generated by imposing a cut-off on the basis of earlier studies of in-vivo response of 1.4-fold. Models were generated using stepwise regression, decision tree and random forest approaches on both the continuous and categorical data. Models were compared by mean squared error (continuous models), or by misclassification rates by nested crossvalidation. RESULTS: From the continuous dataset, stepwise linear regression, regression tree and regression forest methods yielded models with MSE of 0.46, 0.48 and 0.42 respectively. Amino acids 215, 65, 41, 67, 184 and 151 in HIV-1 reverse transcriptase were identified in all three models and amino acid 210 in two. The categorical data yielded logistic regression, classification tree and forest models with misclassification rates of 26, 24 and 23%, respectively. Amino acids 215, 65 and 67 appeared in all; 41, 184, 210 and 151 were also included in the classification forest model. CONCLUSION: The random forests approach has yielded a substantial improvement in the available models to describe the genetic basis of reduced susceptibility to tenofovir in vitro. The most important sites in these models are amino acid sites 215, 65, 41, 67, 184, 151 and 210 in HIV-1 reverse transcriptase.

Adaptation to different human populations by HIV-1 revealed by codon-based analyses.

Several codon-based methods are available for detecting adaptive evolution in protein-coding sequences, but to date none specifically identify sites that are selected differentially in two populations, although such comparisons between populations have been historically useful in identifying the action of natural selection. We have developed two fixed effects maximum likelihood methods: one for identifying codon positions showing selection patterns that persist in a population and another for detecting whether selection is operating differentially on individual codons of a gene sampled from two different populations. Applying these methods to two HIV populations infecting genetically distinct human hosts, we have found that few of the positively selected amino acid sites persist in the population; the other changes are detected only at the tips of the phylogenetic tree and appear deleterious in the long term. Additionally, we have identified seven amino acid sites in protease and reverse transcriptase that are selected differentially in the two samples, demonstrating specific population-level adaptation of HIV to human populations.

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-naive 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

Human immunodeficiency virus type 1 clade B superinfection: evidence for differential immune containment of distinct clade B strains.

Sequential infection with different strains of human immunodeficiency virus type 1 (HIV-1) is a rarely identified phenomenon with important implications for immunopathogenesis and vaccine development. Here, we identify an individual whose good initial control of viremia was lost in association with reduced containment of a superinfecting strain. Subject 2030 presented with acute symptoms of HIV-1 infection with high viremia and an incomplete seroconversion as shown by Western blotting. A low set point of viremia (approximately 1,000 HIV-1 copies/ml) was initially established without drug therapy, but a new higher set point (approximately 40,000 HIV-1 copies/ml) manifested about 5 months after infection. Drug susceptibility testing demonstrated a multidrug-resistant virus initially but a fully sensitive virus after 5 months, and an analysis of pol genotypes showed that these were two phylogenetically distinct strains of virus (strains A and B). Replication capacity assays suggested that the outgrowth of strain B was not due to higher fitness conferred by pol, and env sequences indicated that the two strains had the same R5 coreceptor phenotype. Delineation of CD8+-T-lymphocyte responses against HIV-1 showed a striking pattern of decay of the initial cellular immune responses after superinfection, followed by some adaptation of targeting to new epitopes. An examination of targeted sequences suggested that differences in the recognized epitopes contributed to the poor immune containment of strain B. In conclusion, the rapid overgrowth of a superinfecting strain of HIV-1 of the same subtype raises major concerns for effective vaccine development.

Association of antiretroviral resistance genotypes with response to therapy--comparison of three models.

Genotype-based resistance assays are commonly used to aid treatment in HIV-infected individuals failing antiretroviral therapy. The relationship between genotype and antiretroviral therapy comes mostly from in vitro assays of the response to a single drugs although there is a need for a prediction of clinical response to combination therapy. We have compared three different methods of analysing genotype data as a predictor of clinical response in a small clinical cohort of highly antiretroviral-experienced individuals failing therapy. No method performed well beyond 8 weeks into a new therapeutic regimen. A model based on the number of 'primary' mutations was statistically significant, but a multiple regression model, which identified specific mutations explained threefold more variation in response. Optimal prediction in this dataset was given by a model obtained from a classification tree analysis, in which genotype at amino acid sites 135 and 202 were combined with amino acid site 184, which explained over 50% of the deviance in the data and had a classification success of 86%.

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.