Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil

Background: The outbreak of Zika virus (ZIKV) in the Americas has transformed a previously obscure mosquito-transmitted arbovirus of the Flaviviridae family into a major public health concern. Little is currently known about the evolution and biology of ZIKV and the factors that contribute to the associated pathogenesis. Determining genomic sequences of clinical viral isolates and characterization of elements within these are an important prerequisite to advance our understanding of viral replicative processes and virus-host interactions. Methodology/Principal findings: We obtained a ZIKV isolate from a patient who presented with classical ZIKV-associated symptoms, and used high throughput sequencing and other molecular biology approaches to determine its full genome sequence, including non-coding regions. Genome regions were characterized and compared to the sequences of other isolates where available. Furthermore, we identified a subgenomic flavivirus RNA (sfRNA) in ZIKV-infected cells that has antagonist activity against RIG-I induced type I interferon induction, with a lesser effect on MDA-5 mediated action. Conclusions/Significance: The full-length genome sequence including non-coding regions of a South American ZIKV isolate from a patient with classical symptoms will support efforts to develop genetic tools for this virus. Detection of sfRNA that counteracts interferon responses is likely to be important for further understanding of pathogenesis and virus-host interactions

Estudo da evolução e arquitetura de íntrons mínimos

Eukaryotic introns show a wide span of size, from only 30bp to large 3.6Mbp. However, analysis of intron size distribution in diverse lineages shows a frequent accumulation of introns near the minimum size that is referred to as minimal introns. In this work, structure and evolution of minimal introns were studied based on diverse species of bilaterian animals, especially the platyhelminth Schistosoma mansoni and species from the Vertebrata phylum. Analysis of the distribution of introns size from Schistosoma mansoni shows a minimal intron peak at 34bp, a remarkably short size when compared to other eukaryotic species. Minimal introns from Schistosoma mansoni were preferentially found in some specific chromosomes. While studying intron retention (IR) and splicing signals, it was observed that premature termination codons (PTC) were preferentially found in the second and last codons of minimal introns due to contribution of the splice sites sequences. Symmetric minimal introns display the highest proportion of PTC-containing introns. We speculate that this observation reflects an evolutionary pressure associated with the fact that its retention does not shift the reading frame of translation. Interestingly, the proportion of PTC-containing introns does not increase with size in symmetric minimal introns as observed for non-symmetric minimal introns. We suggest that a large fraction of symmetric minimal introns that do not present PTC may be retained for the production of isoforms with few additional amino acid residues. The lack of preference of minimal introns with PTC for any position along the gene suggests that nonsense-mediated decay of Schistosoma mansoni is independent of exon junction complex (EJC). Study of the evolution of minimal introns from vertebrates shows that the acquisition of homeothermy had a great influence on minimal introns GC%. In high body temperature species, minimal introns can be divided into low and high GC% populations, with peaks of ~30% and ~70% respectively. Analysis of the human genome shows that, although the GC% variation was more prominent in minimal introns, the entire gene sequence varies. Genes without minimal introns do not appear to show GC% variation dependent on the body temperature. This suggests that minimal introns can serve as proxies for detecting temperature-responsive genes in humans. The transition from low to high GC% of some minimal introns may be impaired due to high IR levels in the intermediate GC%. Low GC% minimal intron-containing genes were related to cell division and thus transition to high GC% may be cumbersome due to high levels of IR. Furthermore, genes with low GC% minimal introns were observed to be related to oncogenic transformation and to be highly expressed in the meiosis process. Based on these results, we propose that minimal intron-containing genes could represent a new interesting system for studying diseases related to division defects, such as cancer and infertility. Also, as IR has been observed to be an important factor for selecting minimal introns GC%, the participation of minimal intron-containing genes in diseases in which increase of IR could be associated, such as diabetes type I and cancer, may be further explored.Íntrons de eucariotos possuem diversos tamanhos, desde diminutos 30pb a 3.6Mpb. Entretanto, análises da distribuição de tamanho de íntrons em diversas linhagens mostram um frequente acúmulo de íntrons próximos ao tamanho mínimo, os quais são chamados de íntrons mínimos. Neste trabalho, a estrutura e evolução de íntrons mínimos foram estudadas em diversas espécies de animais bilaterais, especialmente o platelminto Schistosoma mansoni e espécies do filo Vertebrata. Análise de distribuição de tamanho de íntrons de Schistosoma mansoni mostra um pico de íntrons mínimos em 34pb, um tamanho notavelmente pequeno se comparado ao de outras espécies de eucariotos. Íntrons mínimos de Schistosoma mansoni foram preferencialmente encontrados em certos cromossomos. Ao estudar retenção intrônica (IR) e sinais de splicing, observou-se que códons de parada prematura (PTC) são preferencialmente encontrados no segundo e último códons de íntrons mínimos devido à contribuição das sequências dos sítios de splicing. Íntrons mínimos simétricos apresentam maior proporção de íntrons com PTC. Sugerimos que essa constatação reflita pressões evolutivas associadas ao fato da retenção destes íntrons não mudarem o quadro de leitura de tradução. Interessantemente, a proporção de íntron mínimos com PTC não aumenta conforme o tamanho de íntrons mínimos simétricos como visto em íntrons mínimos não simétricos. Sugerimos que grande parte de íntrons mínimos simétricos que não possuam PTC possam ser retidos para produção de isoformas com alguns resíduos de aminoácidos adicionais. A não preferência de íntrons mínimos com PTC por posições ao longo do gene sugere que o decaimento mediado por códons de parada (NMD) independe do complexo de junção de éxons (EJC). Estudo da evolução de íntrons mínimos de vertebrados mostra que a aquisição da homeotermia teve grande influência no conteúdo GC de íntrons mínimos. Em espécies com alta temperatura corpórea, os íntrons mínimos podem ser divididos em populações de baixo e alto GC%, com picos de ~30% e ~70% respectivamente. Análises do genoma humano mostram que, embora variações de GC% sejam proeminentes em íntrons mínimos, sequências gênicas inteiras variam. Genes sem íntrons mínimos não apresentam aparente variação de GC% dependente de temperatura corpórea. Isso sugere que íntrons mínimos possam servir como marca para identificação de genes responsivos à temperatura em humanos. A transição de baixo para alto GC% por alguns íntrons mínimos pode ser comprometida devido aos altos níveis de retenção no GC% intermediário. Genes com íntrons mínimos de baixo GC% são relacionados a divisão celular e, portanto, a transição para alto GC% pode ser complicada devido aos altos níveis de retenção. Ademais, observou-se que genes com íntrons mínimos de baixo GC% estão relacionados à transformação oncogênica e que são altamente expressos na meiose. Baseando-se nestes resultados, propomos que genes com íntron mínimo possam representar um novo sistema de estudo de doenças relacionadas a defeitos de divisão celular, como câncer e infertilidade. Ainda, devido a IR ser um fator importante para seleção de GC% de íntrons mínimos, deve-se melhor explorar a participação de genes com íntrons mínimos em doenças nas quais o aumento da IR possa estar associado, como diabetes tipo I e câncer

La experiencia de enseñar habilidades de programación introductoria a biocientíficos en Brasil

La biología computacional ha ganado fuerza como disciplina científica independiente en los últimos años en América del Sur. Sin embargo, todavía existe una necesidad creciente de biocientíficos, de diferentes orígenes y con diferentes niveles, para adquirir habilidades de programación, lo que podría reducir el tiempo de los datos a los conocimientos y conectar la comunicación entre los científicos de la vida y los informáticos. Python es un lenguaje de programación ampliamente utilizado en bioinformática y ciencia de datos, que es particularmente adecuado para principiantes. Aquí, describimos la concepción, organización e implementación del Taller Brasileño de Python para Datos Biológicos. Este taller ha sido organizado por estudiantes de pregrado y posgrado y apoyado, principalmente en asuntos administrativos, por profesores experimentados desde 2017. El taller fue concebido para enseñar a biocientíficos, principalmente estudiantes en Brasil, sobre cómo programar en un contexto biológico. El objetivo de este artículo fue compartir nuestra experiencia con la edición 2020 del taller en su formato virtual debido a la pandemia de la Enfermedad por Coronavirus 2019 (COVID-19) y comparar y contrastar la experiencia de este año con las ediciones presenciales anteriores. Describimos un modelo de taller práctico y de codificación en vivo para enseñar la programación introductoria de Python. También destacamos las adaptaciones realizadas de formato presencial a online en 2020, la evaluación de los participantes sobre la progresión del aprendizaje y la gestión general del taller. Por último, brindamos un resumen y reflexiones de nuestras experiencias personales de los talleres de los últimos 4 años. Nuestras conclusiones incluyeron los beneficios del aprendizaje a partir de los comentarios de los alumnos (LLF, por sus siglas en inglés) que nos permitieron mejorar el taller en tiempo real, a corto y probablemente a largo plazo. Concluimos que el Taller Brasileño de Python para Datos Biológicos es un modelo de taller altamente efectivo para la enseñanza de un lenguaje de programación que permite a los biocientíficos ir más allá de una exploración inicial de habilidades de programación para el análisis de datos a mediano y largo plazo.Computational biology has gained traction as an independent scientific discipline over the last years in South America. However, there is still a growing need for bioscientists, from different backgrounds, with different levels, to acquire programming skills, which could reduce the time from data to insights and bridge communication between life scientists and computer scientists. Python is a programming language extensively used in bioinformatics and data science, which is particularly suitable for beginners. Here, we describe the conception, organization, and implementation of the Brazilian Python Workshop for Biological Data. This workshop has been organized by graduate and undergraduate students and supported, mostly in administrative matters, by experienced faculty members since 2017. The workshop was conceived for teaching bioscientists, mainly students in Brazil, on how to program in a biological context. The goal of this article was to share our experience with the 2020 edition of the workshop in its virtual format due to the Coronavirus Disease 2019 (COVID-19) pandemic and to compare and contrast this year’s experience with the previous in-person editions. We described a hands-on and live coding workshop model for teaching introductory Python programming. We also highlighted the adaptations made from in-person to online format in 2020, the participants’ assessment of learning progression, and general workshop management. Lastly, we provided a summary and reflections from our personal experiences from the workshops of the last 4 years. Our takeaways included the benefits of the learning from learners’ feedback (LLF) that allowed us to improve the workshop in real time, in the short, and likely in the long term. We concluded that the Brazilian Python Workshop for Biological Data is a highly effective workshop model for teaching a programming language that allows bioscientists to go beyond an initial exploration of programming skills for data analysis in the medium to long term

La experiencia de enseñar habilidades de programación introductoria a biocientíficos en Brasil

La biología computacional ha ganado fuerza como disciplina científica independiente en los últimos años en América del Sur. Sin embargo, todavía existe una necesidad creciente de biocientíficos, de diferentes orígenes y con diferentes niveles, para adquirir habilidades de programación, lo que podría reducir el tiempo de los datos a los conocimientos y conectar la comunicación entre los científicos de la vida y los informáticos. Python es un lenguaje de programación ampliamente utilizado en bioinformática y ciencia de datos, que es particularmente adecuado para principiantes. Aquí, describimos la concepción, organización e implementación del Taller Brasileño de Python para Datos Biológicos. Este taller ha sido organizado por estudiantes de pregrado y posgrado y apoyado, principalmente en asuntos administrativos, por profesores experimentados desde 2017. El taller fue concebido para enseñar a biocientíficos, principalmente estudiantes en Brasil, sobre cómo programar en un contexto biológico. El objetivo de este artículo fue compartir nuestra experiencia con la edición 2020 del taller en su formato virtual debido a la pandemia de la Enfermedad por Coronavirus 2019 (COVID-19) y comparar y contrastar la experiencia de este año con las ediciones presenciales anteriores. Describimos un modelo de taller práctico y de codificación en vivo para enseñar la programación introductoria de Python. También destacamos las adaptaciones realizadas de formato presencial a online en 2020, la evaluación de los participantes sobre la progresión del aprendizaje y la gestión general del taller. Por último, brindamos un resumen y reflexiones de nuestras experiencias personales de los talleres de los últimos 4 años. Nuestras conclusiones incluyeron los beneficios del aprendizaje a partir de los comentarios de los alumnos (LLF, por sus siglas en inglés) que nos permitieron mejorar el taller en tiempo real, a corto y probablemente a largo plazo. Concluimos que el Taller Brasileño de Python para Datos Biológicos es un modelo de taller altamente efectivo para la enseñanza de un lenguaje de programación que permite a los biocientíficos ir más allá de una exploración inicial de habilidades de programación para el análisis de datos a mediano y largo plazo.Computational biology has gained traction as an independent scientific discipline over the last years in South America. However, there is still a growing need for bioscientists, from different backgrounds, with different levels, to acquire programming skills, which could reduce the time from data to insights and bridge communication between life scientists and computer scientists. Python is a programming language extensively used in bioinformatics and data science, which is particularly suitable for beginners. Here, we describe the conception, organization, and implementation of the Brazilian Python Workshop for Biological Data. This workshop has been organized by graduate and undergraduate students and supported, mostly in administrative matters, by experienced faculty members since 2017. The workshop was conceived for teaching bioscientists, mainly students in Brazil, on how to program in a biological context. The goal of this article was to share our experience with the 2020 edition of the workshop in its virtual format due to the Coronavirus Disease 2019 (COVID-19) pandemic and to compare and contrast this year’s experience with the previous in-person editions. We described a hands-on and live coding workshop model for teaching introductory Python programming. We also highlighted the adaptations made from in-person to online format in 2020, the participants’ assessment of learning progression, and general workshop management. Lastly, we provided a summary and reflections from our personal experiences from the workshops of the last 4 years. Our takeaways included the benefits of the learning from learners’ feedback (LLF) that allowed us to improve the workshop in real time, in the short, and likely in the long term. We concluded that the Brazilian Python Workshop for Biological Data is a highly effective workshop model for teaching a programming language that allows bioscientists to go beyond an initial exploration of programming skills for data analysis in the medium to long term

Primers sequences used for 5’/3’ RACE of ZIKV viral termini.

<p>Primers sequences used for 5’/3’ RACE of ZIKV viral termini.</p

Activation of the IFN-β promoter by poly I:C in cells over expressing ZIKV sfRNA.

<p>A549 cells were co-transfected with either pDEST-DENV-3’UTR, pDEST-ZIKV PE243-3‘UTR or pDEST40-MBP (sfRNA over-expression plasmids and MBP-HDVr control, respectively) and p125Luc IFN-β promoter reporter (expressing Firefly luciferase) along with pRL-CMV (internal control, expressing <i>Renilla</i> luciferase). The IFN-β promoter was stimulated by transfecting poly I:C 24 h after the primary transfection. The relative luciferase activity (Firefly/<i>Renilla</i>) was analyzed at 24 h following the second transfection. The mean with standard error is shown for three independent experiments performed in triplicate; values of independent experiments were used for analysis. The data were normalized to cells transfected with pDEST40-MBP without any poly I:C treatment. Asterisk (*) indicates significance (2-way ANOVA, p<0.05).</p

Comparison of African and Asian lineage ZIKV protein coding regions.

<p>The mean pairwise identity of all pairs at a given position is indicated by the identity bar; light blue denotes 100% pairwise identity, dark blue highlights positions possessing less than 100% pairwise identity. Positions and quantity of amino acid substitutions are indicated by black bands within grey sequence bars. Sequences 1–37, highlighted yellow, correspond to the outbreak originating in 2015 in South America. Microcephaly, adult mortality and ZIKV PE243 associated sequences are highlighted as previously described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.g001" target="_blank">Fig 1</a>.</p

Primers used for cloning of ZIKV 3'UTR containing sfRNA.

<p>Primers used for cloning of ZIKV 3'UTR containing sfRNA.</p

Comparison of the 5’UTR nucleotide sequences of Asian and African ZIKV isolates.

<p>The mean pairwise identity of all pairs at a given position is indicated by the identity bar; lilac is indicative of 100% pairwise identity, dark purple highlights positions possessing <100% pairwise identity. Positions and quantity of single nucleotide polymorphisms (SNPs) are represented as black bands within grey sequence bars. Sequences 1–32, highlighted orange, correspond to the outbreak originating in 2015 in Brazil. Microcephaly, adult mortality and ZIKV PE243 associated sequences are highlighted as previously described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005048#pntd.0005048.g001" target="_blank">Fig 1</a>.</p