Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A "naked" rod-like conformation similar but not identical to that observed in vitro

[EN] With a minimal (250-400nt), non-protein-coding, circular RNA genome, viroids rely on sequence/structural motifs for replication and colonization of their host plants. These motifs are embedded in a compact secondary structure whose elucidation is crucial to understand how they function. Viroid RNA structure has been tackled in silico with algorithms searching for the conformation of minimal free energy, and in vitro by probing in solution with RNases, dimethyl sulphate and bisulphite, and with selective 2-hydroxyl acylation analyzed by primer extension (SHAPE), which interrogates the RNA backbone at single-nucleotide resolution. However, in vivo approaches at that resolution have not been assayed. Here, after confirming by 3 termodynamics-based predictions and by in vitro SHAPE that the secondary structure adopted by the infectious monomeric circular (+) RNA of potato spindle tuber viroid (PSTVd) is a rod-like conformation with double-stranded segments flanked by loops, we have probed it in vivo with a SHAPE modification. We provide direct evidence that a similar, but not identical, rod-like conformation exists in PSTVd-infected leaves of Nicotiana benthamiana, verifying the long-standing view that this RNA accumulates in planta as a naked form rather than tightly associated with protecting host proteins. However, certain nucleotides of the central conserved region, including some of the loop E involved in key functions such as replication, are more SHAPE-reactive in vitro than in vivo. This difference is most likely due to interactions with proteins mediating some of these functions, or to structural changes promoted by other factors of the in vivo habitat.This work was supported by grant BFU2014-56812-P (to R.F.) from the Ministerio de Economia y Competitividad of Spain. A.L.C. was the recipient of a predoctoral fellowship from the same organism.López-Carrasco, MA.; Flores Pedauye, R. (2017). Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A "naked" rod-like conformation similar but not identical to that observed in vitro. RNA Biology. 14(8):1046-1054. https://doi.org/10.1080/15476286.2016.1223005S1046105414

Advances in plant virus evolution: translating evolutionary insights into better disease management

[EN] Recent studies in plant virus evolution are revealing that genetic structure and behavior of virus and viroid populations can explain important pathogenic properties of these agents, such as host resistance breakdown, disease severity, and host shifting, among others. Genetic variation is essential for the survival of organisms. The exploration of how these subcellular parasites generate and maintain a certain frequency of mutations at the intra- and inter-host levels is revealing novel molecular virus plant interactions. They emphasize the role of host environment in the dynamic genetic composition of virus populations. Functional genomics has identified host factors that are transcriptionally altered after virus infections. The analyses of these data by means of systems biology approaches are uncovering critical plant genes specifically targeted by viruses during host adaptation. Also, a next-generation re-sequencing approach of a whole virus genome is opening new avenues to study virus recombination and the relationships between intra-host virus composition and pathogenesis. Altogether, the analyzed data indicate that systematic disruption of some specific parameters of evolving virus populations could lead to more efficient ways of disease prevention, eradication, or tolerable virus plant coexistence.Acosta-Leal, R.; Duffy, S.; Xiong, Z.; Hammond, R.; Elena Fito, SF. (2011). Advances in plant virus evolution: translating evolutionary insights into better disease management. Phytopathology. 101(10):1136-1148. doi:10.1094/ PHYTO-01-11-0017S113611481011

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

[EN] Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplastreplicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.This work was supported by the European Research Council (erc.europa.eu; ERC-2011-StG-281191-VIRMUT to RS), the Spanish Ministerio de Economia y Competitividad (www.mineco.gob.es; BFU2013-41329 grant to RS, BFU2014-56812-P grant to RF, and a predoctoral fellowship to ALC), and the Spanish Junta de Comunidades de Castilla-La Mancha (www.castillalamancha.es;postdoctoral fellowship to CB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.López-Carrasco, MA.; Ballesteros Martínez, C.; Sentandreu, V.; Delgado Villar, SG.; Gago Zachert, SP.; Flores Pedauye, R.; Sanjuan Verdeguer, R. (2017). Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing. PLoS Pathogens. 13(9):1-17. https://doi.org/10.1371/journal.ppat.1006547S117139Ganai, R. A., & Johansson, E. (2016). DNA Replication—A Matter of Fidelity. Molecular Cell, 62(5), 745-755. doi:10.1016/j.molcel.2016.05.003Lynch, M. (2010). Evolution of the mutation rate. Trends in Genetics, 26(8), 345-352. doi:10.1016/j.tig.2010.05.003Sanjuán, R., & Domingo-Calap, P. (2016). Mechanisms of viral mutation. Cellular and Molecular Life Sciences, 73(23), 4433-4448. doi:10.1007/s00018-016-2299-6Gago, S., Elena, S. F., Flores, R., & Sanjuan, R. (2009). 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Clinical relevance of timing of assessment of ICU mortality in patients with moderate-to-severe Acute Respiratory Distress Syndrome

Mortality is a frequently reported outcome in clinical studies of acute respiratory distress syndrome (ARDS). However, timing of mortality assessment has not been well characterized. We aimed to identify a crossing-point between cumulative survival and death in the intensive care unit (ICU) of patients with moderate-to-severe ARDS, beyond which the number of survivors would exceed the number of deaths. We hypothesized that this intersection would occur earlier in a successful clinical trial vs. observational studies of moderate/severe ARDS and predict treatment response. We conducted an ancillary study of 1580 patients with moderate-to-severe ARDS managed with lung-protective ventilation to assess the relevance and timing of measuring ICU mortality rates at different time-points during ICU stay. First, we analyzed 1303 patients from four multicenter, observational cohorts enrolling consecutive patients with moderate/severe ARDS. We assessed cumulative ICU survival from the time of moderate/severe ARDS diagnosis to ventilatory support discontinuation within 7-days, 28-days, 60-days, and at ICU discharge. Then, we compared these findings to those of a successful randomized trial of 277 moderate/severe ARDS patients. In the observational cohorts, ICU mortality (487/1303, 37.4%) and 28-day mortality (425/1102, 38.6%) were similar (p = 0.549). Cumulative proportion of ICU survivors and non-survivors crossed at day-7; after day-7, the number of ICU survivors was progressively higher compared to non-survivors. Measures of oxygenation, lung mechanics, and severity scores were different between survivors and non-survivors at each point-in-time (p < 0.001). In the trial cohort, the cumulative proportion of survivors and non-survivors in the treatment group crossed before day-3 after diagnosis of moderate/severe ARDS. In clinical ARDS studies, 28-day mortality closely approximates and may be used as a surrogate for ICU mortality. For patients with moderate-to-severe ARDS, ICU mortality assessment within the first week of a trial might be an early predictor of treatment response

In vitro and in vivo self-cleavage of a viroid RNA with a mutation in the hammerhead catalytic pocket.

Peach latent mosaic viroid (PLMVd) can adopt hammerhead structures in both polarity strands. In the course of a study on the variability of this viroid a natural sequence variant has been characterized in which the hammerhead structure of the plus polarity strand has the sequence CCGA instead of the conserved uridine turn motif CUGA present in the catalytic pocket of all natural hammerhead structures. The viroid RNA containing this mutant hammerhead structure, but not those with the two other possible substitutions, U-->A and U-->G, in the same position of the catalytic pocket, showed significant self-cleavage activity during in vitro transcription. Moreover, the corresponding full-length PLMVd cDNA was infectious and the mutation was retained in a fraction of the viroid progeny. These results indicate that the sequence flexibility of the hammerhead structure, acting in vitro and in vivo , is higher than anticipated and provide relevant data for a deeper insight into the catalytic mechanism of this class of ribozymes and into the structure of the uridine turn motif

Larvas de simulídeos (Diptera, Simuliidae) do centro oeste, sudeste e sul do Brasil, parasitadas por microsporídeos (Protozoa) e mermitídeos (Nematoda) Simulids larvae (Diptera, Simuliidae) from middle western, southeastern and southern Brazil, with microsporids (Protozoa) and mermithids (Nematoda) parasites

<abstract language="eng">A survey of simulid larval parasites was carried out in different localities of the states of Mato Grosso, Minas Gerais, São Paulo, Paraná, Santa Catarina and Rio Grande do Sul, Brazil, from February 1996 to May 1998. Prevalences for the microsporidian Polydispyrenia simulii Lutz & Splendore, 1908 were found in Morungaba and Leme, São Paulo, ranging from around 0.7 to 66.7%, depending mainly on the host simulid species. Microsporidiosis was registered in localities of São Paulo, Paraná, Santa Catarina and Rio Grande do Sul. Parasitism by Isomermis sp. (Nematoda, Mermithidae) was found in Simulium larvae from Serra do Japi, ranging from 0.8 to 45.8%, depending on the simulid species and the larval microhabitat in the stream, whether a cemented ramp in a lake outlet or the natural stream bed. Parasitism by mermithids was also found in ten localities. Mycoses caused by Coelomycidium sp. were for the first time recorded for larvae of Simulium (Chirostilbia) pertinax Kollar, 1832

Conflicto social en jóvenes consumidores de heroína

Presentamos en nuestro Boletín un trabajo sobre la relación del consumo de drogas y la actividad delictiva. Se trata del estudio ITINERE, financiado por la Fundación para la investigación y prevención del SIDA en España (IPSE). El objetivo de este trabajo consiste en descubrir las variables relacionadas con el conflicto social a partir de una cohorte de consumidores habituales de heroína. Para ello se ha entrevistado a 991 jóvenes drogodependientes seleccionados en tres ciudades españolas: Barcelona, Madrid y Sevilla entre los años 2001 y 2003. La captación de estos individuos se realizó mediante personas claves que conocían los escenarios habituales de consumo y por métodos de referencia en cadena a partir de los propios entrevistados. Una vez más se confirma que el consumo de droga no se encuentra estrechamente relacionado con la comisión de delitos. Una versión más completa de este estudio se ha publicado recientemente en la "Revista Española de Salud Pública"