Analysis of the interactors of the Orc1 / Cdc6 protein of Trypanosoma cruzi.

Aproximadamente dez milhões de pessoas, predominantemente na América Latina, são acometidas pela doença de Chagas, causada pelo Trypanosoma cruzi. Apesar disso, essa enfermidade apresenta investimentos reduzidos em pesquisas e produção de medicamentos. As drogas atualmente disponíveis para tratar essa doença têm efeitos tóxicos e não são eficazes contra todas as fases da doença ou cepas de parasitas, tornando necessário estudos que apresentem alvos terapêuticos específicos. Nesse contexto, nosso laboratório estuda a replicação do DNA em Trypanosoma, uma vez que caracterizar a replicação deste parasita pode servir como uma ferramenta para buscar possíveis alvos terapêuticos. Neste estudo, investigamos o papel da proteína DNA Polimerase theta-domínio helicase (Pol-helicase) na replicação do T. cruzi. A DNA polimerase theta (Pol) de eucariontes recentes é um membro da família de polimerase e exibe um domínio C-terminal da polimerase, um domínio central e um domínio de helicase N-terminal. A proteína Pol desempenha importantes papéis no reparo do DNA através de seu domínio polimerase, regulando a integridade do genoma. Além disso, em mamíferos, a Pol modula o tempo de disparo da origem e o recrutamento da helicase MCM para a cromatina. Em contraste, o genoma de T. cruzi exibe dois ortólogos putativos individuais de Pol em diferentes locos do genoma; um homólogo ao domínio da polimerase do terminal C de Pol e o outro homólogo ao domínio da helicase, denominado Pol-polimerase e Pol-helicase, respectivamente. Neste estudo, um ensaio de pull-down usando o componente T. cruzi do complexo de pré-replicação Orc1/Cdc6 como isca capturou Pol-helicase do extrato nuclear. Orc1/Cdc6 e Pol-helicase interagiram diretamente e a Pol-helicase recombinante apresentou atividade de desenrolamento do DNA e de ATPase. Além disso, uma cepa de T. cruzi superexpressando o domínio Pol-helicase exibiu uma quantidade significativamente menor de MCM7 ligado ao DNA e prejudicou o disparo na origem da replicação. Utilizamos a metodologia de CRISPR-Cas9 para gerar uma linhagem que expressa a Pol-helicase sem o domínio de helicase, observamos que essa linhagem, de fato, apresentou mais MCM no DNA. Em conjunto, esses dados sugerem que a Pol-helicase modula a replicação do DNA interagindo diretamente com Orc1/Cdc6, o que reduz a ligação do MCM7 ao DNA e, assim, prejudica o disparo das origens de replicação.Approximately 10 million people, predominantly in Latin America, are affected by Chagas\' disease, caused by Trypanosoma cruzi. Despite this, we observe a reduced investment in research and production of new drugs against this parasite. Drugs available to treat this disease have toxic effects and are not effective against all stages of the disease or strains of parasites. Therefore, studies that have specific therapeutic targets are important and necessary. In this context, our laboratory studies DNA replication in Trypanosoma, since components acting in the replication of this parasite can be viewed as therapeutic targets. In this study, we investigated the role of the protein DNA polymerase theta helicase domain (Pol-helicase) in T. cruzi DNA replication. DNA polymerase theta (Pol) from recent eukaryotes is a member of the polymerase family and exhibits a polymerase C-terminal domain, a central domain and a helicase N-terminal domain. The Pol protein plays important roles in DNA repair, regulating the integrity of the genome. In addition, in mammals, Pol modulates the replication origin firing and recruitment of the MCM helicase to the chromatin. In contrast, the T. cruzi genome exhibits two individual putative orthologues of Pol in different loci of the genome; a polymerase domain homolog of the C-terminal of Pol and a homologous to the helicase domain, designated Pol-polymerase and Pol-helicase, respectively. In this study, a pull-down assay using the T. cruzi component of the prereplication complex Orc1/Cdc6 as bait captured Pol-helicase from the nuclear extract. Orc1/Cdc6 and Pol-helicase directly interacted, and Pol-helicase presented DNA unwinding and ATPase activities. T. cruzi strain overexpressing the Pol-helicase domain exhibited a significantly decreased amount of DNA-bound MCM7 and impaired replication origin firing. Taken together, these data suggest that Pol-helicase modulates DNA replication by directly interacting with Orc1/Cdc6, reducing the binding of MCM7 to DNA and thereby impairs the firing of replication origins

Exploring Viral Metagenomics in Pediatric Patients with Acute Respiratory Infections: Unveiling Pathogens beyond SARS-CoV-2

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

Dynamics of SARS-CoV-2 Variants of Concern in Vaccination Model City in the State of Sao Paulo, Brazil

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

Retrospective Insights of the COVID-19 Epidemic in the Major Latin American City, São Paulo, Southeastern Brazil

São Paulo is the financial center of Brazil, with a population of over 12 million, that receives travelers from all over the world for business and tourism. It was the first city in Brazil to report a case of COVID-19 that rapidly spread across the city despite the implementation of the restriction measures. Despite many reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the city of São Paulo. Thus, in this study, we provide a retrospective overview of the COVID-19 epidemic in São Paulo City, Southeastern, Brazil, by generating a total of 9995 near-complete genome sequences from all the city’s different macro-regions (North, West, Central, East, South, and Southeast). Our analysis revealed that multiple independent introduction events of different variants (mainly Gamma, Delta, and Omicron) occurred throughout time. Additionally, our estimates of viral movement within the different macro-regions further suggested that the East and the Southeast regions were the largest contributors to the Gamma and Delta viral exchanges to other regions. Meanwhile, the North region had a higher contribution to the dispersion of the Omicron variant. Together, our results reinforce the importance of increasing SARS-CoV-2 genomic monitoring within the city and the country to track the real-time evolution of the virus and to detect earlier any eventual emergency of new variants of concern that could undermine the fight against COVID-19 in Brazil and worldwide

Retrospective Insights of the COVID-19 Epidemic in the Major Latin American City, São Paulo, Southeastern Brazil

São Paulo is the financial center of Brazil, with a population of over 12 million, that receives travelers from all over the world for business and tourism. It was the first city in Brazil to report a case of COVID-19 that rapidly spread across the city despite the implementation of the restriction measures. Despite many reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the city of São Paulo. Thus, in this study, we provide a retrospective overview of the COVID-19 epidemic in São Paulo City, Southeastern, Brazil, by generating a total of 9995 near-complete genome sequences from all the city’s different macro-regions (North, West, Central, East, South, and Southeast). Our analysis revealed that multiple independent introduction events of different variants (mainly Gamma, Delta, and Omicron) occurred throughout time. Additionally, our estimates of viral movement within the different macro-regions further suggested that the East and the Southeast regions were the largest contributors to the Gamma and Delta viral exchanges to other regions. Meanwhile, the North region had a higher contribution to the dispersion of the Omicron variant. Together, our results reinforce the importance of increasing SARS-CoV-2 genomic monitoring within the city and the country to track the real-time evolution of the virus and to detect earlier any eventual emergency of new variants of concern that could undermine the fight against COVID-19 in Brazil and worldwide

Correction: Lesbon et al. Nucleocapsid (N) Gene Mutations of SARS-CoV-2 Can Affect Real-Time RT-PCR Diagnostic and Impact False-Negative Results. <i>Viruses</i> 2021, <i>13</i>, 2474

The authors hereby request the inclusion of two authors (Olivia Teixeira and Maria Cristina Nonato) in the recently published article in Viruses entitled “Nucleocapsid (N) gene mutations of SARS-CoV-2 can affect real-time RT-PCR diagnostic and impact false-negative results” [...

Genomic epidemiology reveals how restriction measures shaped the SARS-CoV-2 epidemic in Brazil

Abstract Brazil has experienced some of the highest numbers of COVID-19 infections and deaths globally and made Latin America a pandemic epicenter from May 2021. Although SARS-CoV-2 established sustained transmission in Brazil early in the pandemic, important gaps remain in our understanding of local virus transmission dynamics. Here, we describe the genomic epidemiology of SARS-CoV-2 using near-full genomes sampled from 27 Brazilian states and an adjacent country - Paraguay. We show that the early stage of the pandemic in Brazil was characterised by the co-circulation of multiple viral lineages, linked to multiple importations predominantly from Europe, and subsequently characterized by large local transmission clusters. As the epidemic progressed, the absence of effective restriction measures led to the local emergence and international spread of Variants of Concern (VOC) and under monitoring (VUM), including the Gamma (P.1) and Zeta (P.2) variants. In addition, we provide a preliminary genomic overview of the epidemic in Paraguay, showing evidence of importation from Brazil. These data reinforce the need for the implementation of widespread genomic surveillance in South America as a toolkit for pandemic monitoring and providing a means to follow the real-time spread of emerging SARS-CoV-2 variants with possible implications for public health and immunization strategies