Functional evaluation of extracellular vesicles in human Tcell lymphotropic virus 1 (HTLV-1) infection

O vírus T-linfotrópico humano 1 (HTLV-1) é o agente etiológico da leucemia/linfoma de células T do adulto (ATL) e da paraparesia espástica tropical/mielopatia associada ao HTLV-1 (HAM/TSP). O mecanismo que leva a estas manifestações clínicas tão distintas ainda não está elucidado. No entanto, sabe-se que os genes virais tax e HTLV-1 basic leucine zipper factor (HBZ) estão relacionados à infectividade viral e ao desenvolvimento de complicações neurológicas e hematológicas. Atualmente, existem evidências de que as células infectadas pelo HTLV-1 podem liberar vesículas extracelulares (VE) que participam da disseminação de partículas virais. Visando auxiliar a compreensão da patogênese do HTLV-1, este trabalho avaliou os níveis de expressão de tax e HBZ em VE provenientes do soro de indivíduos infectados, bem como o papel destas na modulação da resposta imune. As VE foram isoladas por precipitação polimérica e caracterizadas quanto ao diâmetro, a presença dos marcadores proteicos Alix, GAPDH e CD9 e ausência da proteína viral p19. A análise da expressão dos genes tax e HBZ foi realizada por PCR quantitativa e comparada à carga proviral (CPV) em células mononucleares de sangue periférico (PBMC) dos portadores assintomáticos e sintomáticos para a HAM/TSP. Os níveis de transcritos dos genes tax e HBZ em VE foram correlacionados positivamente à CPV dos indivíduos HAM/TSP. Apenas os indivíduos com CPV acima de 6 mil cópias para cada 10^5 PBMC apresentaram transcritos virais em VE. Ademais, observou-se que, quando em níveis detectáveis, as unidades relativas de expressão (URE) de HBZ foram de 2 a 12 vezes maiores que as de tax. A seguir, a expressão e secreção de citocinas inflamatórias foi avaliada em PBMC de indivíduos saudáveis e de portadores do HTLV-1, expostas a crescentes doses de VE tax+ e HBZ+. A análise da expressão gênica por PCR quantitativa indicou aumento de transcritos da interleucina 8 (IL-8) em PBMC infectada de CPV baixa decorrentes do tratamento com VE infectadas. Este aumento também foi observado a nível proteico, confirmado pelo ensaio multiplex customizado MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore), que avaliou a presença de citocinas inflamatórias no sobrenadante de cultivo. Interessantemente, o aumento de IL-8 chegou a níveis próximos ao observado em PBMC de CPV elevada. Estes achados indicam que a expressão de transcritos virais em VE derivadas do soro de portadores do HTLV-1 está relacionada à CPV apresentada pelo indivíduo. Adicionalmente, VE tax+ e HBZ+ podem induzir a produção de citocinas inflamatórias em pacientes de CPV reduzida, o que pode estar relacionado ao desenvolvimento de sintomas na infecção pelo HTLV-1.Human T-lymphotropic virus 1 (HTLV-1) is the etiologic agent of adult cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the major questions in HTLV-1 studies is related to the understanding of causes that lead to different clinical manifestations. However, it is well known that the viral genes tax and HTLV-1 basic leucine zipper factor (HBZ) are related to viral infectivity and the development of neurological and hematological diseases. Currently, there is evidence that HTLV-1 infected cells can release extracellular vesicles (EV) involved in mechanisms of viral particles spreading. Therefore, we evaluated the expression levels of tax and HBZ viral transcripts in serum-derived EV from HTLV-1 carriers, as well as the role of these vesicles in the modulation of the immune response. Serum-derived EV were enriched by polymeric precipitation and characterized by size and protein composition (Alix, GAPDH and CD9), as well as the absence of p19 viral protein. The analysis of tax and HBZ gene expression was performed by quantitative PCR (qPCR) and compared to the proviral load (PVL) of asymptomatic carriers and HAM/TSP carriers. Three HAM/TSP carriers presented detectable levels of tax and HBZ transcripts in VE, and it was positively correlated to the PVL in peripheral blood mononuclear cells (PBMC). The detection of viral transcripts was only true in individuals with PVL higher than 6,000/105 PBMC. When detectable, the expression units of HBZ presented a 2 to 12 fold increase over tax expression units. Next, the expression and secretion of inflammatory cytokines were evaluated in PBMC from healthy individuals and HTLV-1 carriers exposed to increasing doses of EV tax+ HBZ+. Gene expression analysis indicated an increase in interleukin 8 (IL-8) transcripts of HTLV-1-infected PBMC with low PVL, due to treatment with EV tax+ HBZ+. The increase was also observed at protein level in cell culture supernatant, confirmed by the multiplex assay MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore). Interestingly, the increase in IL-8 levels were close to that seen in HTLV-1-infected PBMC with high PVL. Taken together, these findings indicate that the expression of viral transcripts in serumderived EV of HTLV-1 carriers is related to the PVL presented by the infected individual. Additionally, EV tax+ HBZ+ can induce the production of inflammatory cytokines in patients with low PVL, which may be related to the development of symptoms in HTLV-1 infection

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” [...