Evolutionary Profile of Mayaro Virus in the Americas: An Update into Genome Variability.

The Mayaro virus (MAYV) is an arbovirus with emerging potential, though with a limited understanding of its epidemiology and evolution due to the lack of studies and surveillance. Here, we investigated 71 MAYV genome sequences from the Americas available at GenBank and characterized the phylogenetic relationship among virus strains. A phylogenetic analysis showed that sequences were grouped according to the genotypes L, D, and N. Genotype D sequences were closely related to sequences collected in adjacent years and from their respective countries, suggesting that isolates may have originated from circulating lineages. The coalescent analysis demonstrated similar results, indicating the continuous circulation of the virus between countries as well. An unidentified sequence from the USA was grouped with genotype D, suggesting the insertion of this genotype in the country. Furthermore, the recombination analysis detected homologous and three heterologous hybrids which presented an insertion into the nsP3 protein. Amino acid substitutions among sequences indicated selective pressure sites, suggesting viral adaptability. This also impacted the binding affinity between the E1-E2 protein complex and the Mxra8 receptor, associated with MAYV entry into human cells. These results provide information for a better understanding of genotypes circulating in the Americas.

ViralFlow v1.0-a computational workflow for streamlining viral genomic surveillance.

ViralFlow v1.0 is a computational workflow developed for viral genomic surveillance. Several key changes turned ViralFlow into a general-purpose reference-based genome assembler for all viruses with an available reference genome. New virus-agnostic modules were implemented to further study nucleotide and amino acid mutations. ViralFlow v1.0 runs on a broad range of computational infrastructures, from laptop computers to high-performance computing (HPC) environments, and generates standard and well-formatted outputs suited for both public health reporting and scientific problem-solving. ViralFlow v1.0 is available at: https://viralflow.github.io/index-en.html.

Spatiotemporal transmission of SARS-CoV-2 lineages during 2020-2021 in Pernambuco-Brazil.

SARS-CoV-2 virus emerged as a new threat to humans and spread around the world, leaving a large death toll. As of January 2023, Brazil is among the countries with the highest number of registered deaths. Nonpharmacological and pharmacological interventions have been heterogeneously implemented in the country, which, associated with large socioeconomic differences between the country regions, has led to distinct virus spread dynamics. Here, we investigate the spatiotemporal dispersion of SARS-CoV-2 lineages in the Pernambuco state (Northeast Brazil) throughout the distinct epidemiological scenarios that unfolded in the first 2 years of the pandemic. We generated a total of 1,389 new SARS-CoV-2 genomes from June 2020 to August 2021. This sampling captured the arrival, communitary transmission, and the circulation of the B1.1, B.1.1.28, and B.1.1.33 lineages; the emergence of the former variant of interest P.2; and the emergence and fast replacement of all previous variants by the more transmissible variant of concern P.1 (Gamma). Based on the incidence and lineage spread pattern, we observed an East-to-West to inner state pattern of transmission, which is in agreement with the transmission of more populous metropolitan areas to medium- and small-size country-side cities in the state. Such transmission patterns may be partially explained by the main routes of traffic across municipalities in the state. Our results highlight that the fine-grained intrastate analysis of lineages and incidence spread can provide actionable insights for planning future nonpharmacological intervention for air-borne transmissible human pathogens.IMPORTANCEDuring the COVID-19 pandemic, Brazil was one of the most affected countries, mainly due its continental-size, socioeconomic differences among regions, and heterogeneous implementation of intervention methods. In order to investigate SARS-CoV-2 dynamics in the state of Pernambuco, we conducted a spatiotemporal dispersion study, covering the period from June 2020 to August 2021, to comprehend the dynamics of viral transmission during the first 2 years of the pandemic. Throughout this study, we were able to track three significant epidemiological waves of transmission caused by B1.1, B.1.1.28, B.1.1.33, P.2, and P.1 lineages. These analyses provided valuable insights into the evolution of the epidemiological landscape, contributing to a deeper understanding of the dynamics of virus transmission during the early years of the pandemic in the state of Pernambuco.

Critical Assessment of Novel Developments in HPGR Technology Using DEM.

Advances in high-pressure grinding roll (HGPR) technology since its first commercial application in the cement industry include new roll wear protection techniques and new confinement systems. The latter contribute to reductions in the edge effects in an attempt to reach a more homogenous product size along the rolls. Additional advances in this technology have been made in recent years, while modeling and simulation tools are also reaching maturity and can now be used to subject such novel developments to detailed scrutiny. This work applies a hybrid approach combining advanced simulations using the discrete element method, the particle replacement model and multibody dynamics to a phenomenological population balance model to critically assess two recent advances in HPGR technology: spring-loaded cheek plates and the offset roller press. Force and torque controllers, included in the EDEM 2022.1 software, were used to describe the responses of the geometries in contact with the granular material processed. Simulations showed that while the former successfully reduced the lateral bypass of the material by as much as 65% when cheek plates became severely worn, the latter demonstrated lower throughput and higher potential wear but an ability to generate a finer product than the traditional design.

Analysis of Haemonchus embryos at single cell resolution identifies two eukaryotic elongation factors as intervention target candidates.

Advances in single cell technologies are allowing investigations of a wide range of biological processes and pathways in animals, such as the multicellular model organism Caenorhabditis elegans - a free-living nematode. However, there has been limited application of such technology to related parasitic nematodes which cause major diseases of humans and animals worldwide. With no vaccines against the vast majority of parasitic nematodes and treatment failures due to drug resistance or inefficacy, new intervention targets are urgently needed, preferably informed by a deep understanding of these nematodes' cellular and molecular biology - which is presently lacking for most worms. Here, we created the first single cell atlas for an early developmental stage of Haemonchus contortus - a highly pathogenic, C. elegans-related parasitic nematode. We obtained and curated RNA sequence (snRNA-seq) data from single nuclei from embryonating eggs of H. contortus (150,000 droplets), and selected high-quality transcriptomic data for > 14,000 single nuclei for analysis, and identified 19 distinct clusters of cells. Guided by comparative analyses with C. elegans, we were able to reproducibly assign seven cell clusters to body wall muscle, hypodermis, neuronal, intestinal or seam cells, and identified eight genes that were transcribed in all cell clusters/types, three of which were inferred to be essential in H. contortus. Two of these genes (i.e. Hc-eef-1A and Hc-eef1G), coding for eukaryotic elongation factors (called Hc-eEF1A and Hc-eEF1G), were also demonstrated to be transcribed and expressed in all key developmental stages of H. contortus. Together with these findings, sequence- and structure-based comparative analyses indicated the potential of Hc-eEF1A and/or Hc-eEF1G as intervention targets within the protein biosynthesis machinery of H. contortus. Future work will focus on single cell studies of all key developmental stages and tissues of H. contortus, and on evaluating the suitability of the two elongation factor proteins as drug targets in H. contortus and related nematodes, with a view to finding new nematocidal drug candidates.

Standing on shoulder of giants: the incredible legacy of the Judet family in orthopaedic surgery.

In orthopaedic surgery, as well as other areas in medicine, it is common for a surgical technique to carry the original authors' name describing the procedure. The Judet family represents a unique history, since several orthopaedic procedures are known as "Judet's technique". The aim of this historic review is to outline the genealogy of the orthopaedic arm of the Judet family, while crediting each surgical procedure to the specific family member that described the technique.

Ankle fractures malreduction rate and its causes in two Brazilian tertiary training hospitals.

INTRODUCTION: Ankle fractures are one of the most frequent injuries managed by any trauma surgeon. Literature has shown that adequate reduction is of utmost importance to achieve satisfactory functional outcomes. However, malreduction rates remain high worldwide. The objective of the present study is to evaluate the quality of ankle fracture reduction in two major trauma centers in a large Brazilian city and to analyze the factors associated with malreduction METHODS: Epidemiologic data of 382 patients (189 men; 193 women) aged between 7 and 87 years who underwent osteosynthesis of the ankle in two trauma centers in a large Brazilian city. Electronic clinical records, preoperative and immediate postoperative radiographs in anteroposterior (AP), lateral and mortise views were evaluated. Pettrone's criteria were used to evaluate the quality of ankle fractures reduction. All radiographs were independently evaluated by two foot and ankle senior surgeons RESULTS: Overall, malreduction rate was 22.2%. Forty-seven (55.2%) fractures classified as malreduced had medial malleolar displacement The results showed three factors that significantly affected the quality of reduction (p<0.05), patients over 60 years, open fractures and fracture-dislocations. Patients aged over 60 years were twice more likely to have poor reduction of ankle fractures than younger ones. The risk of poor fracture reduction among those individuals with open fractures is 2.15 times greater than among patients with closed injuries. Fracture-dislocation imposed a 2.7 higher risk for malreduction DISCUSSION: We found a malreduction rate below most series previously published. Further results agree with the literature. Elderly people aged over 60 years, fracture dislocations and open fractures are associated with worse results, influencing the quality of the reduction, clinical outcomes, and the development of post-traumatic arthrosis CONCLUSION: Ankle fractures malreduction are associated with higher age, open fractures, and fracture-dislocations.

'Bingo'-a large language model- and graph neural network-based workflow for the prediction of essential genes from protein data.

The identification and characterization of essential genes are central to our understanding of the core biological functions in eukaryotic organisms, and has important implications for the treatment of diseases caused by, for example, cancers and pathogens. Given the major constraints in testing the functions of genes of many organisms in the laboratory, due to the absence of in vitro cultures and/or gene perturbation assays for most metazoan species, there has been a need to develop in silico tools for the accurate prediction or inference of essential genes to underpin systems biological investigations. Major advances in machine learning approaches provide unprecedented opportunities to overcome these limitations and accelerate the discovery of essential genes on a genome-wide scale. Here, we developed and evaluated a large language model- and graph neural network (LLM-GNN)-based approach, called 'Bingo', to predict essential protein-coding genes in the metazoan model organisms Caenorhabditis elegans and Drosophila melanogaster as well as in Mus musculus and Homo sapiens (a HepG2 cell line) by integrating LLM and GNNs with adversarial training. Bingo predicts essential genes under two 'zero-shot' scenarios with transfer learning, showing promise to compensate for a lack of high-quality genomic and proteomic data for non-model organisms. In addition, the attention mechanisms and GNNExplainer were employed to manifest the functional sites and structural domain with most contribution to essentiality. In conclusion, Bingo provides the prospect of being able to accurately infer the essential genes of little- or under-studied organisms of interest, and provides a biological explanation for gene essentiality.

High prevalence of GR2 and GR4 plasmids in Acinetobacter baumannii strains from Brazil.

Acinetobacter baumannii is Gram-negative pathogen with extensive role in healthcare-associated infections (HAIs). Plasmids in this species are important carriers of antimicrobial resistance genes. In this work, we investigated the plasmids of 227 Brazilian A. baumannii genomes. A total of 389 plasmid sequences with 424 Rep proteins typed to 22 different homology groups (GRs) were identified. The GR2 plasmid group was the most predominant (40.6%), followed by the GR4 group (16.7%), representing ∼57% of all plasmids. There is a wide distribution of plasmids among the isolates and most strains carry more than one plasmid. Our analyses revealed a significant prevalence of GR4 plasmids in Brazilian A. baumannii genomes carrying several antimicrobial resistance genes, notably to carbapenem (39.43%). These plasmids harbor a MOBQ relaxase that might confer increased spreading potential in the environment. Most plasmids of the predominant groups belong to the same plasmid taxonomic unit (PTU-Pse7) and have a AbkA/AbkB toxin-antitoxin system that has a role in plasmid stability and dissemination of carbapenem resistance genes. The results of this work should contribute to our understanding of the molecular content of plasmids in a large and populous country, highlighting the importance of genomics for enhanced epidemiological surveillance.

Supine Lobenhoffer Approach Optimizes PCL Reconstruction with Tibial Inlay in Knee Dislocations: A Case Report.

CASE: A 47-year-old man suffered a knee dislocation while skiing with associated damage to the anterior cruciate, posterior cruciate, medial collateral, and medial patellofemoral ligaments. The patient underwent a multiple-ligament knee reconstruction using the supine Lobenhoffer approach, which resulted in remarkable outcomes that persisted throughout the 56-month follow-up period. CONCLUSION: The utilization of this approach can prevent inappropriate graft tension and iatrogenic damage, reduce surgical time, and minimize airway complications by eliminating the need for the prone position. Optimizing these factors enhances the patient's chances for long-term outcomes; thus, surgeons should consider this approach in managing multiple-ligament knee injuries.

Reemergence of Dengue Virus Serotype 3, Brazil, 2023.

We characterized 3 autochthonous dengue virus serotype 3 cases and 1 imported case from 2 states in the North and South Regions of Brazil, 15 years after Brazil's last outbreak involving this serotype. We also identified a new Asian lineage recently introduced into the Americas, raising concerns about future outbreaks.

Establishing a Consensus Definition of a Knee Fracture-Dislocation (Schenck Knee Dislocation V) Using a Global Modified Delphi Method.

BACKGROUND: Knee fracture-dislocations are complex injuries; however, there is no universally accepted definition of what constitutes a fracture-dislocation within the Schenck Knee Dislocation (KD) V subcategory. The purpose of this study was to establish a more precise definition for fracture patterns included within the Schenck KD V subcategory. METHODS: A series of clinical scenarios encompassing various fracture patterns in association with a bicruciate knee ligament injury was created by a working group of 8 surgeons. Utilizing a modified Delphi technique, 46 surgeons from 18 countries and 6 continents with clinical and academic expertise in multiligamentous knee injuries undertook 3 rounds of online surveys to establish consensus. Consensus was defined as ≥70% agreement with responses of either "strongly agree" or "agree" for a positive consensus or "strongly disagree" or "disagree" for a negative consensus. RESULTS: There was a 100% response rate for Rounds 1 and 2 and a 96% response rate for Round 3. A total of 11 fracture patterns reached consensus for inclusion: (1) nondisplaced articular fracture of the femur; (2) displaced articular fracture of the femur; (3) tibial plateau fracture involving the weight-bearing surface (with or without tibial spine involvement); (4) tibial plateau peripheral rim compression fracture; (5) posterolateral tibial plateau compression fracture, Bernholt type IIB; (6) posterolateral tibial plateau compression fracture, Bernholt type IIIA; (7) posterolateral tibial plateau compression fracture, Bernholt type IIIB; (8) Gerdy's tubercle avulsion fracture with weight-bearing surface involvement; (9) displaced tibial tubercle fracture; (10) displaced patellar body fracture; and (11) displaced patellar inferior pole fracture. Fourteen fracture patterns reached consensus for exclusion from the definition. Two fracture patterns failed to reach consensus for either inclusion or exclusion from the definition. CONCLUSIONS: Using a modified Delphi technique, this study established consensus for specific fracture patterns to include within or exclude from the Schenck KD V subcategory. LEVEL OF EVIDENCE: Prognostic Level V . See Instructions for Authors for a complete description of levels of evidence.

Extensor Mechanism Disruption Impacts Treatment of Dislocated and Multiligament Injured Knees: Treatment and Schenck Classification Recommendations Based on a Global Delphi Method.

BACKGROUND: Multiligament knee injury (MLKI) with associated extensor mechanism (EM) involvement is a rare injury, with limited evidence to guide optimal treatment. The purpose of this study was to identify areas of consensus among a group of international experts regarding the treatment of patients with MLKI and concomitant EM injury. METHODS: Utilizing a classic Delphi technique, an international group of 46 surgeons from 6 continents with expertise in MLKI undertook 3 rounds of online surveys. Participants were presented with clinical scenarios involving EM disruption in association with MLKI, classified using the Schenck Knee-Dislocation (KD) Classification. Positive consensus was defined as ≥70% agreement with responses of either "strongly agree" or "agree," and negative consensus was defined as ≥70% agreement with "strongly disagree" or "disagree." RESULTS: There was a 100% response rate for rounds 1 and 2 and a 96% response rate for round 3. There was strong positive consensus (87%) that an EM injury in combination with MLKI significantly alters the treatment algorithm. For an EM injury in conjunction with a KD2, KD3M, or KD3L injury, there was positive consensus to repair the EM injury only and negative consensus regarding performing concurrent ligamentous reconstruction at the time of initial surgery. CONCLUSIONS: In the setting of bicruciate MLKI, there was overall agreement on the significant impact of EM injury on the treatment algorithm. We therefore recommend that the Schenck KD Classification be updated with the addition of the modifier suffix "-EM" to highlight this impact. Treatment of the EM injury was judged to have the highest priority, and there was consensus to treat the EM injury only. However, given the lack of clinical outcome data, treatment decisions need to be made on a case-by-case basis with consideration of the numerous clinical factors that are encountered. CLINICAL RELEVANCE: Little clinical evidence exists to guide the surgeon on the management of EM injury in the setting of a multiligament injured or dislocated knee. This survey highlights the impact that EM injury has on the treatment algorithm and provides some guidance for management until a further large case series or prospective studies are undertaken.

Chondrotoxic effects of tranexamic acid and povidone-iodine on the articular cartilage of rabbits.

PURPOSE: To evaluate the chondrotoxic effects of intra-articular use of TXA 20 mg/kg and/or 0.35% PVPI on knee joint cartilage in an experimental model of rabbits. METHODS: Forty-four male New Zealand adult rabbits were randomly assigned to four groups (control, tranexamic acid (TXA), povidone-iodine (PVPI), and PVPI + TXA). The knee joint cartilage was accessed through an arthrotomy and exposed to physiological saline SF 0.9% (control group), TXA, PVPI, and PVPI followed by TXA. Sixty days after surgical procedure, the animals were sacrificed and osteochondral specimens of the distal femur were obtained. Histological sections of cartilage from this area were stained with hematoxylin/eosin and toluidine blue. The following cartilage parameters were evaluated by the Mankin histological/histochemical grading system: structure, cellularity, glycosaminoglycan content in the extracellular matrix, and integrity of the tidemark. RESULTS: The isolated use of PVPI causes statistically significant changes in cartilage cellularity (p-value = 0.005) and decrease glycosaminoglycan content (p = 0.001), whereas the isolated use of TXA decreased significantly the glycosaminoglycan content (p = 0.031). The sequential use of PVPI + TXA causes more pronounced alterations in the structure (p = 0.039) and cellularity (p = 0.002) and decreased content of glycosaminoglycans (p < 0.001) all with statistical significance. CONCLUSION: Data suggest that intra-articular use of tranexamic acid 20 mg/kg and intraoperative lavage with 0.35% povidone-iodine solution for three min are toxic to the articular cartilage of the knee in an experimental in vivo study in rabbits.

Combined reconstruction of medial collateral ligament and posterior cruciate ligament using one femoral tunnel: a technical note and case report applicable to limited-resource settings.

Knee dislocations are severe injuries difficult to treat. Specially in low-resources scenarios, reconstruction of multiple ligaments can be challenging. We describe a technical note that can be reconstruct multi ligaments using ipsilateral hamstrings autograft. A posteromedial knee incision is made to visualise the medial corner of the knee and to reconstruct medial collateral ligament (MCL) and posterior cruciate ligament (PCL) with semitendinosus and gracilis tendon graft, using one femoral tunnel from the anatomic femoral insertion of the MCL to the anatomic femoral insertion of the PCL. After 1-year follow-up, the patient returned to his previous function with a Lysholm score of 86. This technique can reconstruct more than one ligament anatomically with limited graft resource.

Calculation of the Minimal Important Clinical Difference of the Lysholm and IKDC Scores After Anterior Cruciate Ligament Reconstruction.

Objective To calculate the minimal important clinical difference (MICD) value for the Lysholm and International Knee Documentation Committee (IKDC) scores in a sample of patients submitted to anterior cruciate ligament reconstruction. Methods Primary, observational, retrospective, analytical study of participants submitted to anterior cruciate ligament reconstruction from March 2019 to December 2020 by the same surgeon, with a minimum follow-up of 6 months, analysis of knee function in the pre- and postoperative period by the Lysholm and IKDC scores, and answer to an anchor question at 6 months postoperatively for the calculation of the MICD of each score. Results A total of 59 patients participated in the study, with a mean age of 27.1 ± 5.7 years old. In the comparison between pre- and postoperative scores of all groups, there was an increase in values with statistical significance after intervention. The MICD was 5.5 for the Lysholm score, and the MICD value for the IKDC score could not be determined. Conclusion For the Lysholm score, the calculation of the MICD value by the anchor question method in the sample evaluated was 5.5. It was not possible to determine the value of the MICD for the IKDC score.

Calculation of the Minimal Important Clinical Difference of the Lysholm and IKDC Scores After Anterior Cruciate Ligament Reconstruction / Cálculo da mínima diferença clínica importante dos escores Lysholm e IKDC após reconstrução do ligamento cruzado anterior

Abstract Objective To calculate the minimal important clinical difference (MICD) value for the Lysholm and International Knee Documentation Committee (IKDC) scores in a sample of patients submitted to anterior cruciate ligament reconstruction. Methods Primary, observational, retrospective, analytical study of participants submitted to anterior cruciate ligament reconstruction from March 2019 to December 2020 by the same surgeon, with a minimum follow-up of 6 months, analysis of knee function in the pre- and postoperative period by the Lysholm and IKDC scores, and answer to an anchor question at 6 months postoperatively for the calculation of the MICD of each score. Results A total of 59 patients participated in the study, with a mean age of 27.1 ± 5.7 years old. In the comparison between pre- and postoperative scores of all groups, there was an increase in values with statistical significance after intervention. The MICD was 5.5 for the Lysholm score, and the MICD value for the IKDC score could not be determined. Conclusion For the Lysholm score, the calculation of the MICD value by the anchor question method in the sample evaluated was 5.5. It was not possible to determine the value of the MICD for the IKDC score.

Resumo Objetivo Calcular o valor da mínima diferença clinicamente importante (MDCI) para os escores de Lysholm e International Knee Documentation Commitee (IKDC) na amostra de pacientes submetidos a reconstrução de ligamento cruzado anterior. Métodos Estudo primário, observacional, retrospectivo, analítico, de participantes submetidos a reconstrução do ligamento cruzado anterior no período de março de 2019 a dezembro de 2020, pelo mesmo cirurgião, com seguimento mínimo de 6 meses, análise da função do joelho no período pré e pós-operatório pelos escores de Lysholm e IKDC, e resposta a uma pergunta âncora aos 6 meses de seguimento pós-operatório, para o MDCI de cada escore. Resultados Participaram do estudo 59 pacientes, com média de idade de 27,1 ± 5,7 anos. Na comparação dos escores pré- e pós-operatórios de todos os grupos, observa-se aumento dos valores com significância estatística após a intervenção. A MDCI foi de 5,5 para o escore de Lysholm, não tendo sido possível determinar o valor para o IKDC. Conclusão O cálculo do valor da MDCI pelo método da pergunta âncora, na amostra avaliada, foi de 5,5 para o escore de Lysholm. Não foi possível determinar o valor da MDCI para o IKDC.

Real-life evaluation of a rapid antigen test (DPP SARS-CoV-2 Antigen) for COVID-19 diagnosis of primary healthcare patients, in the context of the Omicron-dominant wave in Brazil.

OBJECTIVES: We aimed to investigate the real-life performance of the rapid antigen test in the context of a primary healthcare setting, including symptomatic and asymptomatic individuals that sought diagnosis during an Omicron infection wave. METHODS: We prospectively accessed the performance of the DPP SARS-CoV-2 Antigen test in the context of an Omicron-dominant real-life setting. We evaluated 347 unselected individuals (all-comers) from a public testing centre in Brazil, performing the rapid antigen test diagnosis at point-of-care with fresh samples. The combinatory result from two distinct real-time quantitative PCR (RT-qPCR) methods was employed as a reference and 13 samples with discordant PCR results were excluded. RESULTS: The assessment of the rapid test in 67 PCR-positive and 265 negative samples revealed an overall sensitivity of 80.5% (CI 95% = 69.1%-89.2%), specificity of 99.2% (CI 95% = 97.3%-99.1%) and positive/negative predictive values higher than 95%. However, we observed that the sensitivity was dependent on the viral load (sensitivity in Ct < 31 = 93.7%, CI = 82.8%-98.7%; Ct > 31 = 47.4%, CI = 24.4%-71.1%). The positive samples evaluated in the study were Omicron (BA.1/BA.1.1) by whole-genome sequencing (n = 40) and multiplex RT-qPCR (n = 17). CONCLUSIONS: Altogether, the data obtained from a real-life prospective cohort supports that the rapid antigen test sensitivity for Omicron remains high and underscores the reliability of the test for COVID-19 diagnosis in settings with high disease prevalence and limited PCR testing capability.

Multiple introductions and country-wide spread of DENV-2 genotype II (Cosmopolitan) in Brazil.

Dengue virus serotype 2, genotype Cosmopolitan (DENV-2-GII), is one of the most widespread DENV strains globally. In the USA, DENV-2 epidemics have been dominated by DENV-2 genotype Asian-American (DENV-2-GIII), and the first cases of DENV-2-GII were only described in 2019, in Peru, and in 2021 in Brazil. To gain new information about the circulation of DENV-2-GII in Brazil, we sequenced 237 DENV-2 confirmed cases sampled between March 2021 and March 2023 and revealed that DENV-2-GII is already present in all geographic regions of Brazil. The phylogeographic analysis inferred that DENV-2-GII was introduced at least four times in Brazil, between May 2020 and August 2022, generating multiple clades that spread throughout the country with different success. Despite multiple introductions of DENV-2-GII, analysis of the country-wide laboratory surveillance data showed that the Brazilian dengue epidemic in 2022 was dominated by DENV-1 in most states. We hypothesize that massive circulation of DENV-2-GIII in previous years in Brazil might have created a population immune barrier against symptomatic homotypic reinfections by DENV-2-GII, leading to sustained cryptic circulation in asymptomatic cases and localized outbreaks of this new genotype. In summary, our study stresses the importance of arboviral genomic surveillance to close monitoring and better understanding the potential impact of DENV-2-GII in the coming years.

ViralFlow: A Versatile Automated Workflow for SARS-CoV-2 Genome Assembly, Lineage Assignment, Mutations and Intrahost Variant Detection.

The COVID-19 pandemic is driven by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) that emerged in 2019 and quickly spread worldwide. Genomic surveillance has become the gold standard methodology used to monitor and study this fast-spreading virus and its constantly emerging lineages. The current deluge of SARS-CoV-2 genomic data generated worldwide has put additional pressure on the urgent need for streamlined bioinformatics workflows. Here, we describe a workflow developed by our group to process and analyze large-scale SARS-CoV-2 Illumina amplicon sequencing data. This workflow automates all steps of SARS-CoV-2 reference-based genomic analysis: data processing, genome assembly, PANGO lineage assignment, mutation analysis and the screening of intrahost variants. The pipeline is capable of processing a batch of around 100 samples in less than half an hour on a personal laptop or in less than five minutes on a server with 50 threads. The workflow presented here is available through Docker or Singularity images, allowing for implementation on laptops for small-scale analyses or on high processing capacity servers or clusters. Moreover, the low requirements for memory and CPU cores and the standardized results provided by ViralFlow highlight it as a versatile tool for SARS-CoV-2 genomic analysis.