Appearances can be deceptive: Revealing a hidden viral infection with deep sequencing in a plant quarantine context

Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share .91% genomewide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations

First report on detection of three Bunya-Like Viruses in apples in Brazil.

Apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), and apple stem pitting virus (ASPV) cause significant losses to Brazilian (BR) apple production. Looking beyond these latent viruses, high-throughput sequencing (HTS) of three samples (Vacaria, Brazil) was performed on an Illumina HiSeq X Ten platform (USA), cv. Braeburn (BB), and a BGISEQ-500 platform (China), cvs. Royal Gala (RG) and Mishima (MI)

Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context

Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus, Family Geminiviridae), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share .91% genomewide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations

A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.Peer reviewe

Complete genomic sequence of Raphanus sativus cryptic virus 4 (RsCV4), a novel alphapartitivirus from radish

The present work reports the discovery and complete genome sequencing of a virus from symptomless radish seedlings, classifiable as a novel member of the genus Alphapartitivirus, family Partitiviridae. Total RNA extracted from germinating seedlings was sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed two contigs representing the near full-length genomic sequences of two genomic RNAs representing a new virus. Analysis of the genome sequence (excluding the polyA tail, RNA1: 1976 nt and RNA2: 1751 nt, respectively) showed a genomic organization typical of viruses classed within the Partitiviridae, with each genomic RNA encoding a single open reading frame (ORF). Phylogenetic analysis of the RNA dependent RNA polymerase (RNA1 ORF) and of the capsid protein (RNA2 ORF) clearly showed the new virus can be classified within the genus Alphapartitivirus, but sequence divergence establishes it as a new species, for which the name “Raphanus sativus cryptic virus 4” is proposed

Pest categorisation of Ips cembrae

The Panel on Plant Health performed a pest categorisation of the large larch bark beetle, Ips cembrae (Heer) (Coleoptera: Curculionidae, Scolytinae), for the EU. I. cembrae is a well-defined and distinguishable species, native to Europe and recognised mainly as a pest of larch (Larix spp.) and occasionally of pine (Pinus spp.) and spruce (Picea spp.). It is distributed in 16 Member States of the EU and listed in Annex IIB of Council Directive 2000/29/EC. Protected zones are in place in Greece, Ireland and the United Kingdom (Northern Ireland and Isle of Man). Wood, wood products, bark and wood packaging material are considered as pathways for this pest, which is also able to disperse by flight. The insects normally establish on fallen or weakened trees but, when their populations are high, can also mass-attack healthy trees. The males produce aggregation pheromones that attract conspecifics of both sexes. The insects also inoculate pathogenic fungi to their hosts. There are one to two generations per year. Before establishing their broods, the young adults need to proceed to maturation feeding either within the bark of the tree where they developed or in 2–18 years old twigs. I. cembrae has been expanding its geographical range in Europe during the second half of the 20th century. Sanitary thinning or clear felling is the major control methods. Quarantine measures are implemented to prevent entry in the protected zones. All criteria for consideration as potential protected zone quarantine pest are met. The criteria for considering I. cembrae as a potential regulated non-quarantine pest are not met since plants for planting are not viewed as a major pathway

Susceptibility of Phoenix roebelenii to Xylella fastidiosa

Following a request from the European Commission, the EFSA Plant Health Panel analysed a dossier submitted by Costa Rica Authorities to reach a conclusion on the host status of Phoenix roebelenii for Xylella fastidiosa. The Panel wishes first to stress the difficulties faced in providing compelling evidence for the non-susceptibility status of any particular plant species. The Panel acknowledges that the listing of P. roebelenii as a host of X. fastidiosa rests on a single report from California. Because isolation of X. fastidiosa from some hosts can be difficult, the Panel considers that the failure to isolate X. fastidiosa from P. roebelenii cannot be used to totally discard the detection of X. fastidiosa by ELISA and PCR. The Panel concludes that the detection of X. fastidiosa by two independent techniques provides sufficient evidence, although not totally conclusive, for the listing of P. roebelenii as a X. fastidiosa host plant. Concerning the survey data provided in the Costa Rican dossier, the Panel wishes to stress that such surveys cannot demonstrate the non-host status but can only provide a probability bound, upper estimate of the proportion of infected plants in the field. In the present case, and assuming all survey parameters to be optimal, the 95% confidence incidence threshold obtained is 0.2%, leaving the possibility that close to 25,000 P. roebelenii plants could be infected but undetected in the country. Accepting a scenario of local, non-systemic infection of P. roebelenii by X. fastidiosa would further increase uncertainties. In addition, the absence of data on the vector infection pressure further affects the ability to derive meaningful information on the P. roebelenii host status from the survey data. Appropriately conducted mechanical and/or vector-mediated inoculation experiments are critical to reach a more solid conclusion on the X. fastidiosa host status of P. roebelenii

Guidance on the environmental risk assessment of plant pests

The European Food Safety Authority (EFSA) requested the Panel on Plant Health to develop a methodology for assessing the environmental risks posed by harmful organisms that may enter, establish and spread in the European Union. To do so, the Panel first reviewed the methods for assessing the environmental risks of plant pests that have previously been used in pest risk assessment. The limitations identified by the review led the Panel to define the new methodology for environmental risk assessment which is described in this guidance document. The guidance is primarily addressed to the EFSA PLH Panel and has been conceived as an enhancement of the relevant parts of the “Guidance on a harmonised framework for pest risk assessment and the identification and evaluation of pest risk management options by EFSA”. Emphasizing the importance of assessing the consequences on both the structural (biodiversity) and the functional (ecosystem services) aspects of the environment, this new approach includes methods for assessing both aspects for the first time in a pest risk assessment scheme. A list of questions has been developed for the assessor to evaluate the consequences for structural biodiversity and for ecosystem services in the current area of invasion and in the risk assessment area. To ensure the consistency and transparency of the assessment, a rating system has also been developed based on a probabilistic approach with an evaluation of the degree of uncertainty. Finally, an overview of the available risk reduction options for pests in natural environments is presented, minimum data requirements are described, and a glossary to support the common understanding of the principles of this opinion is provided