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

Abstract

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.

Figure 1. SWSV genome coverage following NGS.

The genomic organization of SWSV is schematically shown above the graph. While relative degrees of coverage achieved after a posteriori mapping of reads produced by Illumina-based siRNA sequencing against the SWSV genome is indicated in green, the coverage achieved after mapping reads produced by 454 GS FLX Titanium-based VANA sequencing is indicated in blue.

Figure 2. Size distribution of sequenced siRNAs obtained from the VARX plant.

The histograms represent the numbers of siRNA reads in each size class. (A) The size distributions of total reads, (B) The size distributions of reads mapping to the rep gene C-sense intronic region of SWSV, (C) The size distributions of reads mapping to the V1–V2 ORFs region of SWSV and (D) The size distributions of reads mapping to the C1 ORF region of SWSV.

Figure 3. Maximum-likelihood phylogenetic tree of 63 virus isolates representing each known mastrevirus species (including major strains) and the 6 SWSV isolates determined in this study.

Tree branches are coloured according to the geographical origins of the viruses. Branches marked with filled and open circles respectively have >95% and 80–94% approximate likelihood ratio test support; branches having <80% support were collapsed. The phylogenetic tree is rooted using the full genome sequence of Dicot-infecting mastreviruses.

Figure 4. Maximum-likelihood phylogenetic tree of Rep (A) and CP (B) proteins.

Tree branches are coloured according to the geographical origins of the viruses. Branches marked with filled and open circles are respectively have >95% and 80–94% approximate likelihood ratio test support; branches having <80% support were collapsed.