Assessing transmission attribution risk from simulated sequencing data in HIV molecular epidemiology.

BACKGROUND: HIV molecular epidemiology (ME) is the analysis of sequence data together with individual-level clinical, demographic, and behavioral data to understand HIV epidemiology. The use of ME has raised concerns regarding identification of the putative source in direct transmission events. This could result in harm ranging from stigma to criminal prosecution in some jurisdictions. Here we assessed the risks of ME using simulated HIV genetic sequencing data. METHODS: We simulated social networks of men-who-have-sex-with-men, calibrating the simulations to data from San Diego. We used these networks to simulate consensus and next-generation sequence (NGS) data to evaluate the risks of identifying direct transmissions using different HIV sequence lengths, and population sampling depths. To identify the source of transmissions, we calculated infector probability and used phyloscanner software for the analysis of consensus and NGS data, respectively. RESULTS: Consensus sequence analyses showed that the risk of correctly inferring the source (direct transmission) within identified transmission pairs was very small and independent of sampling depth. Alternatively, NGS analyses showed that identification of the source of a transmission was very accurate, but only for 6.5% of inferred pairs. False positive transmissions were also observed, where one or more unobserved intermediaries were present when compared to the true network. CONCLUSION: Source attribution using consensus sequences rarely infers direct transmission pairs with high confidence but is still useful for population studies. In contrast, source attribution using NGS data was much more accurate in identifying direct transmission pairs, but for only a small percentage of transmission pairs analyzed.

A species-level timeline of mammal evolution integrating phylogenomic data.

High-throughput sequencing projects generate genome-scale sequence data for species-level phylogenies1-3. However, state-of-the-art Bayesian methods for inferring timetrees are computationally limited to small datasets and cannot exploit the growing number of available genomes4. In the case of mammals, molecular-clock analyses of limited datasets have produced conflicting estimates of clade ages with large uncertainties5,6, and thus the timescale of placental mammal evolution remains contentious7-10. Here we develop a Bayesian molecular-clock dating approach to estimate a timetree of 4,705 mammal species integrating information from 72 mammal genomes. We show that increasingly larger phylogenomic datasets produce diversification time estimates with progressively smaller uncertainties, facilitating precise tests of macroevolutionary hypotheses. For example, we confidently reject an explosive model of placental mammal origination in the Palaeogene8 and show that crown Placentalia originated in the Late Cretaceous with unambiguous ordinal diversification in the Palaeocene/Eocene. Our Bayesian methodology facilitates analysis of complete genomes and thousands of species within an integrated framework, making it possible to address hitherto intractable research questions on species diversifications. This approach can be used to address other contentious cases of animal and plant diversifications that require analysis of species-level phylogenomic datasets.

Evaluating whole HIV-1 genome sequence for estimation of incidence and migration in a rural South African community.

Background: South Africa has the largest number of people living with HIV (PLWHIV) in the world, with HIV prevalence and transmission patterns varying greatly between provinces. Transmission between regions is still poorly understood, but phylodynamics of HIV-1 evolution can reveal how many infections are attributable to contacts outside a given community. We analysed whole genome HIV-1 genetic sequences to estimate incidence and the proportion of transmissions between communities in Hlabisa, a rural South African community. Methods: We separately analysed HIV-1 for gag, pol, and env genes sampled from 2,503 PLWHIV. We estimated time-scaled phylogenies by maximum likelihood under a molecular clock model. Phylodynamic models were fitted to time-scaled trees to estimate transmission rates, effective number of infections, incidence through time, and the proportion of infections imported to Hlabisa. We also partitioned time-scaled phylogenies with significantly different distributions of coalescent times. Results: Phylodynamic analyses showed similar trends in epidemic growth rates between 1980 and 1990. Model-based estimates of incidence and effective number of infections were consistent across genes. Parameter estimates with gag were generally smaller than those estimated with pol and env. When estimating the proportions of new infections in Hlabisa from immigration or transmission from external sources, our posterior median estimates were 85% (95% credible interval (CI) = 78%-92%) for gag, 62% (CI = 40%-78%) for pol, and 77% (CI = 58%-90%) for env in 2015. Analysis of phylogenetic partitions by gene showed that most close global reference sequences clustered within a single partition. This suggests local evolving epidemics or potential unmeasured heterogeneity in the population. Conclusions: We estimated consistent epidemic dynamic trends for gag, pol and env genes using phylodynamic models. There was a high probability that new infections were not attributable to endogenous transmission within Hlabisa, suggesting high inter-connectedness between communities in rural South Africa.

Genetic evidence for the association between COVID-19 epidemic severity and timing of non-pharmaceutical interventions.

Unprecedented public health interventions including travel restrictions and national lockdowns have been implemented to stem the COVID-19 epidemic, but the effectiveness of non-pharmaceutical interventions is still debated. We carried out a phylogenetic analysis of more than 29,000 publicly available whole genome SARS-CoV-2 sequences from 57 locations to estimate the time that the epidemic originated in different places. These estimates were examined in relation to the dates of the most stringent interventions in each location as well as to the number of cumulative COVID-19 deaths and phylodynamic estimates of epidemic size. Here we report that the time elapsed between epidemic origin and maximum intervention is associated with different measures of epidemic severity and explains 11% of the variance in reported deaths one month after the most stringent intervention. Locations where strong non-pharmaceutical interventions were implemented earlier experienced much less severe COVID-19 morbidity and mortality during the period of study.

Genomic epidemiology of a densely sampled COVID-19 outbreak in China.

Analysis of genetic sequence data from the SARS-CoV-2 pandemic can provide insights into epidemic origins, worldwide dispersal, and epidemiological history. With few exceptions, genomic epidemiological analysis has focused on geographically distributed data sets with few isolates in any given location. Here, we report an analysis of 20 whole SARS- CoV-2 genomes from a single relatively small and geographically constrained outbreak in Weifang, People's Republic of China. Using Bayesian model-based phylodynamic methods, we estimate a mean basic reproduction number (R 0) of 3.4 (95% highest posterior density interval: 2.1-5.2) in Weifang, and a mean effective reproduction number (Rt) that falls below 1 on 4 February. We further estimate the number of infections through time and compare these estimates to confirmed diagnoses by the Weifang Centers for Disease Control. We find that these estimates are consistent with reported cases and there is unlikely to be a large undiagnosed burden of infection over the period we studied.

Reduction in mobility and COVID-19 transmission.

In response to the COVID-19 pandemic, countries have sought to control SARS-CoV-2 transmission by restricting population movement through social distancing interventions, thus reducing the number of contacts. Mobility data represent an important proxy measure of social distancing, and here, we characterise the relationship between transmission and mobility for 52 countries around the world. Transmission significantly decreased with the initial reduction in mobility in 73% of the countries analysed, but we found evidence of decoupling of transmission and mobility following the relaxation of strict control measures for 80% of countries. For the majority of countries, mobility explained a substantial proportion of the variation in transmissibility (median adjusted R-squared: 48%, interquartile range - IQR - across countries [27-77%]). Where a change in the relationship occurred, predictive ability decreased after the relaxation; from a median adjusted R-squared of 74% (IQR across countries [49-91%]) pre-relaxation, to a median adjusted R-squared of 30% (IQR across countries [12-48%]) post-relaxation. In countries with a clear relationship between mobility and transmission both before and after strict control measures were relaxed, mobility was associated with lower transmission rates after control measures were relaxed indicating that the beneficial effects of ongoing social distancing behaviours were substantial.

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant.

Phylodynamic analysis of HIV-1 subtypes B, C and CRF 02_AG in Senegal.

Surveillance of HIV epidemics in key populations and in developing countries is often challenging due to sparse, incomplete, or low-quality data. Analysis of HIV sequence data can provide an alternative source of information about epidemic history, population structure, and transmission patterns. To understand HIV-1 dynamics and transmission patterns in Senegal, we carried out model-based phylodynamic analyses using the structured-coalescent approach using HIV-1 sequence data from three different subgroups: reproductive aged males and females from the adult Senegalese population and men who have sex with other men (MSM). We fitted these phylodynamic analyses to time-scaled phylogenetic trees individually for subtypes C and CRF 02_AG, and for the combined data for subtypes B, C and CRF 02_AG. In general, the combined analysis showed a decreasing proportion of effective number of infections among all reproductive aged adults relative to MSM. However, we observed a nearly time-invariant distribution for subtype CRF 02_AG and an increasing trend for subtype C on the proportion of effective number of infections. The population attributable fraction also differed between analyses: subtype CRF 02_AG showed little contribution from MSM, while for subtype C and combined analyses this contribution was much higher. Despite observed differences, results suggested that the combination of high assortativity among MSM and the unmet HIV prevention and treatment needs represent a significant component of the HIV epidemic in Senegal.

A biologist's guide to Bayesian phylogenetic analysis.

Bayesian methods have become very popular in molecular phylogenetics due to the availability of user-friendly software implementing sophisticated models of evolution. However, Bayesian phylogenetic models are complex, and analyses are often carried out using default settings, which may not be appropriate. Here, we summarize the major features of Bayesian phylogenetic inference and discuss Bayesian computation using Markov chain Monte Carlo (MCMC), the diagnosis of an MCMC run, and ways of summarising the MCMC sample. We discuss the specification of the prior, the choice of the substitution model, and partitioning of the data. Finally, we provide a list of common Bayesian phylogenetic software and provide recommendations as to their use.

Computational Evaluation of the Strict Master and Random Template Models of Endogenous Retrovirus Evolution.

Transposable elements (TEs) are DNA sequences that are able to replicate and move within and between host genomes. Their mechanism of replication is also shared with endogenous retroviruses (ERVs), which are also a type of TE that represent an ancient retroviral infection within animal genomes. Two models have been proposed to explain TE proliferation in host genomes: the strict master model (SMM), and the random template (or transposon) model (TM). In SMM only a single copy of a given TE lineage is able to replicate, and all other genomic copies of TEs are derived from that master copy. In TM, any element of a given family is able to replicate in the host genome. In this paper, we simulated ERV phylogenetic trees under variations of SMM and TM. To test whether current phylogenetic programs can recover the simulated ERV phylogenies, DNA sequence alignments were simulated and maximum likelihood trees were reconstructed and compared to the simulated phylogenies. Results indicate that visual inspection of phylogenetic trees alone can be misleading. However, if a set of statistical summaries is calculated, we are able to distinguish between models with high accuracy by using a data mining algorithm that we introduce here. We also demonstrate the use of our data mining algorithm with empirical data for the porcine endogenous retrovirus (PERV), an ERV that is able to replicate in human and pig cells in vitro.

Deciphering unusual uncultured magnetotactic multicellular prokaryotes through genomics.

Candidatus Magnetoglobus multicellularis (Ca. M. multicellularis) is a member of a group of uncultured magnetotactic prokaryotes that possesses a unique multicellular morphology. To better understand this organism's physiology, we used a genomic approach through pyrosequencing. Genomic data analysis corroborates previous structural studies and reveals the proteins that are likely involved in multicellular morphogenesis of this microorganism. Interestingly, some detected protein sequences that might be involved in cell adhesion are homologues to phylogenetically unrelated filamentous multicellular bacteria proteins, suggesting their contribution in the early development of multicellular organization in Bacteria. Genes related to the behavior of Ca. M. multicellularis (chemo-, photo- and magnetotaxis) and its metabolic capabilities were analyzed. On the basis of the genomic-physiologic information, enrichment media were tested. One medium supported chemoorganoheterotrophic growth of Ca. M. multicellularis and allowed the microorganisms to maintain their multicellular morphology and cell cycle, confirming for the first time that the entire life cycle of the MMP occurs in a multicellular form. Because Ca. M. multicellularis has a unique multicellular life style, its cultivation is an important achievement for further studies regarding the multicellular evolution in prokaryotes.

The role of historical barriers in the diversification processes in open vegetation formations during the Miocene/Pliocene using an ancient rodent lineage as a model.

The Neotropics harbors a high diversity of species and several hypotheses have been proposed to account for this pattern. However, while species of forested domains are frequently studied, less is known of species from open vegetation formations occupying, altogether, a larger area than the Amazon Forest. Here we evaluate the role of historical barriers and the riverine hypothesis in the speciation patterns of small mammals by analyzing an ancient rodent lineage (Thrichomys, Hystricomorpha). Phylogenetic and biogeographic analyses were carried out with mitochondrial and nuclear DNA markers to analyze the evolutionary relationships between Thrichomys lineages occurring in dry domains along both banks of the Rio São Francisco. This river is one of the longest of South America whose course and water flow have been modified by inland tectonic activities and climate changes. Molecular data showed a higher number of lineages than previously described. The T. inermis species complex with 2n = 26, FN = 48 was observed in both banks of the river showing a paraphyletic arrangement, suggesting that river crossing had occurred, from east to west. A similar pattern was also observed for the T. apereoides complex. Thrichomys speciation occurred in Late Miocene when the river followed a different course. The current geographic distribution of Thrichomys species and their phylogenetic relationships suggested the existence of frequent past connections between both banks in the middle section of the Rio São Francisco. The extensive palaeodune region found in this area has been identified as a centre of endemism of several vertebrate species and is likely to be a center of Thrichomys diversification.

Biogeographic determinants of genetic diversification in the mouse opossum Gracilinanus agilis (Didelphimorphia: Didelphidae).

The genetic variation of Brazilian populations of the mouse opossum Gracilinanus agilis was analyzed on the basis of the mitochondrial Cytochrome b gene (mt-Cytb) and the exon 28 of the nuclear Von Willenbrand factor (e28-vWF). The radiation of Gracilinanus was dated at 4.80 Ma, with the appearance of G. agilis around 1.93 Ma. Gracilinanus aceramarcae appeared as the first offshoot of the genus, followed by Gracilinanus emiliae and Gracilinanus microtarsus, which composed a sister clade of G. agilis. Phylogeographic analyses and genetic distance estimates indicate G. agilis as a single species, with haplotypes grouping in three well-supported clades, one from midwestern Brazil, a second one from northeastern Brazil, and a third one from eastern Brazil. Phylogeographic patterns in G. agilis were interpreted in search for congruence between genetic breaks and historic geomorphologic events documented for the region stretching northeastern to central-western of the Brazilian shield. The Rio São Francisco and the Serra Geral de Goiás were found to represent relevant geographic barriers to gene flow for G. agilis populations as well as for populations of several other widespread taxa.

Evolution of endogenous retroviruses in the Suidae: evidence for different viral subpopulations in African and Eurasian host species.

BACKGROUND: Porcine endogenous retroviruses (PERVs) represent remnants of an exogenous form that have become integrated in the domestic pig (Sus scrofa) genome. Although they are usually inactive, the capacity of γ1 ERVs to infect human cells in vitro has raised concerns about xenotransplantation because the viruses could cross the species barrier to humans. Here we have analyzed the evolution of γ1 ERVs in ten species of Suidae (suids, pigs and hogs) from Eurasia and Africa using DNA sequences for their coding domains (gag, pro/pol and env genes). For comparison with γ1 PERVs, we have also analysed γ2 ERVs which in domestic pigs are known to be inactive and do not pose a risk to xenotransplantation. RESULTS: Phylogenetic analysis using Bayesian inference showed that γ1 and γ2 ERVs have distinctive evolutionary histories. Firstly, two different viral lineages of γ1 ERVs were found and a coevolutionary analysis demonstrated that they correspond broadly to their host phylogeny, one of Eurasian and another of African species, and show no evidence of horizontal transmission. γ2 ERVs, however, show a bush-like evolution, suggesting a rapid viral radiation from a single common ancestor with no correspondence between host and viral evolutionary trees. Furthermore, though γ1 ERV env genes do not possess frequent stop codons, γ2 env genes do. To understand whether γ1 suid ERVs may be still replicating, we have also evaluated their likely mechanism of proliferation by statistically testing internal to terminal branches using nonsynonymous versus synonymous substitution ratios. Our results suggest that γ1 ERVs are increasing in copy number by reinfection, which requires the translocation of the virus from one cell to another. CONCLUSIONS: Evidence of at least two viral subpopulations was observed in γ1 ERVs from Eurasian and African host species. These results should be taken into account in xenotransplantation since γ1 ERVs appear to be codiverging with their host and maintaining ongoing capacity to infect somatic and germ cells.

Common ancestry of iron oxide- and iron-sulfide-based biomineralization in magnetotactic bacteria.

Magnetosomes are prokaryotic organelles produced by magnetotactic bacteria that consist of nanometer-sized magnetite (Fe(3)O(4)) or/and greigite (Fe(3)S(4)) magnetic crystals enveloped by a lipid bilayer membrane. In magnetite-producing magnetotactic bacteria, proteins present in the magnetosome membrane modulate biomineralization of the magnetite crystal. In these microorganisms, genes that encode for magnetosome membrane proteins as well as genes involved in the construction of the magnetite magnetosome chain, the mam and mms genes, are organized within a genomic island. However, partially because there are presently no greigite-producing magnetotactic bacteria in pure culture, little is known regarding the greigite biomineralization process in these organisms including whether similar genes are involved in the process. Here using culture-independent techniques, we now show that mam genes involved in the production of magnetite magnetosomes are also present in greigite-producing magnetotactic bacteria. This finding suggest that the biomineralization of magnetite and greigite did not have evolve independently (that is, magnetotaxis is polyphyletic) as once suggested. Instead, results presented here are consistent with a model in which the ability to biomineralize magnetosomes and the possession of the mam genes was acquired by bacteria from a common ancestor, that is, the magnetotactic trait is monophyletic.

Population genetic studies of Alouatta caraya (Alouattinae, Primates): inferences on geographic distribution and ecology.

Cytochrome b DNA sequence data (ca. 1,140 bp) of 44 Alouatta caraya, including 42 specimens from three localities of Brazil and two from Bolivia, were used for phylogenetic reconstructions and population studies. Seventeen haplotypes were identified, eight of which were present in more than one individual. Seven of these eight haplotypes were shared by individuals from a same locality and one by individuals from two localities. We found 26 variable sites along the entire gene, consisting of 18 transitions and eight transversions; most replacements occurring at the third codon position (65.39%) in contrast to first and second positions (26.92 and 7.69%, respectively). In the sample collected at Chapada dos Guimarães (Brazil), nucleotide and haplotype diversity estimates were pi=0.002325 and h=0.8772, respectively. Maximum parsimony analysis grouped all haplotypes in two clades, separating Bolivian haplotypes from Brazilian haplotypes, the grouping of which did not show a straightforward correspondence with geographic distribution. Median-joining and TCS network pointed to haplotypes 11 or 12 as the most likely ancestral ones. Mismatch distribution and the goodness-of-fit test (SSD estimate=0.0027; P=0.6999) indicated that the population from Chapada dos Guimarães experienced a demographic expansion, in agreement with the median-joining star-like pattern, although this finding could not be confirmed by Fu's F(s) test.

The lowest diploid number (2n = 16) yet found in any primate: Callicebus lugens (Humboldt, 1811).

Morphologic, molecular and karyologic analyses of Callicebus lugens (Humboldt, 1811) of known geographic origin supported the proposition that this is a valid species. Morphologic and morphometric analyses showed evident differences between C. lugens and two other related taxa of the same group (Callicebus purinus and Callicebus torquatus). Cytochrome b DNA analyses (maximum parsimony, neighbour joining and maximum likelihood) were congruent in showing a strong association between C. lugens and Callicebus sp. of the torquatus group in one branch and a sister branch further divided into two clades: one with species of the personatus group and another, with species of the moloch group. Karyotypic analysis showed that C. lugens has the lowest diploid chromosome number of the primate order (2n = 16). Comparisons with other congeneric species clearly supported the proposition that C. lugens is karyotypically similar to others of the torquatus group.