Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported, but little attention has been paid so far to their sensitivity and reliability for diagnostic purposes. We therefore compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large scale performance test ten datasets of 21-24 nt small (s)RNA sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty to detect viral agents when they are novel and/or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases and (iv) the significant level of scientific expertise needed when interpreting pipelines results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

SummaryThe ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway.

The ecology of plant viruses began to be explored at the end of the 19th century. Since then, major advances have revealed mechanisms of virus-host-vector interactions in various environments. These advances have been accelerated by new technlogies for virus detection and characterization, most recently including high throughput sequencing (HTS). HTS allows investigators, for the first time, to characterize all or nearly all viruses in a sample without a priori information about which viruses might be present. This powerful approach has spurred new investigation of the viral metagenome (virome). The rich virome datasets accumulated illuminate important ecological phenomena such as virus spread among host reservoirs (wild and domestic), effects of ecosystem simplification caused by human activities (and agriculture) on the biodiversity and the emergence of new viruses in crops. To be effective, however, HTS-based virome studies must successfully navigate challenges and pitfalls at each procedural step, from plant sampling to library preparation and bioinformatic analyses. This review summarizes major advances in plant virus ecology associated with technological developments, and then presents important considerations and best practices for HTS use in virome studies.

a b s t r a c tBeyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR-induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in.

Over the last decade, viral metagenomic studies have resulted in the discovery of thousands of previously unknown viruses. These studies are likely to play a pivotal role in obtaining an accurate and robust understanding of how viruses affect the stability and productivity of ecosystems. Among the metagenomics-based approaches that have been developed since the beginning of the 21st century, shotgun metagenomics applied specifically to virion-associated nucleic acids (VANA) has been used to disentangle the diversity of the viral world. We summarize herein the results of 24 VANA-based studies, focusing on plant and insect samples conducted over the last decade (2010-2020). Collectively, viruses from 85 different families were reliably detected in these studies, including capsid-less RNA viruses that replicate in fungi, oomycetes and plants. Finally, strengths and weaknesses of the VANA approach are summarized and perspectives of applications in detection, epidemiological surveillance, environmental monitoring and ecology of plant viruses are provided.

High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health.

A 42-year-old man was admitted to the intensive care unit in October 2020 for coronavirus disease-19 (COVID-19)-related acute respiratory distress syndrome requiring mechanical ventilation. He presented with multi-organ failure and died 41 days later. Comorbidities included a sleeve gastrectomy in 2016 and cholecystectomy in April 2020.Autopsy revealed diffuse alveolar damage (Fig. 1A) and presence of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in the carotid body (Fig. 1B, C). Detection of SARS-CoV-2 in the carotid body was performed by real-time reverse transcription polymerase chain reaction (Fig. 1D).The carotid body plays a role in peripheral arterial chemoreception, in metabolic and immune sensing, and could also be a route of nervous system invasion by SARS-CoV-2. Involvement of the carotid body by SARS-CoV-2 may explain silent hypoxemia and thus could also contribute to increased morbidity and mortality in COVID-19 patients.