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.

Deep Sequencing of Virus-Derived Small Interfering RNAs and RNA from Viral Particles Shows Highly Similar Mutational Landscapes of a Plant Virus Population

“…Different explanations have been suggested for these observations, including biological explanations, e.g., connecting the hotspots with predicted secondary structures in viral genomes (23,36), as well as library preparation-connected ligation biases (34). In other cases (37, 38) such patterns were not observed.…”

“…To fill this gap, we have used a sequenceindependent, preamplification-free deep sequencing approach to investigate and compare two different pools of RNA virus sequences from the infected plants: single-stranded RNA (ssRNA) isolated from purified viral particles (VP pool) and vsiRNAs (sRNA pool). Deep sequencing of viral RNA directly from purified viral particles was performed only in a few cases and was shown to be highly efficient for enrichment for viral nucleic acids (4,22,23). Here, we have used the recently developed CIM monolithic chromatography-based approach for fast purification of viral particles (24).…”

Capulavirus and Grablovirus: two new genera in the family Geminiviridae

“…Introns are present in several geminivirus genes. Rep and RepA proteins in becurtoviruses, capulaviruses, grabloviruses, mastreviruses, MMCaV and CCDaV are either proven to be, or inferred to be, expressed from alternatively spliced complementary-sense transcripts [8,13,40] (Fig. 1).…”

Plant Disease Diagnosis: Technological Advancements and Challenges

“…Detection protocols used for the diagnosis or quarantine measures should be reproducible, repeatable and should have minimum false results. All detection methods should be sensitive to pathogen concentration, genetic variability within a target pathogen population, and similarities between the target and other organisms (Candresse et al, 2014;Martin et al, 2016).…”