General Time Reversible Model Research Articles

A Molecular Temporal Evolutionary Framework of Land Plants and the Age of Angiosperms

A series of Bayesian molecular clock analyses with varying model combinations, parameters, and tree topologies were conducted to establish a temporal evolutionary framework of land plants and to estimate the age of angiosperms. The data consisted of five organellar and nuclear genes, 633 taxa, and 23 fossil calibration points (FCPs). The analyses were evaluated using an internal search thoroughness measure, effective sample sizes, and two external criteria: the consensus land plant phylogeny and a newly developed method that used the Hennigian reciprocal illumination principle to compare molecular age estimates of 21 nodes representing major land plant clades and the root split in green plants with those of their fossils in an a posteriori fashion. The results show that an analysis using a general time reversible (GTR) DNA substitution model, log normal branch-rate distribution, and Yule tree prior represented the best model combination, with the Birth-Death tree prior being equally optimal. This analysis estimated the age of the land plant crown group as 496.95 million years (MY), with a 95% highest posterior density (HPD) of 465.66–531.43 MY, and the age of the angiosperm crown group as 219.93 MY, with a 95% HPD of 184.09–256.82 MY. The former agrees with the fossil evidence, but the latter is significantly older than the fossil ages. A thorough examination of fossil evidence of angiosperms, as well as their evolutionary history in light of recent knowledge of morphology, physiology, and atmospheric CO2 concentration in the Mesozoic era, suggests that angiosperm radiation might have been delayed after their origin and that the current fossil record probably reflects only the products of that radiation, not the origin of angiosperms. Overall, the study shows that both molecular clock analyses and fossil studies, while agreeing on some aspects but disagreeing on others, can help develop a complete understanding of the temporal evolutionary history of land plants.

Epitope prediction of malarial MSP-1 protein of plasmodium sp. using in silico method

Due to its pivotal function in parasite adherence to the RBC membrane during erythrocyte invasion, Msp1 represents a potential vaccine candidate. Three or four fragments of MSP1 are produced during the proteolytic maturation process, and these fragments stay together as a complex on the cell surface. Msp1 fragments have been investigated as potential vaccine candidate, and malaria antibody immunoassays have made use of the fragmented polypeptide, which exhibits a considerable degree of intra-species conservation. Because of its polymorphism, Msp1 is also useful in strain differentiation and carries a crucial genetic marker. To comprehend genetic variation among species and find epitopes for the development of a disease vaccine, Msp1 needs to be investigated. The preliminary investigation on the evolutionary history using the Maximum Likelihood method and the General Time Reversible model (GTR) models were used. The phylogenetic relationship of the MSP1 gene was inferred and it represents the level of similarity between the MSP1 gene of Plasmodium species from various locations in South-East Asia. The top 30 MHC class II epitopic regions were predicted using Bepipred server with epitope mean score above 0.05.

GTRpmix: A Linked General Time-Reversible Model for Profile Mixture Models.

Profile mixture models capture distinct biochemical constraints on the amino acid substitution process at different sites in proteins. These models feature a mixture of time-reversible models with a common matrix of exchangeabilities and distinct sets of equilibrium amino acid frequencies known as profiles. Combining the exchangeability matrix with each profile generates the matrix of instantaneous rates of amino acid exchange for that profile. Currently, empirically estimated exchangeability matrices (e.g. the LG matrix) are widely used for phylogenetic inference under profile mixture models. However, these were estimated using a single profile and are unlikely optimal for profile mixture models. Here, we describe the GTRpmix model that allows maximum likelihood estimation of a common exchangeability matrix under any profile mixture model. We show that exchangeability matrices estimated under profile mixture models differ from the LG matrix, dramatically improving model fit and topological estimation accuracy for empirical test cases. Because the GTRpmix model is computationally expensive, we provide two exchangeability matrices estimated from large concatenated phylogenomic-supermatrices to be used for phylogenetic analyses. One, called Eukaryotic Linked Mixture (ELM), is designed for phylogenetic analysis of proteins encoded by nuclear genomes of eukaryotes, and the other, Eukaryotic and Archaeal Linked mixture (EAL), for reconstructing relationships between eukaryotes and Archaea. These matrices, combined with profile mixture models, fit data better and have improved topology estimation relative to the LG matrix combined with the same mixture models. Starting with version 2.3.1, IQ-TREE2 allows users to estimate linked exchangeabilities (i.e. amino acid exchange rates) under profile mixture models.

First report of Tomato interveinal chlorosis virus infecting Rhynchosia minima plants in Brazil.

Tomato interveinal chlorosis virus (ToICV; Begomovirus solanumintervenae, genus Begomovirus, family Geminiviridae) has been described infecting tomato (Solanum lycopersicum) and Macroptilium lathyroides in Northeastern (NE) Brazil for more than a decade (Albuquerque et al., 2012; Silva et al., 2012). During a survey in 2020, plants of the leguminous weed Rhynchosia minima exhibiting virus-like symptoms such as mosaic and interveinal chlorosis were observed in the state of Alagoas, NE Brazil. Symptomatic leaf samples of R. minima were randomly collected (n=15; supplementary figure 1). Total DNA from each sample was used as a template for PCR amplification of partial begomoviral DNA-A sequences using the degenerate primer pair PAL1v1978 and PAR1c496, universal for geminiviruses (Rojas et al., 1993). Amplicons of ~1.2 kbp were observed from 12 samples, although this should not be considered as incidence since only symptomatic plants were collected. To identify the begomovirus associated with R. minima, viral genomes were amplified from PCR-positive samples using rolling circle amplification (RCA) (Inoue-Nagata et al., 2004). The RCA products were digested with HindIII, cloned into the pBluescript II KS+ plasmid vector and bidirectionally Sanger-sequenced (Macrogen Inc., Seoul). BLASTn searches indicated that the clones (n=4) reported here corresponded to a begomovirus DNA-A component, and pairwise comparisons showed that they shared the highest identity with ToICV, at 92.4-94.7% nucleotide sequence identity. Based on the species demarcation criteria of ≥91% nucleotide identity for the genus Begomovirus (Brown et al., 2015), the begomoviruses obtained from R. minima are new isolates of ToICV. The new DNA-A sequences of 2,619-2,623 nt in length were deposited in GenBank under accession numbers PP639092 to PP639095. Multiple nucleotide sequence alignments were prepared using the MUSCLE algorithm implemented in MEGA v.11 (Kumar et al., 2018), and a maximum likelihood (ML) tree was reconstructed in RaxML-NG (Kozlov et al., 2019), assuming a general time reversible (GTR) nucleotide substitution model with a gamma (G) model of rate heterogeneity and 1,000 bootstrap replicates. The DNA-A-based tree showed that the ToICV sequences clustered into a monophyletic group, additionally supporting these isolates as members of the species Begomovirus solanumintervenae. At least two independent interspecies recombination events were predicted among the ToICV isolates, with breakpoints located in the Rep-encoding region and ToICV (GenBank Accession JF803253), tomato mottle leaf curl virus (JF803248) and soybean blistering mosaic virus (MN486865) detected as putative parents. To the best of our knowledge, this is the first report of ToICV infecting R. minima worldwide, expanding the host range of this begomovirus. Non-cultivated plants such as R. minima play a crucial role as reservoirs and sources of inoculum for begomoviruses (Paz-Carrasco et al., 2014), reinforcing their relevance to socioeconomically important crops.

Genomic Landscape of Epstein-Barr Virus in Endemic Burkitt Lymphoma

Introduction: Endemic Burkitt lymphoma (eBL) is a fast-growing B-cell malignancy strongly linked to Epstein-Barr virus (EBV) infection, a member of the herpes virus family. The virus is widespread, infecting 95% of the global population, and has also been linked to several other cancers, including nasopharyngeal carcinoma (NPC), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), natural killer T-cell lymphoma (NKTCL), gastric carcinoma (GC) and leiomyosarcomas. The distribution of these cancers is inconsistent in different regions, raising the hypothesis of whether different strains are linked to specific cancers. Using high throughput sequencing, we obtained EBV whole genome sequences from the plasma of children with eBL diagnosis from a large cohort study in East Africa. Methods: Twenty-one EBV-positive patients (Sixteen BL, one diffuse large B-cell lymphoma, one lymphoblastoid lymphoma, one clear cell sarcoma, one small blue cell tumour and one alveolar rhabdomyosarcoma) were recruited from St Mary's Hospital Lacor, in Uganda, between 2020 and 2023. Venous blood samples were collected in appropriate DNA tubes, and ~ 5 ml of plasma was used for cfDNA extraction using QIAamp circulating Nucleic acid kit (Qiagen, USA). The cfDNA libraries were prepared using the Thruplex Tag- Seq kit (Takara Bio Inc. Japan), target capture and enrichment was performed using an EBV-custom panel (IDT Inc. USA) before sequencing. The Illumina fastq files were processed using an in-house pipeline, and paired-end reads were aligned to a bespoke hybrid EBV reference genome (NC_007605.1 and EBNA genes from NC_009334) using BWA with an alt-ware method. The average sequencing depth and EBV genome coverage were 5164 and 97.8%, respectively. Following the GATK best practice workflows (version 4.0), 15,318 variants were called across all samples using Varscan and Vardict after base and variant recalibration. Functional annotation was performed using the SNPEff package (v5.1d) according to the reference genome (NC_007605.1, NCBI annotation, Nov 2013). For the phylogenetic analysis, 322 public EBV genomes were downloaded from the GenBank and aligned with 21 EBV genomes from the current study using a multiple sequence alignment program (MAFFT v7.52), and the maximum likelihood of the phylogenetic relationship was inferred using RAxML (v8.0), assuming a general time reversible (GTR) model. The inferred phylogeny was subsequently rooted using the evolutionary placement (EPA) algorithm from RAxML using a Macacine herpesvirus 4 genome sequence (NC_006146) as an outer group. The tree was annotated using FigTree software (v1.4.4). The study was approved by the Oxford Tropical Research Ethics Committee (OxTREC Ref: 15-19), National Institute of Medical Research (NIMR/HQ/R.8a/Vol.IX/3408), Uganda National Council of Science and Technology (Ref: HS529ES), and St Mary's Hospital Lacor Institutional Research Ethics Committee. Participants provided written informed consent and assent for minors. Results: We examined the genomes of 21 EBV samples, including 18 type-1 and 3 type-2 strains. Our analysis revealed an average of 729 mutations per sample, comprised of 707 SNVs and 26 indels (<50bp) per sample (Figure 1). Within 16 eBL tumours, we observed the 30-bp deletion in LMP-1 in 43.8% (7/16) cases. This deletion has been previously reported in NPC in Southeast Asia and is associated with high oncogenic potential and weak immunogenicity. We also identified a novel frameshift variant (15-bp deletion) in LMP-1 in 66.7% (14/21) of tumour samples. The LMP-1 gene encodes a 356-amino acid polypeptide with a cytoplasmic amino terminus, a transmembrane domain, and a cytoplasmic carboxy terminus. The C-terminus interacts with cellular proteins, activating the transcriptional nuclear factor-kB (NF-kB) pathway associated with antiapoptotic cell signalling. Additionally, we observed a high degree of polymorphism in EBNA-1, EBNA3s, LMP-2A, BALF1, and BOLF-1 viral genes. Our phylogenetic analysis revealed distinct clustering of African EBV genomes compared to Asian strains and NPC versus eBL (Figure 2). Conclusions: Overall, our study provides the first-ever comprehensive mutational profile of EBV in eBL clinical samples, offering valuable insights into the somatic events of EBV in tumour biology.

Embeddability of centrosymmetric matrices capturing the double-helix structure in natural and synthetic DNA

In this paper, we discuss the embedding problem for centrosymmetric matrices, which are higher order generalizations of the matrices occurring in strand symmetric models. These models capture the substitution symmetries arising from the double helix structure of the DNA. Deciding whether a transition matrix is embeddable or not enables us to know if the observed substitution probabilities are consistent with a homogeneous continuous time substitution model, such as the Kimura models, the Jukes-Cantor model or the general time-reversible model. On the other hand, the generalization to higher order matrices is motivated by the setting of synthetic biology, which works with different sizes of genetic alphabets.

First Report of Epicoccum sorghinum Causing leaf spot on Bothriochloa ischaemum in Malaysia.

Bothriochloa ischaemum (family Poaceae) is a perennial weed that can be found in borders of agricultural fields, pastures and roadsides in Malaysia. B. ischaemum is an important phytoremediation species in copper tailings dams (Jia et al. 2020). In December 2021, chlorotic spots with brown halos were observed on leaf samples of B. ischaemum with an incidence of approximately 80% in Penampang, Sabah province (5°56'50.4"N, 116°04'32.8"E). On older leaves, the spots coalesced into larger chlorotic spots. Small pieces (5 x 5 mm) of infected leaves collected from three plants were excised, and then surface sterilized according to Khoo et al. (2022). The fungus was isolated (one isolate was obtained) and cultured on potato dextrose agar (PDA) at 25°C. After 3 days, the colony had cottony aerial mycelia with light purple concentric rings appearing on the underside of the colony. Chlamydospores were produced, either unicellular or multicellular. Conidia were unicellular, hyaline, oval, and were 3.7 to 5.1 x 1.8 to 2.6 μm (n=20). Pycnidia were spheroid, and were 66.4 to 115.3 x 43.1 to 87.4 μm (n=20). Genomic DNA was extracted from fresh mycelia of the fungus based on the extraction method described by Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region and large subunit (LSU) of rDNA, and actin (ACT), tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, T10/Bt2b and RPB2-5F2/RPB2-7cR primers, respectively (O'Donnell and Cigelnik, 1997; Liu et al. 1999; Sung et al. 2007; Chen et al. 2021). The PCR products were sequenced at Apical Scientific Sdn. Bhd.. Sequences were deposited in GenBank as OM453926 (ITS), OM453925 (LSU), OM451236 (ACT), OM451237 (TUB) and OM863567 (RPB2). Sequences of our isolate had 100% homology to ITS of isolate UMS (OK626271) (507/507 bp), LSU of isolate UMS (OM238129) (1328/1328 bp), ACT of isolate CZ01 (MN956831) (275/275 bp), TUB of isolate BJ-F1 (MF987525) (556/556 bp) and RPB2 of isolate HYCX2 (MK836295) (596/596 bp) sequences. Phylogenetic analysis was performed using the maximum likelihood method based on the general time reversible model with a gamma distribution and invariant sites (GTR + G + I) generated from the combined ITS, TUB, LSU and RPB2 sequences, indicating that the isolates formed a supported clade to the related Epicoccum sorghinum type sequences. Morphological and molecular characterization matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were performed by spray inoculation (106 spores/ml) on the leaves of three healthy B. ischaemum plants, using isolate BPL01, while sterilized water was sprayed on three additional B. ischaemum which served as the control. Symptoms similar to those occurred after 6 days post inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported previously on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021) in China. To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. ischaemum in Malaysia. Our findings expand the geographic range and host range of E. sorghinum in Malaysia. B. ischaemum which is a weed in agricultural fields is a host of the pathogen and therefore could be a potential threat to Brassica parachinensis, Camellia sinensis, Oryza sativa and Zea mays in Malaysia. Weed management could be an effective way to eliminate inoculum sources of E. sorghinum.

Inference from Open-Source Sequence Data on the Genetic Epidemiology of COVID-19 Infection in Africa

Between late December 2019 when it was first reported, to early September 2020, severe acute respiratory syndrome coronavirus-2 (SARSCoV- 2), the causative agent of coronavirus disease-2019 (COVID-19), reportedly infected over 10 million people globally. In Africa, more than 300,000 infections occurred within the period, from which several genetic sequences of the virus were generated. During disease outbreak, phylogenetic reconstruction of genomic data can assist in making epidemiological inferences about time of pathogen introduction, epidemic growth rate and temporal-spatial spread of the infection. In this work, we studied the genetic epidemiology of COVID-19 in Africa. Genetic sequence data of SARS-CoV-2 and metadata from African countries were obtained from publicly accessible sequence database hosted by the GISAID initiative. Whole genome sequences were subjected to multiple sequence alignment and the aligned sequences used to construct a Maximum Likelihood phylogenetic tree based on the general time reversible model. Of the 227 genetic sequences obtained for 9 African countries (DRC=133, Senegal=23, South Africa=20, Ghana=15, Tunisia=6, Algeria=3, Gambia=3, Egypt=2 and Nigeria=2), 220 were whole genome sequences while 7 were partial genome sequences of the surface glycoprotein S. Majority of the viruses (58.1%) belonged to the G clade of GISAID classification. Phylogenetic analysis confirmed multiple introductions of the virus to the continent from multiple external sources prior to local adaptation and spread. The very close alignment of three viruses - Ghana/1659_S14/2020|EPI_ISL_422405, DRC/KN0054/2020|EPI_ ISL_417437, and South_Africa/R05475/2020|EPI_ISL_435059 – to the reference Wuhan strain on the time tree, suggests possible introduction and circulation of the virus into the continent much earlier than when the first case in the continent was announced in February 15 2020. In conclusion, this study provided evidence to support multiple introductions of SARSCoV- 2 into Africa, and further suggests that the virus may have already been circulating in the continent prior to official reporting of the first case. Also, there is strong impression to infer likely genetic adaptation of the virus in the continent that may account for the close clustering of isolates from different countries of the continent.

Persistent Transmission of HCV among Men Who Have Sex with Men despite Widespread Screening and Treatment with Direct-Acting Antivirals.

Background: In the Netherlands, unrestricted access to direct-acting antivirals (DAAs) halved the incidence of acute hepatitis C virus (HCV) infections among HIV-infected men who have sex with men (MSM). To develop strategies that can further reduce the spread of HCV, it is important to understand the transmission dynamics of HCV. We used phylogenetic analysis of a dense sample of MSM to provide insight into the impact of unrestricted access to DAAs on HCV transmission in the Netherlands and in Belgium. Methods: We included 89 MSM that were recently infected with HCV genotype 1a in ten Dutch and one Belgian HIV treatment centers. Sequences were generated using next gene sequencing and Sanger sequencing. Maximum likelihood phylogenetic analysis (general time reversible model) was performed on concatenated NS5A and NS5B sequences and a reference set of 389 highly similar control sequences selected from GenBank. A cluster was based on a minimum bootstrap support of 90% and a 3% genetic distance threshold. Results: We found that 78 (88%) of individuals were part of seven major clusters. All clusters included individuals from across the study region, however, different cities were part of different clusters. In three clusters, HIV-negative MSM clustered with sequences from HIV-positive MSM. All clusters that were observed before the introduction of DAAs persisted after unrestricted access to DAAs became available. Conclusion: Recently acquired HCV infections among MSM in the Netherlands and Belgium are strongly clustered and therefore highly suitable for targeted prevention strategies, such as contact tracing and partner notification. Importantly, despite an HCV incidence reduction after high DAA uptake and continuously monitoring, HCV transmission persisted in the same clusters.

Molecular revision of Rhagiini sensu lato (Coleoptera, Cerambycidae): Paraphyly, intricate evolution and novel taxonomy

Due to classical taxonomy, the subfamily Lepturinae is divided into two large tribes Rhagiini s.l. and Lepturini s.l. However, this division is clearly artificial and does not correspond to the evolutionary relationships between the groups of genera with different morphologies that are part of these two tribes. However, there is a consensus among researchers supporting the monophyly of Lepturini s.l. while there is no such consensus for Rhagiini s.l. Over the past three decades, there have been several attempts to revise the tribe Rhagiini s.l. and divide it into smaller tribes. These attempts were somewhat successful based on larval and adult morphology. In general, eight tribes are distinguished within Rhagiini s.l. These include Teledapini, Xylosteini, Encyclopini, Oxymirini, Enoploderini, Rhamnusiini, Rhagiini and Sachalinobiini. However, the new system of Rhagiini s.l. is not always unambiguously accepted by different researchers, which causes discussions among experts. First of all, this is due to the fact that this system is only partially natural and far from fully reflects the phylogeny not only of Rhagiini s.l., but also of entire subfamily Lepturinae. In addition to the classical morphologic methods of studies, the use of the modern molecular phylogenetic methods opens up wide prospects for solving this puzzle. However, there have been very few such studies. Moreover, all of them were based on the use of only one gene. In this study, I used a general time-reversible (GTR) model of sequence evolution based on three mitochondrial (12S rRNA, 16S rRNA, COI) and two nuclear (18S rRNA, 28S rRNA) genes. My analysis yielded a well-resolved phylogenetic maximum likelihood tree, which clearly demonstrates the paraphyly of Rhagiini s.l. It consists of at least three clades representing different and distantly related evolutionary branches: 1) PaRh, 2) OSaXyR-SEP, 3) GAC. The extant Rhagiini s.l. are mostly heterogeneous relict groups with an intricate evolutionary and phylogeographic history. Most of these groups are represented by one or very few species, often isolated geographically on different continents. In particular, numerous cases of parallel and convergent evolution and homoplasy, a chimeric combination of plesiomorphic and apomorphic morphological characters, were found in all clades. Therefore, the evolutionary "tree" of Rhagiini s.l. is more like a "bush" with numerous relict branches. Finally, I proposed a new taxonomic model of Rhagiini s.l., which is the most consistent with their natural phylogeny, with new developments in nomenclature.

Using a GTR+Γ substitution model for dating sequence divergence when stationarity and time-reversibility assumptions are violated.

As the number and diversity of species and genes grow in contemporary datasets, two common assumptions made in all molecular dating methods, namely the time-reversibility and stationarity of the substitution process, become untenable. No software tools for molecular dating allow researchers to relax these two assumptions in their data analyses. Frequently the same General Time Reversible (GTR) model across lineages along with a gamma (+Γ) distributed rates across sites is used in relaxed clock analyses, which assumes time-reversibility and stationarity of the substitution process. Many reports have quantified the impact of violations of these underlying assumptions on molecular phylogeny, but none have systematically analyzed their impact on divergence time estimates. We quantified the bias on time estimates that resulted from using the GTR + Γ model for the analysis of computer-simulated nucleotide sequence alignments that were evolved with non-stationary (NS) and non-reversible (NR) substitution models. We tested Bayesian and RelTime approaches that do not require a molecular clock for estimating divergence times. Divergence times obtained using a GTR + Γ model differed only slightly (∼3% on average) from the expected times for NR datasets, but the difference was larger for NS datasets (∼10% on average). The use of only a few calibrations reduced these biases considerably (∼5%). Confidence and credibility intervals from GTR + Γ analysis usually contained correct times. Therefore, the bias introduced by the use of the GTR + Γ model to analyze datasets, in which the time-reversibility and stationarity assumptions are violated, is likely not large and can be reduced by applying multiple calibrations. All datasets are deposited in Figshare: https://doi.org/10.6084/m9.figshare.12594638.

Phyloevolutionary analysis of SARS-CoV-2 in Nigeria.

Phyloepidemiological approaches have provided specific insight into understanding the emergence and evolution of infection. Knowledge on the outbreak and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Nigeria would assist in provision of preventive measures to reduce transmission among populations at risk. This study aimed to investigate the evolution of SARS-CoV-2 in Nigeria. A total of 39 complete genomes of SARS-CoV-2 were retrieved from the GISAID EpiFlu™ database on 29 March 2020 to investigate its evolution in Nigeria. Sequences were selected based on the travel history of the individual and the collection date. Other sequences were not selected because they were short, contained artefacts, were not from an original source or had insufficient information. Evolutionary history was inferred using the maximum likelihood method based on the general time reversible model. A phylogenetic tree was constructed to determine the common ancestor of each strain. The phylogenetic analysis showed that the strain in Nigeria clustered in a monophyletic clade with a Wuhan sublineage. Nucleotide alignment also showed a 100% similarity indicating a common origin of evolution. Comparative analysis showed 27 972 (93.6%) identical sites and 97.6% pairwise identity with the consensus. The study evidently showed the entire outbreak of SARS-CoV-2 infection in Nigeria stemmed from a single introduction sharing consensus similarity with the reference SARS-CoV-2 human genome from Wuhan. Preventive measures that can limit the spread of the infection among populations at risk should be implemented.

Evaluating probabilistic programming and fast variational Bayesian inference in phylogenetics.

Recent advances in statistical machine learning techniques have led to the creation of probabilistic programming frameworks. These frameworks enable probabilistic models to be rapidly prototyped and fit to data using scalable approximation methods such as variational inference. In this work, we explore the use of the Stan language for probabilistic programming in application to phylogenetic models. We show that many commonly used phylogenetic models including the general time reversible substitution model, rate heterogeneity among sites, and a range of coalescent models can be implemented using a probabilistic programming language. The posterior probability distributions obtained via the black box variational inference engine in Stan were compared to those obtained with reference implementations of Markov chain Monte Carlo (MCMC) for phylogenetic inference. We find that black box variational inference in Stan is less accurate than MCMC methods for phylogenetic models, but requires far less compute time. Finally, we evaluate a custom implementation of mean-field variational inference on the Jukes–Cantor substitution model and show that a specialized implementation of variational inference can be two orders of magnitude faster and more accurate than a general purpose probabilistic implementation.

A51 Genetic variability of small ruminant lentiviruses in sheep and goats from single-species flocks from Poland

Previous phylogenetic analyses of small ruminant lentivirus (SRLV) sequences found in Poland revealed the circulation of subtype A1 in both sheep and goats, subtypes B1 in goats, and subtypes B2, A12, and A13 in sheep only. This study aimed to analyze the genetic nature of SRLV circulating in sheep and goats from single-species flocks. In order to analyze the degree of genetic variability, the fragments of gag and env genes of 24 SRLV strains were amplified by PCR, cloned into plasmid vectors, sequenced, and consensus sequences were aligned to each other and to reference sequences available from GenBank. Phylogenetic analysis was performed using the Geneious tree-builder tool, and phylogenetic trees were constructed using Mr Bayes (using the general time reversible substitution model) within Geneious Pro 5.3. Pairwise genetic distances were calculated in MEGA 6. Phylogenetic analysis revealed that the strains were highly heterogeneous and represented ovine strains belonging to subtypes A12 and B2 and caprine strains grouped in subtypes B1, B2, A1, and A12. In addition, two novel subtypes, A16 and A17, were found in goats. The mean pairwise genetic distances of gag and env sequences of both clusters were above 15 per cent nucleotide divergence when compared to all other subtypes within group A, which is a criterion required to distinguish a new subtype. Additionally, the existence of two separated clusters was confirmed by high bootstrap values. Co-infections with strains belonging to different subtypes within A and B groups were detected in one sheep and four goats originating from four flocks. Since the co-infection with more than one lentivirus genotype offers an opportunity for viral recombination, the possible recombination events were tested based on RDP analysis. For all co-infected animals, no evidence of recombination was found within the gag gene; however, env sequences showed some recombination patterns in three samples. In conclusion, we have demonstrated extended genetic variability of SRLV in sheep and goats from Poland with the existence of co-infection and recombination events.

A66 Tracing the evolutionary history of an emerging Salmonella 4,[5],12:i:- clone in the United States

Salmonellosis is one of the leading causes of foodborne disease worldwide, with an estimated one million cases a year in the United States. Salmonella 4,[5],12: i:-, a monophasic variant of Salmonella typhimurium, is an emerging serovar that has been associated with multiple foodborne outbreaks throughout the world, mostly attributed to pig and pig products. Recently, we have demonstrated that two distinct groups of Salmonella 4,[5],12:i:- circulate in the USA and Europe, with the majority of isolates recovered during recent years belonging to an emerging multidrug-resistant clade (Elnekave et al. 2018). We applied Bayesian phylodynamic reconstruction to uncover the evolutionary history of this clade. We used a dataset of whole-genome sequences of 1446 4,[5],12:i:- isolates from different sources (livestock, human, food products, and others) from the USA (n = 752) and Europe (n = 694), collected between 2008 and 2017 and belonging to the Multilocus Subtype 34, which was predominant in the emerging clade (Elnekave et al. 2018). A subset (n = 110) of Salmonella 4,[5],12:i: isolates was then randomly selected after stratifying by location and year of isolation in order to achieve balanced sampling. Evidence of temporal signal was confirmed by looking at root-to-tip divergences using TempEst. Evolutionary hypotheses using strict and relaxed-clock models were tested using BEAST for a variety of demographic models and assuming a general time reversible substitution model. Model selection was performed by estimating Bayes Factors using path sampling and stepping-stone sampling. The selected model was then used for applying discrete trait models comparing different scenarios of transmission between locations (i.e. bidirectional symmetric/asymmetric or unidirectional). Our preliminary phylodynamic inference results indicate that the origin of this subtype was in Europe and dates back to 1990 (HPD 95%: 1984–2001). We report an exponential growth rate of 0.362 per year, which corresponds to a doubling time of 1.43 years. Our results suggest that this subtype was introduced to the US in the year 2000 (HPD 95%: 1994–2006). Phylodynamic analysis suggests that the recent increase in isolation of Salmonella 4,[5],12:i:- from different sources in the USA may be due to the exponential expansion of an emerging clone which originated in Europe and then expanded to the USA. The emergence and expansion of this serovar is of great public health importance due to the high prevalence of multidrug resistance traits found in USA isolates from this group and especially due to the presence of plasmid-mediated resistance genes for quinolones and extended spectrum cephalosporins, key antimicrobials used for the treatment of invasive Salmonella infections.

A2 Phylogenetic investigation of transmitted HIV-1 drug resistance mutations in Denmark, 2009–17

Transmission of HIV-1 resistance mutations among therapy-naïve patients impairs the efficiency of antiretroviral therapy (ART). Therefore, genotypic resistance testing of patients is recommended at baseline, as this both allows for the selection of the correct ART regimen and for surveillance of transmitted drug resistance mutations (TDRM) among therapy naive HIV-1 patients. In Denmark, the occurrence of TDRM in newly diagnosed and therapy naïve HIV-1 patients is monitored through the SERO project. Here, we investigated if the prevalence of TDRM differed between patients within and outside of phylogenetically identified transmission clusters. Samples from 1,227 newly diagnosed HIV-1 patients were sent along with epidemiological information to the Virological Surveillance and Research group at Statens Serum Institut. HIV-1 RNA extraction, RT-PCR and Sanger sequencing of the pol gene was performed using an in-house assay. The sequences were analyzed using BioNumerics v. 6.6 and manually checked for the presence of mixed mutations and analyzed for mutations using the HIVDB 8.4 algorithm implemented at the Stanford database. Sequence alignments were performed in Mafft, and phylogenetic analysis was performed using Mega 6.0 using the Maximum likelihood general time reversible model with 100 bootstrap replicates. Clusters were identified with ClusterPicker at default settings (cluster support = 90%, genetic distance 4.5%). Active clusters contained newly diagnosed patients from the 2015 to 2017 period. HIV-1 sequences from 588 patients belonged to one of 154 clusters, and sequences from 639 patients did not belong to a cluster. Patients in clusters were significantly more likely to be men who have sex with men and subtype B and significantly less likely to be late presenters (Fisher’s test P < 0.05). The TDRM prevalence was significantly higher for patients outside of clusters than within clusters, 16.6 per cent versus 12.1 per cent, respectively (Fisher’s test P < 0.05); however, no significant differences were found in the TDRM prevalence between the 75 active and 79 inactive clusters, nor between small (<3 patients) and large (≥3 patients) clusters. E138A, V179D, and K103N were the three most prevalent TDRMs for both patient groups, whereas M41L differed between them. In Denmark, the TDRM prevalence is lower within clusters than outside, indicating that TDRM cases are either imported and/or belong to yet unidentified clusters.

Does the choice of nucleotide substitution models matter topologically?

BackgroundIn the context of a master level programming practical at the computer science department of the Karlsruhe Institute of Technology, we developed and make available an open-source code for testing all 203 possible nucleotide substitution models in the Maximum Likelihood (ML) setting under the common Akaike, corrected Akaike, and Bayesian information criteria. We address the question if model selection matters topologically, that is, if conducting ML inferences under the optimal, instead of a standard General Time Reversible model, yields different tree topologies. We also assess, to which degree models selected and trees inferred under the three standard criteria (AIC, AICc, BIC) differ. Finally, we assess if the definition of the sample size (#sites versus #sites × #taxa) yields different models and, as a consequence, different tree topologies.ResultsWe find that, all three factors (by order of impact: nucleotide model selection, information criterion used, sample size definition) can yield topologically substantially different final tree topologies (topological difference exceeding 10 %) for approximately 5 % of the tree inferences conducted on the 39 empirical datasets used in our study.ConclusionsWe find that, using the best-fit nucleotide substitution model may change the final ML tree topology compared to an inference under a default GTR model. The effect is less pronounced when comparing distinct information criteria. Nonetheless, in some cases we did obtain substantial topological differences.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-0985-x) contains supplementary material, which is available to authorized users.

Morphological and molecular characterization of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic)

Morphological and molecular characterization of the cyanobiont-bearing dinoflagellate Sinophysis canaliculata from the Canary Islands (eastern central Atlantic)

Phylogenetic Analysis of HIV-1 CRF65_CPX Reveals Yunnan Province Is Still a Source Contributing to the Spread of HIV-1 in China

Phylogenetic Analysis of HIV-1 CRF65_CPX Reveals Yunnan Province Is Still a Source Contributing to the Spread of HIV-1 in China

The Influence of Hepatitis C Virus Genetic Region on Phylogenetic Clustering Analysis

Sequencing is important for understanding the molecular epidemiology and viral evolution of hepatitis C virus (HCV) infection. To date, there is little standardisation among sequencing protocols, in-part due to the high genetic diversity that is observed within HCV. This study aimed to develop a novel, practical sequencing protocol that covered both conserved and variable regions of the viral genome and assess the influence of each subregion, sequence concatenation and unrelated reference sequences on phylogenetic clustering analysis. The Core to the hypervariable region 1 (HVR1) of envelope-2 (E2) and non-structural-5B (NS5B) regions of the HCV genome were amplified and sequenced from participants from the Australian Trial in Acute Hepatitis C (ATAHC), a prospective study of the natural history and treatment of recent HCV infection. Phylogenetic trees were constructed using a general time-reversible substitution model and sensitivity analyses were completed for every subregion. Pairwise distance, genetic distance and bootstrap support were computed to assess the impact of HCV region on clustering results as measured by the identification and percentage of participants falling within all clusters, cluster size, average patristic distance, and bootstrap value. The Robinson-Foulds metrics was also used to compare phylogenetic trees among the different HCV regions. Our results demonstrated that the genomic region of HCV analysed influenced phylogenetic tree topology and clustering results. The HCV Core region alone was not suitable for clustering analysis; NS5B concatenation, the inclusion of reference sequences and removal of HVR1 all influenced clustering outcome. The Core-E2 region, which represented the highest genetic diversity and longest sequence length in this study, provides an ideal method for clustering analysis to address a range of molecular epidemiological questions.