Binding Evolution of the Dengue Virus Envelope Against DC-SIGN: A Combined Approach of Phylogenetics and Molecular Dynamics Analyses Over 30 Years of Dengue Virus in Brazil.

The Red Queen Hypothesis (RQH), derived from Lewis Carroll's "Through the Looking-Glass", postulates that organisms must continually adapt in response to each other to maintain relative fitness. Within the context of host-pathogen interactions, the RQH implies an evolutionary arms race, wherein viruses evolve to exploit hosts and hosts evolve to resist viral invasion. This study delves into the dynamics of the RQH in the context of virus-cell interactions, specifically focusing on virus receptors and cell receptors. We observed multiple virus-host systems and noted patterns of co-evolution. As viruses evolved receptor-binding proteins to effectively engage with cell receptors, cells countered by altering their receptor genes. This ongoing mutual adaptation cycle has influenced the molecular intricacies of receptor-ligand interactions. Our data supports the RQH as a driving force behind the diversification and specialization of both viral and host cell receptors. Understanding this co-evolutionary dance offers insights into the unpredictability of emerging viral diseases and potential therapeutic interventions. Future research is crucial to dissect the nuanced molecular changes and the broader ecological consequences of this ever-evolving battle. Here, we combine phylogenetic inferences, structural modeling, and molecular dynamics analyses to describe the epidemiological characteristics of major Brazilian DENV strains that circulated from 1990 to 2022 from a combined perspective, thus providing us with a more detailed picture on the dynamics of such interactions over time.

Pathophysiology of chikungunya virus infection associated with fatal outcomes.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes acute, subacute, and chronic human arthritogenic diseases and, in rare instances, can lead to neurological complications and death. Here, we combined epidemiological, virological, histopathological, cytokine, molecular dynamics, metabolomic, proteomic, and genomic analyses to investigate viral and host factors that contribute to chikungunya-associated (CHIK) death. Our results indicate that CHIK deaths are associated with multi-organ infection, central nervous system damage, and elevated serum levels of pro-inflammatory cytokines and chemokines compared with survivors. The histopathologic, metabolite, and proteomic signatures of CHIK deaths reveal hemodynamic disorders and dysregulated immune responses. The CHIKV East-Central-South-African lineage infecting our study population causes both fatal and survival cases. Additionally, CHIKV infection impairs the integrity of the blood-brain barrier, as evidenced by an increase in permeability and altered tight junction protein expression. Overall, our findings improve the understanding of CHIK pathophysiology and the causes of fatal infections.

Chikungunya: a decade of burden in the Americas.

In the Americas, one decade following its emergence in 2013, chikungunya virus (CHIKV) continues to spread and cause epidemics across the region. To date, 3.7 million suspected and laboratory-confirmed chikungunya cases have been reported in 50 countries or territories in the Americas. Here, we outline the current status and epidemiological aspects of chikungunya in the Americas and discuss prospects for future research and public health strategies to combat CHIKV in the region.

Potential use of high-resolution melting analyses for SARS-CoV-2 genomic surveillance.

The pandemic caused by COVID-19 and the emergence of new variants of SARS-CoV-2 have generated clinical and epidemiological impacts on a global scale. The use of strategies for monitoring viral circulation and identifying mutations in genomic regions involved in host interaction are important measures to mitigate viral dissemination and reduce its likely complications on population health. In this context, the objective of this work was to explore the potential of high-resolution melting (HRM) analysis combined with one-step real-time reverse transcription PCR in a closed-tube system, as a fast and convenient method of screening for SARS-CoV-2 mutations with possible implications on host-pathogen interactions. The HRM analyses allowed the distinction of the Gamma, Zeta, Alpha, Delta, and Omicron variants against the predecessors (B.1.1.28, B.1.1.33) of occurrence in Brazil. It is concluded that the molecular tool standardized here has the potential to optimize the genomic surveillance of SARS-CoV-2, and could be adapted for genomic surveillance of other pathogens, due to its ability to detect, prior to sequencing, samples suggestive of new variants, selecting them more assertively and earlier for whole genome sequencing when compared to random screening.

Spatiotemporal dynamics and recurrence of chikungunya virus in Brazil: an epidemiological study.

BACKGROUND: Chikungunya virus (CHIKV) is an Aedes mosquito-borne virus that has caused large epidemics linked to acute, chronic, and severe clinical outcomes. Currently, Brazil has the highest number of chikungunya cases in the Americas. We aimed to investigate the spatiotemporal dynamics and recurrence pattern of chikungunya in Brazil since its introduction in 2013. METHODS: In this epidemiological study, we used CHIKV genomic sequencing data, CHIKV vector information, and aggregate clinical data on chikungunya cases from Brazil. The genomic data comprised 241 Brazilian CHIKV genome sequences from GenBank (n=180) and the 2022 CHIKV outbreak in Ceará state (n=61). The vector data (Breteau index and House index) were obtained from the Brazilian Ministry of Health for all 184 municipalities in Ceará state and 116 municipalities in Tocantins state in 2022. Epidemiological data on laboratory-confirmed cases of chikungunya between 2013 and 2022 were obtained from the Brazilian Ministry of Health and Laboratory of Public Health of Ceará. We assessed the spatiotemporal dynamics of chikungunya in Brazil via time series, mapping, age-sex distribution, cumulative case-fatality, linear correlation, logistic regression, and phylogenetic analyses. FINDINGS: Between March 3, 2013, and June 4, 2022, 253 545 laboratory-confirmed chikungunya cases were reported in 3316 (59·5%) of 5570 municipalities, mainly distributed in seven epidemic waves from 2016 to 2022. To date, Ceará in the northeast has been the most affected state, with 77 418 cases during the two largest epidemic waves in 2016 and 2017 and the third wave in 2022. From 2016 to 2022 in Ceará, the odds of being CHIKV-positive were higher in females than in males (odds ratio 0·87, 95% CI 0·85-0·89, p<0·0001), and the cumulative case-fatality ratio was 1·3 deaths per 1000 cases. Chikungunya recurrences in the states of Ceará, Tocantins (recurrence in 2022), and Pernambuco (recurrence in 2021) were limited to municipalities with few or no previously reported cases in the previous epidemic waves. The recurrence of chikungunya in Ceará in 2022 was associated with a new East-Central-South-African lineage. Population density metrics of the main CHIKV vector in Brazil, Aedes aegypti, were not correlated spatially with locations of chikungunya recurrence in Ceará and Tocantins. INTERPRETATION: Spatial heterogeneity of CHIKV spread and population immunity might explain the recurrence pattern of chikungunya in Brazil. These results can be used to inform public health interventions to prevent future chikungunya epidemic waves in urban settings. FUNDING: Global Virus Network, Burroughs Wellcome Fund, Wellcome Trust, US National Institutes of Health, São Paulo Research Foundation, Brazil Ministry of Education, UK Medical Research Council, Brazilian National Council for Scientific and Technological Development, and UK Royal Society. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.

Genomic Surveillance of Monkeypox Virus, Minas Gerais, Brazil, 2022.

Phylogenetic analysis of 34 monkeypox virus genome sequences isolated from patients in Minas Gerais, Brazil, revealed initial importation events in early June 2022, then community transmission within the state. All generated genomes belonged to the B.1 lineage responsible for a global mpox outbreak. These findings can inform public health measures.

Molecular dynamics simulations of the SARS-CoV-2 Spike protein and variants of concern: structural evidence for convergent adaptive evolution.

The Spike protein's structure of the SARS-CoV-2 provides a unique opportunity to consider perturbations at the atomic level. We used the cryo-electron microscopy structure of the open conformation of the Spike protein to assess the impact of the mutations observed in the variants of concern at the molecular level. Molecular dynamics were subsequently performed with both the wt and the mutated forms to compare the flexibility and variation data for each residue of the three-dimensional fluctuations in the region associated with each alpha carbon. Additionally, protein-protein docking was used to investigate the interaction of each mutated profile with the ACE-2 receptor. After the molecular dynamics, the results show that the mutations increased the stability of the trimeric protein, with greater stability observed in the Gamma variant harboring the 10 characteristic mutations. The results of molecular dynamics, as shown by RMSF demonstrated for the residues that comprise the binding domain receptor (RBD), exhibited a reduction in flexibility, which was more pronounced in the Gamma variant. Finally, protein-protein docking experiments revealed an increase in the number of hydrophobic interactions and hydrogen bonds in the Gamma variant against the ACE-2 receptor, as opposed to the other variants. Taken together, these in silico experiments suggest that the evolution of the mutations favored the increased stability of Spike protein while potentially improving its interaction with the ACE-2 receptor, which in turn may indicate putative structural outcomes of the selection of these mutations in the convergent adaptive evolution as it has been observed for SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Machine learning models exploring characteristic single-nucleotide signatures in yellow fever virus.

Yellow fever virus (YFV) is the agent of the most severe mosquito-borne disease in the tropics. Recently, Brazil suffered major YFV outbreaks with a high fatality rate affecting areas where the virus has not been reported for decades, consisting of urban areas where a large number of unvaccinated people live. We developed a machine learning framework combining three different algorithms (XGBoost, random forest and regularized logistic regression) to analyze YFV genomic sequences. This method was applied to 56 YFV sequences from human infections and 27 from non-human primate (NHPs) infections to investigate the presence of genetic signatures possibly related to disease severity (in human related sequences) and differences in PCR cycle threshold (Ct) values (in NHP related sequences). Our analyses reveal four non-synonymous single nucleotide variations (SNVs) on sequences from human infections, in proteins NS3 (E614D), NS4a (I69V), NS5 (R727G, V643A) and six non-synonymous SNVs on NHP sequences, in proteins E (L385F), NS1 (A171V), NS3 (I184V) and NS5 (N11S, I374V, E641D). We performed comparative protein structural analysis on these SNVs, describing possible impacts on protein function. Despite the fact that the dataset is limited in size and that this study does not consider virus-host interactions, our work highlights the use of machine learning as a versatile and fast initial approach to genomic data exploration.

Retrospective Genomic Surveillance of Chikungunya Transmission in Minas Gerais State, Southeast Brazil.

Brazil accounted for a total number of 1,276,194 reported cases of chikungunya fever between 2014 and 2022. Additionally, since 2015, the country has experienced an increasing death toll, in which the Northeast and Southeast regions appear to report the worst scenarios. Although the CHIKV transmission dynamics have been studied in many parts of the country since its introduction in 2014, little is still known about chikungunya virus (CHIKV) transmission and genetic diversity in the state of Minas Gerais, located in southeast Brazil. Moreover, no studies have been published characterizing CHIKV genomic surveillance in this state. Thus, to retrospectively explore the CHIKV epidemic in Minas Gerais, we generated 40 genomes from clinical samples using Nanopore sequencing. Phylogenetic analysis indicated that multiple introductions of CHIKV occurred, likely from the northeastern Brazilian states, with the most recent common ancestral strain dating to early March 2016, which is in agreement with local epidemiological reports. Additionally, epidemiological data reveals a decline in the number of reported cases from 2017 to 2021, indicating that population immunity or changes in vector activity may have contributed to the decreasing waves of CHIKV infection. Together, our results shed light on the dispersion dynamics of CHIKV and show that infections decreased from March 2017 to January 2021 despite multiple introductions into Minas Gerais State. In conclusion, our study highlights the importance of combining genomic and epidemiological data in order to assist public health laboratories in monitoring and understanding the patterns and diversity of mosquito-borne viral epidemics. IMPORTANCE Arbovirus infections in Brazil, including chikungunya, dengue, yellow fever, and Zika, result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by the limited availability of genomic data. In this study, we combine epidemiological analysis and portable genome sequencing to retrospectively describe the CHIKV epidemic in Minas Gerais between 2017 and 2021. Our results indicate that the East/Central/South African (ECSA) CHIKV lineage was introduced into Minas Gerais by three distinct events, likely from the North and Northeast regions of Brazil. Our study provides an understanding of how CHIKV initiates transmission in the region and illustrates that genomics in the field can augment traditional approaches to infectious disease surveillance and control.

Retrospective Investigation in Horses with Encephalitis Reveals Unnoticed Circulation of West Nile Virus in Brazil.

During these past years, several studies have provided serological evidence regarding the circulation of West Nile virus (WNV) in Brazil. Despite some reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the country. Recently, genomic monitoring activities in horses revealed the circulation of WNV in several Brazilian regions. These findings on the paucity of genomic data reinforce the need for prompt investigation of WNV infection in horses, which may precede human cases of encephalitis in Brazil. Thus, in this study, we retrospectively screened 54 suspicious WNV samples collected between 2017 and 2020 from the spinal cord and brain of horses with encephalitis and generated three new WNV genomes from the Ceará and Bahia states, located in the northeastern region of Brazil. The Bayesian reconstruction revealed that at least two independent introduction events occurred in Brazil. The first introduction event appears to be likely related to the North American outbreak, and was estimated to have occurred in March 2013.The second introduction event appears to have occurred in September 2017 and appears to be likely related to the South American outbreak. Together, our results reinforce the importance of increasing the priority of WNV genomic monitoring in equines with encephalitis in order to track the dispersion of this emerging pathogen through the country.

Field and classroom initiatives for portable sequence-based monitoring of dengue virus in Brazil.

Brazil experienced a large dengue virus (DENV) epidemic in 2019, highlighting a continuous struggle with effective control and public health preparedness. Using Oxford Nanopore sequencing, we led field and classroom initiatives for the monitoring of DENV in Brazil, generating 227 novel genome sequences of DENV1-2 from 85 municipalities (2015-2019). This equated to an over 50% increase in the number of DENV genomes from Brazil available in public databases. Using both phylogenetic and epidemiological models we retrospectively reconstructed the recent transmission history of DENV1-2. Phylogenetic analysis revealed complex patterns of transmission, with both lineage co-circulation and replacement. We identified two lineages within the DENV2 BR-4 clade, for which we estimated the effective reproduction number and pattern of seasonality. Overall, the surveillance outputs and training initiative described here serve as a proof-of-concept for the utility of real-time portable sequencing for research and local capacity building in the genomic surveillance of emerging viruses.

Chikungunya virus ECSA lineage reintroduction in the northeasternmost region of Brazil.

The Northeast region of Brazil registered the second-highest incidence proportion of Chikungunya fever in 2019. In that year, an outbreak consisting of patients presenting with febrile disease associated with joint pain was reported by the public primary health care service in the city of Natal, in the state of Rio Grande do Norte, in March 2019. At first, the aetiological agent of the disease was undetermined. Since much is still unknown about chikungunya virus' (CHIKV) genomic diversity and evolutionary history in this northeasternmost state, we used a combination of portable whole-genome sequencing, molecular clock, and epidemiological analyses that revealed the reintroduction of the CHIKV East-Central-South-African (ECSA) lineage into Rio Grande do Norte. We estimated that the CHIKV ECSA lineage was first introduced into Rio Grande do Norte in early June 2014, while the 2019 outbreak clade diverged around April 2018, during a period of increased Chikungunya incidence in the Southeast region, which might have acted as a source of virus dispersion towards the Northeast region. Together, these results confirm that the ECSA lineage continues to spread across the country through interregional importation events, likely mediated by human mobility.

Fatal Outcome of Chikungunya Virus Infection in Brazil.

BACKGROUND: Chikungunya virus (CHIKV) emerged in the Americas in 2013 and has caused approximately 2.1 million cases and >600 deaths. A retrospective investigation was undertaken to describe clinical, epidemiological, and viral genomic features associated with deaths caused by CHIKV in Ceará state, northeast Brazil. METHODS: Sera, cerebrospinal fluid (CSF), and tissue samples from 100 fatal cases with suspected arbovirus infection were tested for CHIKV, dengue virus (DENV), and Zika virus (ZIKV). Clinical, epidemiological, and death reports were obtained for patients with confirmed CHIKV infection. Logistic regression analysis was undertaken to identify independent factors associated with risk of death during CHIKV infection. Phylogenetic analysis was conducted using whole genomes from a subset of cases. RESULTS: Sixty-eight fatal cases had CHIKV infection confirmed by reverse-transcription quantitative polymerase chain reaction (52.9%), viral antigen (41.1%), and/or specific immunoglobulin M (63.2%). Co-detection of CHIKV with DENV was found in 22% of fatal cases, ZIKV in 2.9%, and DENV and ZIKV in 1.5%. A total of 39 CHIKV deaths presented with neurological signs and symptoms, and CHIKV-RNA was found in the CSF of 92.3% of these patients. Fatal outcomes were associated with irreversible multiple organ dysfunction syndrome. Patients with diabetes appear to die at a higher frequency during the subacute phase. Genetic analysis showed circulation of 2 CHIKV East-Central-South African (ECSA) lineages in Ceará and revealed no unique virus genomic mutation associated with fatal outcome. CONCLUSIONS: The investigation of the largest cross-sectional cohort of CHIKV deaths to date reveals that CHIKV-ECSA strains can cause death in individuals from both risk and nonrisk groups, including young adults.

The ongoing COVID-19 epidemic in Minas Gerais, Brazil: insights from epidemiological data and SARS-CoV-2 whole genome sequencing.

The recent emergence of a coronavirus (SARS-CoV-2), first identified in the Chinese city of Wuhan in December 2019, has had major public health and economic consequences. Although 61,888 confirmed cases were reported in Brazil by 28 April 2020, little is known about the SARS-CoV-2 epidemic in this country. To better understand the recent epidemic in the second most populous state in southeast Brazil - Minas Gerais (MG) - we sequenced 40 complete SARS-CoV-2 genomes from MG cases and examined epidemiological data from three Brazilian states. Both the genome analyses and the geographical distribution of reported cases indicate for multiple independent introductions into MG. Epidemiological estimates of the reproductive number (R) using different data sources and theoretical assumptions suggest the potential for sustained virus transmission despite a reduction in R from the first reported case to the end of April 2020. The estimated date of SARS-CoV-2 introduction into Brazil was consistent with epidemiological data from the first case of a returned traveller from Lombardy, Italy. These findings highlight the nature of the COVID-19 epidemic in MG and reinforce the need for real-time and continued genomic surveillance strategies to better understand and prepare for the epidemic spread of emerging viral pathogens..

Sistema Gerenciador de Ambiente Laboratorial - GAL: Avaliação de uma ferramenta para a vigilância sentinela de síndrome gripal, Brasil, 2011-2012 / Sistema Gerenciador de Ambiente Laboratorial- GAL: Evaluación de una herramienta para la vigilancia centinela del síndrome gripal, Brasil, 2011-201 / Laboratory Environment Management System - GAL: Assessment of a tool for sentinel surveillance of influenza-like illness, Brazil, 2011-2012

OBJETIVO: avaliar o sistema Gerenciador de Ambiente Laboratorial (GAL) como fonte de dados para a Vigilância Sentinela da Síndrome Gripal (VSSG), no Brasil, nos anos de 2011 e 2012. MÉTODOS: estudo de avaliação do GAL a partir da análise dos atributos simplicidade, flexibilidade, qualidade dos dados, aceitabilidade, representatividade, oportunidade, estabilidade do sistema e sua utilidade para a VSSG, com base no guia de avaliação de sistema de vigilância em saúde pública do Centers for Disease Control and Prevention (Atlanta/GA, Estados Unidos). RESULTADOS: nos anos de 2011 e 2012, foram registrados 13.765 exames de vírus respiratórios. O GAL mostrou-se simples de estrutura, flexível às mudanças, com boa qualidade de dados, aceitabilidade e oportunidade no acesso aos resultados dos exames, tendo sido representativo e estável em 23 estados. CONCLUSÃO: o sistema é útil no atendimento aos objetivos da VSSG; contudo, recomenda-se realizar ajustes e incentivar a adesão dos estados que não o utilizam.

OBJETIVO: evaluar el Sistema Gerenciador de Ambiente Laboratorial (GAL) como fuente de datos para la vigilancia centinela de la enfermedad tipo influenza (VCETI). MÉTODOS: estudio de evaluación de GAL a partir del análisis de los atributos simplicidad, flexibilidad, calidad de datos, aceptabilidad, representatividad, oportunidad, estabilidad del sistema y su utilidad para VCETI, utilizando los criterios de la guía de evaluación del Centers for Disease Control and Prevention (Atlanta/GA, EUA). RESULTADOS: el análisis utilizó datos de 2011-2012, en total 13.765 exámenes de virus respiratorios. El GAL demostró tener una estructura simple, flexibilidad al cambio, buena calidad de datos, aceptabilidad y oportunidad en el acceso a los resultados de las pruebas, siendo representativo y estable en 23 de los 27 estados. CONCLUSIÓN: el sistema es útil en el cumplimiento de los objetivos de la vigilancia, sin embargo, se recomienda hacer ajustes y fomentar la adhesión de los Estados que no lo utilizan.

OBJECTIVE: to evaluate the Brazilian Laboratorial Environment Management System (GAL) as a data source for the Sentinel Surveillance of Influenza-Like Illness (SSILI). METHODS: this is an evaluation study of GAL, through the analysis of its simplicity, flexibility, data quality, acceptability, representativeness, timeliness, stability and usefulness for SSILI, based on the guide for system evaluation in public health of the Centers for Disease Control and Prevention (Atlanta/GA, USA). RESULTS: in 2011 and 2012, a total of 13,765 exams for respiratory viruses were registered. GAL presented simple structure, flexibility to changes, good data quality, acceptability and timeliness in the access to test results, being representative and stable in 23 Brazilian states. CONCLUSION: the system is useful in meeting the goals of SSILI; however, there are some recommendations for adjustments and for encouraging the adherence by the states that do not use the system yet.

Laboratory Environment Management System - GAL: Assessment of a tool for sentinel surveillance of influenza-like illness, Brazil, 2011-2012. / Sistema Gerenciador de Ambiente Laboratorial - GAL: Avaliação de uma ferramenta para a vigilância sentinela de síndrome gripal, Brasil, 2011-2012.

OBJECTIVE: to evaluate the Brazilian Laboratorial Environment Management System (GAL) as a data source for the Sentinel Surveillance of Influenza-Like Illness (SSILI). METHODS: this is an evaluation study of GAL, through the analysis of its simplicity, flexibility, data quality, acceptability, representativeness, timeliness, stability and usefulness for SSILI, based on the guide for system evaluation in public health of the Centers for Disease Control and Prevention (Atlanta/GA, USA). RESULTS: in 2011 and 2012, a total of 13,765 exams for respiratory viruses were registered. GAL presented simple structure, flexibility to changes, good data quality, acceptability and timeliness in the access to test results, being representative and stable in 23 Brazilian states. CONCLUSION: the system is useful in meeting the goals of SSILI; however, there are some recommendations for adjustments and for encouraging the adherence by the states that do not use the system yet.

Sistema Gerenciador de Ambiente Laboratorial: relato de experiência de uma ferramenta transformadora para a gestão laboratorial e vigilância em saúde / Laboratory Environment Management System: account of an experience with a transformational tool for laboratory management and health surveillance

Objetivo: apresentar o relato da experiência de implantação do sistema Gerenciador de Ambiente Laboratorial (GAL) como ferramenta de monitoramento e controle de exames laboratoriais, essencial à gestão e ao acompanhamento dos programas de saúde pública brasileira. Métodos: o GAL foi proposto como ferramenta de monitoramento e controle de exames laboratoriais, essencial à gestão e ao acompanhamento dos programas de saúde pública brasileira. O relato foi elaborado a partir de pesquisa documental. Resultados: o GAL tem favorecido a comunicação da informação, fornecendo subsídios para a melhoria na divulgação dos resultados dos ensaios e exames diagnósticos e planos estratégicos na área de saúde. Conclusão: após a implantação do GAL, houve uma melhora substancial na forma de gestão da informação adotada pelos laboratórios de saúde pública.

Objective: to present a report on the experience of implanting the Laboratory Environment Management (GAL) system as a tool for monitoring and controlling laboratory tests. Methods: GAL has been proposed as a tool for monitoring and controlling laboratory tests, vital to the management and monitoring of public health programs in Brazil. The report was compiled from documentary research. Results: GAL has favoured the communication of information, providing input for improving the dissemination of assay and diagnostic test results as well as strategic plans for health. Conclusion: following GAL’s implantation, there has been a substantial improvement in information management by public health laboratories.