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PLoS One. 2014 Jul 25;9(7):e102945. doi: 10.1371/journal.pone.0102945. eCollection 2014.

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

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INRA, UMR 1332 Biologie du Fruit et Pathologie, CS 20032, 33882 Villenave d'Ornon Cedex, France; Université de Bordeaux, UMR 1332 Biologie du Fruit et Pathologie, CS 20032, 33882 Villenave d'Ornon Cedex, France.
CIRAD, UMR BGPI, Campus International de Montferrier-Baillarguet, 34398 Montpellier Cedex-5, France.
Computational Biology Group, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Christchurch, New Zealand; Department of Plant Pathology and Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of America; Electron Microscope Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, South Africa.


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% genome-wide 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.

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