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

A Modular Integration of SAT/SMT Solvers to Coq through Proof Witnesses

International audienceWe present a way to enjoy the power of SAT and SMT provers in Coq without compromising soundness. This requires these provers to return not only a yes/no answer, but also a proof witness that can be independently rechecked. We present such a checker, written and fully certified in Coq. It is conceived in a modular way, in order to tame the proofs' complexity and to be extendable. It can currently check witnesses from the SAT solver ZChaff and from the SMT solver veriT. Experiments highlight the efficiency of this checker. On top of it, new reflexive Coq tactics have been built that can decide a subset of Coq's logic by calling external provers and carefully checking their answers

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

Human peritoneal mesothelial cells respond to bacterial ligands through a specific subset of Toll-like receptors

Background. Bacterial infection remains a major cause of morbidity and mortality in peritoneal dialysis (PD) patients worldwide. Previous studies have identified a key role for mesothelial cells, lining the peritoneal cavity, in coordinating inflammation and host defense. Toll-like receptor (TLR) involvement in early activation events within the mesothelium, however, remains poorly defined. To investigate the initiation of bacterial peritonitis, we characterized TLR activation by bacterial ligands in human peritoneal mesothelial cells (HPMC)

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

IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity