Luiz Max Carvalho

Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen)

Gene sequences sampled at different points in time can be used to infer molecular phylogenies on a natural timescale of months or years, provided that the sequences in question undergo measurable amounts of evolutionary change between sampling times. Data sets with this property are termed heterochronous and have become increasingly common in several fields of biology, most notably the molecular epidemiology of rapidly evolving viruses. Here we introduce the cross-platform software tool, TempEst (formerly known as Path-O-Gen), for the visualization and analysis of temporally sampled sequence data. Given a molecular phylogeny and the dates of sampling for each sequence, TempEst uses an interactive regression approach to explore the association between genetic divergence through time and sampling dates. TempEst can be used to (1) assess whether there is sufficient temporal signal in the data to proceed with phylogenetic molecular clock analysis, and (2) identify sequences whose genetic divergence and sampling date are incongruent. Examination of the latter can help identify data quality problems, including errors in data annotation, sample contamination, sequence recombination, or alignment error. We recommend that all users of the molecular clock models implemented in BEAST first check their data using TempEst prior to analysis.

Virus genomes reveal factors that spread and sustained the Ebola epidemic

The 2013–2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic ‘gravity’ model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

Higher incidence of Zika in adult women than adult men in Rio de Janeiro suggests a significant contribution of sexual transmission from men to women

OBJECTIVES The recent emergence of Zika in Brazil and its association with an increased rate of congenital malformations has raised concerns over its impact on the birth rate in the country. Using data on the incidence of Zika in 2015-2016 and dengue in 2013 and 2015-2016 for the city of Rio de Janeiro (population 6.4 million), a massive increase of Zika in women compared to men was documented. METHODS The age-adjusted incidence was compared between men and women. A negative binomial Poisson generalized linear model was fitted to the Zika incidence data to determine the significance of sexual transmission statistically. RESULTS Even after correcting for the bias due to the systematic testing of pregnant women for Zika, there were found to be 90% more registered cases per 100000 women than men in the sexually active age group (15-65 years); this was not the case for age groups <15 years and >65 years. Assuming that infected men transmit the disease to women in their semen, but that the converse is not true, some extra incidence in women is to be expected. An alternate hypothesis would be that women visit doctors more often than men. To test this, the incidence of dengue fever was compared in men and women in 2015 and in 2013 (before Zika reached Rio de Janeiro): in both years, women were 30% more likely to be reported with dengue. CONCLUSION Women in the sexually active age group are far more likely to get Zika than men (+90% increase); sexual transmission is the most probable cause. Women in the 15-65 years age group are also 30% more likely to be reported with dengue than men, which is probably due to women being more careful with their health.

Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013–2016 Epidemic

Summary The magnitude of the 2013–2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013–2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.

πBUSS: a parallel BEAST/BEAGLE utility for sequence simulation under complex evolutionary scenarios

BackgroundSimulated nucleotide or amino acid sequences are frequently used to assess the performance of phylogenetic reconstruction methods. BEAST, a Bayesian statistical framework that focuses on reconstructing time-calibrated molecular evolutionary processes, supports a wide array of evolutionary models, but lacked matching machinery for simulation of character evolution along phylogenies.ResultsWe present a flexible Monte Carlo simulation tool, called πBUSS, that employs the BEAGLE high performance library for phylogenetic computations to rapidly generate large sequence alignments under complex evolutionary models. πBUSS sports a user-friendly graphical user interface (GUI) that allows combining a rich array of models across an arbitrary number of partitions. A command-line interface mirrors the options available through the GUI and facilitates scripting in large-scale simulation studies. πBUSS may serve as an easy-to-use, standard sequence simulation tool, but the available models and data types are particularly useful to assess the performance of complex BEAST inferences. The connection with BEAST is further strengthened through the use of a common extensible markup language (XML), allowing to specify also more advanced evolutionary models. To support simulation under the latter, as well as to support simulation and analysis in a single run, we also add the πBUSS core simulation routine to the list of BEAST XML parsers.ConclusionsπBUSS offers a unique combination of flexibility and ease-of-use for sequence simulation under realistic evolutionary scenarios. Through different interfaces, πBUSS supports simulation studies ranging from modest endeavors for illustrative purposes to complex and large-scale assessments of evolutionary inference procedures. Applications are not restricted to the BEAST framework, or even time-measured evolutionary histories, and πBUSS can be connected to various other programs using standard input and output format.

Zika in Rio de Janeiro: Assessment of basic reproductive number and its comparison with dengue

Zika virus infection was declared a public health emergency of international concern in February 2016 in response to the outbreak in Brazil and its suspected link with congenital anomalies. In this study we use notification data and disease natural history parameters to estimate the basic reproduction number (R0) of Zika in Rio de Janeiro, Brazil. We also obtain estimates of R0 of dengue from time series of dengue cases in the outbreaks registered in 2002 and 2012 in the city, when DENV-3 and DENV-4 serotypes respectively, had just emerged. Our estimates of the basic reproduction number for Zika in Rio de Janeiro based on surveillance notifications (R0 = 2.33, 95% CI: 1.97 − 2.97) were higher than those obtained for dengue in the city (year 2002: R0 = 1.70 [1.50 − 2.02]; year 2012: Ro = 1.25 [1.18 − 1.36]). Given the role of Aedes aegypti as vector of both the Zika and dengue viruses, we also derive Ro of Zika as a function of both dengue reproduction number and entomological and epidemiological parameters for dengue and Zika. Using the dengue outbreaks from previous years allowed us to estimate the potential R0 of Zika. Our estimates were closely in agreement with our first Zika’s R0 estimation from notification data. Hence, these results validate deriving the potential risk of Zika transmission in areas with recurring dengue outbreaks. Whether transmission routes other than vector-based can sustain a Zika epidemic still deserves attention, but our results suggest that the Zika outbreak in Rio de Janeiro emerged due to population susceptibility and ubiquitous presence of Ae. aegypti.

Estimating the Attack Ratio of Dengue Epidemics under Time-varying Force of Infection using Aggregated Notification Data

Quantifying the attack ratio of disease is key to epidemiological inference and public health planning. For multi-serotype pathogens, however, different levels of serotype-specific immunity make it difficult to assess the population at risk. In this paper we propose a Bayesian method for estimation of the attack ratio of an epidemic and the initial fraction of susceptibles using aggregated incidence data. We derive the probability distribution of the effective reproductive number, Rt, and use MCMC to obtain posterior distributions of the parameters of a single-strain SIR transmission model with time-varying force of infection. Our method is showcased in a data set consisting of 18 years of dengue incidence in the city of Rio de Janeiro, Brazil. We demonstrate that it is possible to learn about the initial fraction of susceptibles and the attack ratio even in the absence of serotype specific data. On the other hand, the information provided by this approach is limited, stressing the need for detailed serological surveys to characterise the distribution of serotype-specific immunity in the population.

Spatiotemporal Dynamics of DENV-2 Asian-American Genotype Lineages in the Americas

The Asian/American (AS/AM) genotype of dengue virus type 2 (DENV-2) has been evolving in the Americas over the last 30 years, leading to several waves of dengue epidemics and to the emergence of different viral lineages in the region. In this study, we investigate the spatiotemporal dissemination pattern of the DENV-2 lineages at a regional level. We applied phylogenetic and phylogeographic analytical methods to a comprehensive data set of 582 DENV-2 E gene sequences of the AS/AM genotype isolated from 29 different American countries over a period of 30 years (1983 to 2012). Our study reveals that genetic diversity of DENV-2 AS/AM genotype circulating in the Americas mainly resulted from one single founder event and can be organized in at least four major lineages (I to IV), which emerged in the Caribbean region at the early 1980s and then spread and die out with different dynamics. Lineages I and II dominate the epidemics in the Caribbean region during the 1980s and early 1990s, lineage III becomes the prevalent DENV-2 one in the Caribbean and South America during the 1990s, whereas lineage IV dominates the epidemics in South and Central America during the 2000s. Suriname and Guyana seem to represent important entry points for DENV-2 from the Lesser Antilles to South America, whereas Venezuela, Brazil and Nicaragua were pointed as the main secondary hubs of dissemination to other mainland countries. Our study also indicates that DENV-2 AS/AM genotype was disseminated within South America following two main routes. The first route hits Venezuela and the western side of the Andes, while the second route mainly hits Brazil and the eastern side of the Andes. The phenomenon of DENV-2 lineage replacement across successive epidemic outbreaks was a common characteristic in all American countries, although the timing of lineage replacements greatly vary across locations.

Sexual transmission causes a marked increase in the incidence of Zika in women in Rio de Janeiro, Brazil.

The recent emergence of Zika in Brazil and its association with increased congenital malformation rates has raised concerns over its impact on the birth rates in the country. Using data on the incidence of Zika in 2015-2016 and dengue in 2013 and 2015-16 for the city of Rio de Janeiro (pop: 6.4 million), we document a massive increase of Zika in women compared to men. Even after correcting for the bias due to the systematic testing of pregnant women for Zika, there are 90% more registered cases per 100,000 women in the sexually active age group (15-65 years) than for men but not before 15 or after 65. Assuming that infected men transmit the disease to women in their semen but that the converse is not true, some extra incidence in women is to be expected. An alternate hypothesis would be that women visit doctors more often than men. To test this, we compared the incidence of dengue fever in men and women in 2015 and in 2013 (before Zika reached Rio de Janeiro): in both years, women are 30% more likely to be reported with dengue. Summing up, women in the sexually active age bracket are far more likely to get Zika than men (+90% increase); sexual transmission is the most probable cause. Women in the 15-65 age group are also 30% more likely to be reported with dengue than men, which is probably due to women being more careful with their health.

Phylogeography of foot-and-mouth disease virus serotype O in Ecuador

Foot-and-mouth disease virus (FMDV) is the causative agent of the most important disease of domestic cattle, foot-and-mouth disease. In Ecuador, FMDV is maintained at an endemic state, with sporadic outbreaks. To unravel the tempo and mode of FMDV spread within the country we conducted a Bayesian phylogeographic analysis using a continuous time Markov chain (CTMC) to model the diffusion of FMDV between Ecuadorian provinces. We implement this framework through Markov chain Monte Carlo available in the BEAST package to study 90 FMDV serotype O isolates from 17 provinces in the period 2002-2010. The Bayesian approach also allowed us to test hypotheses on the mechanisms of viral spread by incorporating environmental and epidemiological data in our prior distributions and perform Bayesian model selection. Our analyses suggest an intense flow of viral strains throughout the country that is possibly coupled to animal movements and ecological factors, since most of inferred viral spread routes were in Coast and Highland regions. Moreover, our results suggest that both short- and long-range spread occur within Ecuador. The province of Esmeraldas, in the border with Colombia and where most animal commerce is done, was found to be the most probable origin of the circulating strains, pointing to a transboundary behavior of FMDV in South America. These findings suggest that uncontrolled animal movements can create a favorable environment for FMDV maintenance and pose a serious threat to control programmes. Also, we show that phylogeographic modeling can be a powerful tool in unraveling the spatial dynamics of viruses and potentially in controlling the spread of these pathogens.