Biology subjects

Trovao, N. S.

Publications and source records attributed to Trovao, N. S..

11 recordsLinked to original sources

bioRxiv2026

Phylodynamic Analysis of the Global Dispersal and Spatiotemporal Dynamics of HIV-1 Subtype C

HIV-1 subtype C is the most prevalent HIV clade globally, yet its cross-continental transmission dynamics remain incompletely resolved due to sampling biases and unquantified analytical uncertainty. We performed a large-scale phylodynamic analysis of 1,221 near full-length genomic sequences from 32 countries (1986-2019). Employing a stratified subsampling strategy to mitigate bias, we systematically benchmarked 16 method combinations, pairing four maximum likelihood phylogenetic tools (FastTree, IQ-TREE, PhyML, RAxML-NG) with four temporal dating methods (TempEst, LSD, treedater, TreeTime). Topological analysis revealed significant discordance between rapid heuristics and higher-precision estimators. The estimated time to the most recent common ancestor (TMRCA) and the timing of individual introductions were highly sensitive to the temporal dating method, while evolutionary rates varied across both phylogenetic and temporal tools. Crucially, counts of viral introductions and the reconstruction of spatial transmission networks were statistically robust across all 16 combinations. Phylogeographic reconstruction confirmed a dominant, recurring dispersal corridor from Africa to Europe, with independent secondary reservoirs in South America and Asia. Ancestral trait reconstruction demonstrated transmission is primarily driven by heterosexual networks, within which "Not Recorded" risk groups were phylogenetically nested. This multi-method benchmarking reveals that while exact chronologies are sensitive to methodological choices, spatial network reconstructions and source-sink classifications remain remarkably consistent. These findings provide a robust map of subtype Cs global expansion and establish highly reliable, scalable workflows for resolving spatial dissemination routes for real-time genomic epidemiology.

evolutionary biology↗

bioRxiv2026

Multi-omic analysis identifies mitochondrial dysfunction as a conserved driver of acute severity and long-term complications in RSV infection

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants, older adults, and immunocompromised individuals. The molecular mechanisms linking acute RSV infection to disease severity and long-term complications remain incompletely understood. Herein, we conducted a comprehensive multi-omic analysis of 12 independent datasets encompassing epigenomics, transcriptomics, proteomics, and metabolomics across diverse systems, including in vitro infection models, clinical cohorts, longitudinal pediatric studies, vaccination models, and multiple viral strains. Across these experimental platforms and omic analysis, RSV consistently triggered suppression of oxidative phosphorylation (OXPHOS), alongside HIF-1-driven glycolytic metabolism and mitochondrial stress response. This coordinated reprogramming was consistent across transcriptomic, proteomic, and chromatin datasets. In adult challenge studies, symptomatic individuals exhibited prolonged OXPHOS suppression and greater activation of HIF-1 immune signaling than asymptomatic individuals. Similarly, pediatric intensive care cohorts showed comparable signatures associated with severe disease. Vaccinated mice showed attenuation of infection-induced metabolic disruption, further supporting a link between mitochondrial dysfunction and disease severity. Longitudinal analyses in pediatric samples revealed that these metabolic alterations persist for up to 1-year post-infection, with sustained metabolic dysfunction, persistent epigenetic remodeling, and single-cell evidence of epithelial remodeling, including depletion of multiciliated cells, expansion of secretory populations, and prolonged OXPHOS suppression, in children who developed wheezing. Comparative analysis across RSV strains revealed variable OXPHOS suppression and variable HIF-1 activation, indicating strain-specific differences in metabolic reprogramming. Together, these findings establish mitochondrial dysfunction as a central and conserved feature of RSV pathogenesis, encompassing acute severity, viral strain variation, and long-term complications, and highlight mitochondrial pathways as promising therapeutic targets to mitigate both acute disease severity and post-viral sequelae. Ultimately, demonstrating that distinct viral lineages drive unique bioenergetic phenotypes establishes a foundation for predictive molecular epidemiology and gaining insight into host-pathogen dynamics in response to novel interventions. HighlightsO_LIMulti-omic integration of 13 independent RSV datasets reveals mitochondrial dysfunction as a conserved hallmark of infection. C_LIO_LIRSV consistently suppresses oxidative phosphorylation (OXPHOS) while activating HIF-1 signaling, glycolysis, mitochondrial stress responses, and immune pathways. C_LIO_LIGreater mitochondrial dysfunction correlates with increased disease severity, persists in children who develop wheezing, and is partially ameliorated by vaccination. C_LIO_LIDistinct RSV strains display variable patterns of metabolic reprogramming, linking viral genetic diversity to differential host mitochondrial responses. C_LI

systems biology↗

bioRxiv2025

Emergence of D1.1 reassortant H5N1 avian influenza viruses in North America

Since 2021, highly pathogenic avian influenza viruses (HPAIVs) belonging to H5N1 clade 2.3.4.4b have circulated widely in North American wild birds and repeatedly spilled over into mammals. In 2025, the first H5N1-associated deaths in humans were recorded in the Western hemisphere, raising questions about how the ongoing evolution of the virus in wild birds impacts spillover risk. Here, our analysis of 21,471 H5N1 genomes identified an evolutionary shift in mid-2024, driven by interhemispheric migration from Asia and reassortment with new antigens. The genotypes that dominated the early years of North Americas H5N1 epizootic traced their ancestry back to Europe, but Asia was the source of new "D1.1" genotype viruses that (a) spread faster, (b) have higher reassortment potential, (c) a broader host range, (d) repeatedly spill over to bovines, and (e) cause severe disease in humans, including non-farm workers.

evolutionary biology↗

bioRxiv2025

Integrating phylodynamics and historical records reveals decades-old introductions of PRRSV into Costa Rica via international swine trade

BackgroundIn August 1995, necropsies on post-weaning piglets from the CA-CART farm in the province of Cartago, Costa Rica, revealed respiratory lesions, pleuritis, peritonitis, and arthritis. Skin lesions were also observed, progressing to scabs. A subsequent outbreak in 1996 prompted antibiotic administration. Mortality analysis from 1990 to 1995 showed no significant seasonal patterns, but yearly variations were noted. Piglets born in Costa Rica from imported gilts had a higher average mortality rate (10.65%) than the 8.11% mortality rate for piglets born from non-imported gilts and sows or imported sows (p = 0.002). MethodsIn March 1996, serum samples were sent for potential PRRS virus (PRRSV) diagnosis, and 10 PRRSV-2 ORF5 sequences collected during a prospective study from 2019-2021 were obtained from various locations in the country. In this article, we seek to investigate the evolutionary and spatio-temporal dynamics of PRRSV-2 in Costa Rica in the context of international swine movements using the phylodynamic framework integrated with the BEAST package. Results and DiscussionThe phylodynamic modeling estimated at least two independent PRRSV-2 introductions into the country. The earliest introduction occurred around 1978 (95% highest posterior density interval: 1959.01-1997.54) and led to the viruses circulating in the farm 1CRC with an origin from Japan, possibly via US swine exports. A second cluster, 4CRC, subsequently emerged within Costa Rica from the earlier 1CRC lineage (1990.24; 95% HPD interval = 1976.15-2010.30). Another viral introduction from the US occurred around 1991 (95% highest posterior density interval: 1974.34-2011.35) and established the 3CRC cluster. Though the viral introduction was traced back to the US, the limited genomic surveillance of PRRSV leaves room for considering alternative origins. ConclusionOur findings provide a high-resolution model of how international swine trade drives the introduction and evolution of a major livestock pathogen, highlighting the critical need for integrating genomic surveillance into biosecurity protocols.

microbiology↗

bioRxiv2024

From North to South: Transmission Dynamics of H1N1pdm09 Swine Influenza A Viruses in Italy

The influenza A H1N1pdm09 virus continues to be a significant pathogen, posing potential risks to both animal and human health due to its zoonotic potential. Italy, which has one of the largest swine populations in Europe, plays a crucial role in monitoring the evolution of influenza viruses in livestock. This study aims to address the existing knowledge gaps regarding the genetic diversity and transmission dynamics of H1N1pdm09 circulating in Italian swine populations. Utilizing whole genome sequencing and dynamic modeling, we conducted a comprehensive analysis of virus samples collected from swine farms across Italy. Our results reveal that multiple independent viral introductions have occurred into the country, with most cases resulting in self-limited infections and limited onward transmission. However, six distinct transmission clusters were identified, suggesting instances of sustained viral spread. These clusters were found across multiple regions of Italy, highlighting the broad geographic distribution of virus lineages. Our findings indicate that while many introductions led to localized containment, certain virus lineages were able to spread within specific regions of Italy. Through a detailed examination of selective pressures, we observed that most viral genes are under strong purifying selection in both swine and human hosts, as reflected by dN/dS ratios well below 1. The hemagglutinin (HA) gene exhibited a notably higher dN/dS ratio in swine ([~]0.28) compared to humans ([~]0.22), indicating slightly relaxed selection in swine. In contrast, other genes, such as neuraminidase (NA) and non-structural protein (NS), showed similarly strong purifying selection across both hosts. These results reflect a general trend of selective pressures affecting multiple viral components, rather than emphasizing specific genes. Our study emphasizes the importance of ongoing genomic surveillance in detecting viral circulation and mitigating risks to both animal and public health. Italys efforts contribute significantly to global influenza monitoring and highlight the importance of a One Health approach that integrates human, animal, and environmental health. These findings provide essential data to inform public health policies and enhance preparedness against future zoonotic influenza outbreaks.

microbiology↗

bioRxiv2023

LETHAL COVID-19 ASSOCIATES WITH RAAS-INDUCED INFLAMMATION FOR MULTIPLE ORGAN DAMAGE INCLUDING MEDIASTINAL LYMPH NODES

Lethal COVID-19 outcomes are most often attributed to classic cytokine storm and attendant excessive immune signaling. We re-visit this question using RNA sequencing in nasopharyngeal and 40 autopsy samples from COVID-19-positive and negative individuals. In nasal swabs, the top 100 genes which significantly correlated with COVID-19 viral load, include many canonical innate immune genes. However, 22 much less studied "non-canonical" genes are found and despite the absence of viral transcripts, subsets of these are upregulated in heart, lung, kidney, and liver, but not mediastinal lymph nodes. An important regulatory potential emerges for the non-canonical genes for over-activating the renin-angiotensin-activation-system (RAAS) pathway, resembling this phenomenon in hereditary angioedema (HAE) and its overlapping multiple features with lethal COVID-19 infections. Specifically, RAAS overactivation links increased fibrin deposition, leaky vessels, thrombotic tendency, and initiating the PANoptosis death pathway, as suggested in heart, lung, and especially mediastinal lymph nodes, with a tightly associated mitochondrial dysfunction linked to immune responses. For mediastinal lymph nodes, immunohistochemistry studies validate the transcriptomic findings showing abnormal architecture, excess fibrin and collagen deposition, and pathogenic fibroblasts. Further, our findings overlap findings in SARS-CoV-2 infected hamsters, C57BL/6 and BALB/c mouse models, and importantly peripheral blood mononuclear cell (PBMC) and whole blood samples from COVID-19 patients infected with early variants and later SARS-CoV-2 strains. We thus present cytokine storm in lethal COVID-19 disease as an interplay between upstream immune gene signaling producing downstream RAAS overactivation with resultant severe organ damage, especially compromising mediastinal lymph node function.

immunology↗

bioRxiv2023

Synchrony of Bird Migration with Avian Influenza Global Spread; Implications for Vulnerable Bird Orders

Highly pathogenic avian influenza virus (HPAIV) A H5 clade 2.3.4.4 has caused worldwide outbreaks in domestic poultry, occasional spillover to humans, and increasing deaths of diverse species of wild birds since 2014. Wild bird migration is currently acknowledged as an important ecological process contributing to the global dispersal of HPAIV H5. However, it is unclear how seasonal bird migration facilitates global virus dispersal, and which avian species are exposed to HPAI H5 clade 2.3.4.4 viruses and where. To shed light on ongoing global outbreaks, we sought to explore these questions through phylodynamic analyses based on empirical data of bird movement tracking and virus genome sequences. First, based on viral phylogeography and bird migration networks, we demonstrate that seasonal bird migration can explain salient features of the global dispersal of clade 2.3.4.4. Second, we detect synchrony between the seasonality of bird annual cycle phases and virus lineage movements. We reveal the differing vulnerable bird orders at geographical origins and destinations of HPAIV H5 lineage movements. Notably, we highlight the potential importance of relatively under-discussed Suliformes and Ciconiiformes, in addition to Anseriformes and Charadriiformes, in virus lineage movements. Our study provides a phylodynamic framework that links the bird movement ecology and genomic epidemiology of avian influenza; it highlights the importance of integrating bird behaviour and life history in avian influenza studies.

ecology↗

bioRxiv2022

Multiple-trait subsampling for optimized ancestral trait reconstruction

Large datasets along with sampling bias represent a challenge for phylodynamic reconstructions, particularly when the study data are obtained from various heterogeneous sources and/or through convenience sampling. In this study, we evaluate the presence of unbalanced sampled distribution by collection date, location, and risk group of HIV-1 subtype C using a compre-hensive subsampling strategy, and assess their impact on the reconstruction of the viral spatial and risk group dynamics using phylogenetic comparative methods. Our study shows that the most suitable dataset for ancestral trait reconstruction can be obtained through subsampling by collection date, location, and risk group, particularly using multigene datasets. We also demonstrate that sampling bias is inflated when considerable information for a given trait is unavailable or of poor quality, as we observed for the risk group in the analysis of HIV-1 subtype C. In conclusion, we suggest that, even if traits are not well recorded, including them deliberately optimizes the representativeness of the original dataset rather than completely excluding them. Therefore, we advise the inclusion of as many traits as possible with the aid of subsampling approaches in order to optimize the dataset for phylodynamic analysis while reducing the computational burden. This will benefit research communities investigating the evolutionary and spatiotemporal patterns of infectious diseases.

bioinformatics↗

bioRxiv2022

Origins and Evolution of Seasonal Human Coronaviruses

Four seasonal human coronaviruses (sHCoVs) are endemic globally (229E, NL63, OC43, and HKU1), accounting for 5-30% of human respiratory infections. However, the epidemiology and evolution of these CoVs remain understudied due to their association with mild symptomatology. Using a multigene and complete genomes analysis approach, we find the evolutionary histories of sHCoVs to be more complex than previously recognized, owing to frequent recombination of CoVs, including within and between sHCoVs. Within sHCoV recombination rate was highest for 229E and OC43, and within genus highest for betaCoVs, whereas substitutions per recombination event inversely highest in NL63 and HKU1, and the alphaCoVs. Depending on the gene studied, OC43 may have ungulate, canine, or rabbit CoV ancestors, while 229E may have origins in a bat, camel or an unsampled intermediate host. HKU1 had the earliest most recent common ancestor (MRCA: 1809-1899), comprised two genetically divergent genotypes (A and B) possibly representing two independent transmission events from murine CoVs, and genotype B was genetically more diverse than all the other sHCoVs. Finally, we found shared amino acid substitutions in multiple proteins along the non-human to sHCoV host-jump branches. The complex evolution of CoVs and their frequent host switches could benefit from continued surveillance of CoVs across non-human hosts.

evolutionary biology↗

bioRxiv2022

Evolution of influenza A virus hemagglutinin H1 and H3 across host species

The Influenza A virus (IAV) hemagglutinin protein (HA) has been studied extensively, but its evolution has not been thoroughly compared among major host species. We compared H3 and H1 evolutionary rates among 49 lineages differentiated by host (avian, canine, equine, human, swine), phylogeny, and geography. Rates of nonsynonymous evolution, relative to synonymous rates, were higher in mammalian than avian hosts. Human seasonal HA and classical swine H1 accumulated 11-13 glycosylation sites, primarily in the antigenically important head domain, whereas lower numbers were maintained in other hosts. Canines had the highest ratio of nonsynonymous to synonymous changes in the more conserved stalk domain. Amino acid changes in canine viruses occurred disproportionately at residues located at protein interfaces. This suggests that they were adaptations affecting the major structural rearrangement of HA, which is critical for cell entry. These findings invite further study of how host ecology and physiology affect natural selection. AUTHOR SUMMARYInfluenza virus evolution is of practical importance to health in addition to being an excellent system for the study of parasite/host evolution. Our work explores a largely untapped aspect of influenza evolution: sequence evolution in non-human hosts. This is important in its own right, in terms of both science and domestic animal health. It also puts the evolution of human influenza in a larger, comparative context. Our results also provide evidence concerning the evolution of the hemagglutinin stem domain, which has not been a focus of study but has new importance due to the development of stem-based universal influenza vaccines.

evolutionary biology↗

bioRxiv2017

Hemagglutinin Glycan Clock Guides Human Influenza A Virus Evolution

Adaptive immunity to influenza A virus is limited by frequent mutations in the immunodominant head of hemagglutinin (HA). Over the last century, the upward trend in HA-head glycosylation indicates glycan addition can increase fitness, but its role in viral evolution remains unclear. Here, we report glycan evolution follows a clock-like rhythm, pacing the timeline, trajectory, and replacement of HA. Following pandemic introduction, glycans are added to HA at 4- to 6-year intervals, until a functional glycan limit is reached, after which, at 9- to 12-year intervals, glycans are either swapped between different sites, or the HA is replaced by a novel pandemic virus. Using this, we predicted the appearance of the newest glycan on pH1N1 HA. Phylogeographic reconstruction suggests these highly fit strains originated in the Middle East, before rapidly replacing all strains globally. Going forward, we can use this simple algorithm to forecast future glycan evolution and identify seasons with higher pandemic potential.

microbiology↗