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Acute respiratory infections (ARIs) remain a leading cause of global morbidity and mortality, disproportionately affecting vulnerable populations such as children, the elderly, and immunocompromised individuals. Despite the availability of traditional diagnostic tools, including viral culture and highly elaborated PCR respiratory panels, many cases of ARI remain without an identified etiological agent. This is due to the vast diversity of viral agents that can be involved in cases of ARI, which represents a major limitation of the targeted diagnosis. In this context, viral metagenomics has emerged as a powerful, unbiased approach for detecting both known and novel pathogens directly from clinical samples. This review highlights the application of metagenomic next-generation sequencing for the investigation of etiological causes of ARIs, emphasizing its relevance in complex cases—particularly among immunocompromised patients—where standard methods might fail. We highlight the main viruses involved in respiratory infections, the strengths and limitations of metagenomic next-generation sequencing approaches, their role in genomic surveillance of respiratory viruses, and their potential to build public health responses to potentially emerging respiratory threats. Ultimately, integrating viral metagenomics into clinical and surveillance frameworks could enhance the early detection and control of respiratory viral diseases worldwide.

Trypanosoma cruzi , responsible for Chagas disease, affects millions globally, particularly in Latin America. Lack of vaccine or treatment underscores the need for research. Parasite’s genome, with virulent antigen-coding multigenic families, resides in the rapidly evolving Disruptive compartment. Study sheds light on the parasite’s dynamic DNA replication, discussing the evolution of the Disruptive compartment. Therefore, the findings represent a significant stride in comprehending T. cruzi ’s biology and the molecular bases that contribute to the success of infection caused by this parasite.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here, we propose adding 2 sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present assignment tools to show that the proposed lineages are useful for regional, national, and subnational discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org .

Respiratory illness affects individuals across all age demographics on a global scale, often precipitated by viral infections. The symptomatic manifestations of these diseases bear clinical resemblance, complicating the accurate determination of their etiological origins. Furthermore, the diagnostic panels for respiratory pathogens used within local medical practices, may not encompass the full spectrum of viral agents responsible for such ailments. Consequently, a significant number of clinically important viral pathogens may remain undetected. In the light of this, we conducted a metagenomic examination of 66 nasopharyngeal swab specimens, obtained from patients presenting with acute respiratory conditions yet tested negative by the standard diagnostic panels available locally. These specimens were obtained from the Public Health Laboratory, Maceio, State of Alagoas. Our findings indicate a predominant diagnostic escape of rhinoviruses and notably enterovirus D68. Moreover, our study identified a substantial quantity of sequence reads attributed to human respirovirus 3 (human parainfluenza 3) along with various herpresviruses including human herpesvirus-1, Epstein-Barr virus (Human herpesvirus-4), Human herpesviruses 6 and 7 and human parvovirus B19 (B19V). Notably, the metagenomic analysis uncovered a widespread presence of the emerging human vientovirus FB in most of sample pools, though its clinical importance remains to be elucidated. The obtained results in this study underscore the invaluable role of viral metagenomics in the identification of underrecognized viruses bearing clinical relevance. Furthermore, it offers insights into the dissemination of these pathogens within the studied area, thereby informing public health strategies aimed at enhancing diagnostic accuracy and improving patient care.

Enterovirus D68 (EV-D68) is a leading cause of acute respiratory disease outbreaks, especially among children. EV-D68 infections can rapidly progress to severe clinical complications and potentially fatal outcomes. In Brazil, no diagnostic or genomic surveillance of this virus is currently performed. Between July and September 2023, cases of acute EV-D68 infection were identified among pediatric patients in several municipalities within the State of Alagoas, Northeast Brazil. Infections were confirmed by RT-qPCR using nasopharyngeal samples, and the complete EV-D68 genomes were sequenced and analyzed through phylogenetic inference. EV-D68 RNA was identified in four children aged 1–9 years from four geographically distinct municipalities in Alagoas. All infections were associated with lower respiratory tract symptoms, including dyspnea and wheezing; however, no fatalities were reported. Complete genomic sequencing revealed that the samples belonged to genotype B, subgenotype B3. This is the first study to report complete genomic data on EV-D68 infections from Brazil and South America. Enhanced genomic surveillance and focused EV-D68 diagnosis are critical to better understanding and managing the regional and national dissemination of this virus.

Background Viral metagenomics has expanded significantly in recent years due to advancements in next-generation sequencing, establishing it as the leading method for identifying emerging viruses. A crucial step in metagenomics is taxonomic classification, where sequence data is assigned to specific taxa, thereby enabling the characterization of species composition within a sample. Various taxonomic classifiers have been developed in recent years, each employing distinct classification approaches that produce varying results and abundance profiles, even when analyzing the same sample. Methods In this study, we propose using the identification of Torque Teno Viruses (TTVs), from the Anelloviridae family, as indicators to evaluate the performance of four short-read-based metagenomic classifiers: Kraken2, Kaiju, CLARK and DIAMOND, when evaluating human plasma samples. Results Our results show that each classifier assigns TTV species at different abundance levels, potentially influencing the interpretation of diversity within samples. Specifically, nucleotide-based classifiers tend to detect a broader range of TTV species, indicating higher sensitivity, while amino acid-based classifiers like DIAMOND and CLARK display lower abundance indices. Interestingly, despite employing different algorithms and data types (protein-based vs. nucleotide-based), Kaiju and Kraken2 performed similarly. Conclusion Our study underscores the critical impact of classifier selection on diversity indices in metagenomic analyses. Kaiju effectively assigned a wide variety of TTV species, demonstrating it did not require a high volume of reads to capture diversity. Nucleotide-based classifiers like CLARK and Kraken2 showed superior sensitivity, which is valuable for detecting emerging or rare viruses. At the same time, protein-based approaches such as DIAMOND and Kaiju proved robust for identifying known species with low variability.

Background DNA replication in trypanosomatids operates in a uniquely challenging environment, since most of their genomes are constitutively transcribed. Trypanosoma cruzi , the etiological agent of Chagas disease, presents high variability in both chromosomes size and copy number among strains, though the underlying mechanisms are unknown. Results Here we have mapped sites of DNA replication initiation across the T. cruzi genome using Marker Frequency Analysis, which has previously only been deployed in two related trypanosomatids. The putative origins identified in T. cruzi show a notable enrichment of GC content, a preferential position at subtelomeric regions, coinciding with genes transcribed towards the telomeres, and a pronounced enrichment within coding DNA sequences, most notably in genes from the Dispersed Gene Family 1 (DGF-1). Conclusions These findings suggest a scenario where collisions between DNA replication and transcription are frequent, leading to increased genetic variability, as seen by the increase SNP levels at chromosome subtelomeres and in DGF-1 genes containing putative origins.

Background Genomic organization and gene expression regulation in trypanosomes are remarkable because protein-coding genes are organized into codirectional gene clusters with unrelated functions. Moreover, there is no dedicated promoter for each gene, resulting in polycistronic gene transcription, with posttranscriptional control playing a major role. Nonetheless, these parasites harbor epigenetic modifications at critical regulatory genome features that dynamically change among parasite stages, which are not fully understood. Results Here, we investigated the impact of chromatin changes in a scenario commanded by posttranscriptional control exploring the parasite Trypanosoma cruzi and its differentiation program using FAIRE-seq approach supported by transmission electron microscopy. We identified differences in T. cruzi genome compartments, putative transcriptional start regions, and virulence factors. In addition, we also detected a developmental chromatin regulation at tRNA loci (tDNA), which could be linked to the intense chromatin remodeling and/or the translation regulatory mechanism required for parasite differentiation. We further integrated the open chromatin profile with public transcriptomic and MNase-seq datasets. Strikingly, a positive correlation was observed between active chromatin and steady-state transcription levels. Conclusion Taken together, our results indicate that chromatin changes reflect the unusual gene expression regulation of trypanosomes and the differences among parasite developmental stages, even in the context of a lack of canonical transcriptional control of protein-coding genes.

From a country with one of the highest SARS-CoV-2 morbidity and mortality rates, Brazil has implemented one of the most successful vaccination programs. Brazil’s first model city vaccination program was performed by the CoronaVac vaccine (Sinovac Biotech) in the town of Serrana, São Paulo State. To evaluate the vaccination effect on the SARS-CoV-2 molecular dynamics and clinical outcomes, we performed SARS-CoV-2 molecular surveillance on 4375 complete genomes obtained between June 2020 and April 2022 in this location. This study included the period between the initial SARS-CoV-2 introduction and during the vaccination process. We observed that the SARS-CoV-2 substitution dynamics in Serrana followed the viral molecular epidemiology in Brazil, including the initial identification of the ancestral lineages (B.1.1.28 and B.1.1.33) and epidemic waves of variants of concern (VOC) including the Gamma, Delta, and, more recently, Omicron. Most probably, as a result of the immunization campaign, the mortality during the Gamma and Delta VOC was significantly reduced compared to the rest of Brazil, which was also related to lower morbidity. Our phylogenetic analysis revealed the evolutionary history of the SARS-CoV-2 in this location and showed that multiple introduction events have occurred over time. The evaluation of the COVID-19 clinical outcome revealed that most cases were mild (88.9%, 98.1%, 99.1% to Gamma, Delta, and Omicron, respectively) regardless of the infecting VOC. In conclusion, we observed that vaccination was responsible for reducing the death toll rate and related COVID-19 morbidity, especially during the gamma and Delta VOC; however, it does not prevent the rapid substitution rate and morbidity of the Omicron VOC.

The emergence of SARS-CoV-2 and the subsequent pandemic have prompted extensive diagnostic and clinical efforts to mitigate viral spread. However, these strategies have largely overlooked the presence of other respiratory viruses. Acute respiratory diseases in pediatric patients can be caused by a diverse range of viral agents, and metagenomics represents a powerful tool for their characterization. This study aimed to investigate the viral abundance in pediatric patients with acute respiratory symptoms who tested negative for SARS-CoV-2 during the Omicron pandemic wave. To achieve this, viral metagenomics and next-generation sequencing were employed on 96 nasopharyngeal swab samples, which were organized into 12 pools, with each pool consisting of eight individual samples. Metagenomic analysis revealed that the most prevalent viruses associated with acute disease in pediatric patients were respiratory syncytial virus (detected in all pools) and enteroviruses, which are known to cause significant morbidity and mortality in children. Additionally, clinically significant viruses such as mumps orthorubulavirus, human metapneumovirus, influenza A, and a wide array of human herpesviruses (1, 3–7) were identified. These findings highlight the extensive potential of viral metagenomics in identifying viruses other than SARS-CoV-2 that contribute to acute infections in children. Consequently, this methodology should garner clinical attention in terms of differential diagnosis and the development of public policies to address such conditions in the global pediatric population.