Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

Background Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. Results Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. Conclusions The fast and efficient strategies described here should have broad applications, in particular for the study "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents

Rancang Bangun Sistem Pengukuran Posisi Target dengan Kamera Stereo untuk Pengarah Senjata Otomatis

Salah satu hal yang penting dalam mengarahkan senjata secara otomatis ke target adalah informasi posisi dari target terhadap senjata. Terdapat banyak metode untuk mengetahui posisi target. Salah satunya adalah dengan metode pengukuran triangulasi. Metode ini membutuhkan minimal dua citra untuk medapatkan informasi jarak target terhadap kamera. Kemudian, informasi jarak tersebut bisa diolah untuk mengetahui posisi target terhadap senjata. Di dalam sistem ini, stereo visual digunakan untuk mendukung proses pengukuran triangulasi. Stereo visual menggunakan dua kamera untuk menghasilkan dua citra. Dalam sistem ini, salah satu kamera bertindak sebagai pemilih target. Citra yang ditangkap dua kamera tersebut akan diproses oleh processing unit untuk mendapatkan informasi posisi target terhadap senjata. Informasi ini digunakan untuk menggerakkan motor pada platform senjata agar senjata mengarah ke target. Hasil pengujian yang dilakukan pada sistem ini adalah sistem dapat menentukan posisi target yang dipilih oleh operator dan juga dapat mengarahkan senjata ke arah target tersebut. Akurasi tertinggi dalam penentuan posisi target dicapai ketika jarak antar dua kamera sekitar 30 cm

Natural Infection of Pomegranate (Punica Granatum) by Apple Dimple Fruit Viroid

The analysis by high throughput sequencing (HTS) and RT-PCR of Spanish pomegranate fruits showing yellow rings revealed the presence of viroid isolates closely related to fig isolates of apple dimple fruit viroid (ADFVd). The analysis of pomegranate public RNASeq data (Sequence Reads Archives, SRAs) from Israel provided evidence for the presence of similar ADFVd isolates in pomegranate trees in this country. In addition, reads or contigs of plum viroid I (PVd-I) isolates were also identified in two of the analyzed SRA datasets from Israel, suggesting the presence of this second viroid in pomegranate. Full length ADFVd genomic sequences have been recovered, increasing knowledge on the diversity of this viroid and on the pomegranate virome in which only four viruses and one viroid had previously been reported

The Expanding Menagerie of Prunus-Infecting Luteoviruses

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.This study was funded by the European Union through the Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-60 ITN) project “INEXTVIR” (grant agreement number 813542). The ChLVA research part was financed by the Academy of Sciences of the Czech Republic (RVO60077344). The plant indexing biological tests conducted by CTIFL were funded by INTERFEL (fresh fruit and vegetable interprofessional association). D. Safarova and M. Navratil received support from the Ministry of Agriculture of the Czech Republic, National Agency for Agricultural Research (project QK1920124). M. Glasa and D. Mihálik received support from the Slovak Research & Development Agency (project APVV-18-0005)Peer reviewe

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties