Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard

Abstract

Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.

Extracción y purificación de dsRNAs largos de plantas infectadas con virus y hongos; aplicación en la detección e identificación de virus

“…It is possible that methods for the extraction of viral dsRNAs will have a place in future implementation of NGS for virus detection and identification. This has been demonstrated with the use of dsRNAs to conduct the complete sequencing of viral RNA genomes from plants and fungi and to identify virus-like elements in aquatic microbial populations using NGS (Nerva et al, 2016), deep sequencing analysis of viruses infecting grapevines (Al Rwahnih et al, 2011;Coetzee et al, 2010), and a metagenomics study of environmental samples (Roossinck, 2012).…”

“…El ensamble De novo comparando con secuencias de genomas de virus disponibles en la base de datos del NCBI ha permitido identificar nuevas especies de endornavirus y micovirus (Espach et al, 2012;Jo et al, 2015;Khalifa et al, 2016). Utilizando dsRNAs virales, en combinación con secuenciación profunda, los investigadoress han podido obtener secuencias genómicas completas de fitovirus y micovirus (Al Rwahnih et al, 2011;Candresse et al, 2013;Coetzee et al, 2010;Deker y Parker, 2014;Espach et al, 2012;Quito-Avila et al, 2011;Magae, 2012), e identificar elementos que semejan virus en poblaciones microbianas acuáticas (Nerva et al, 2016). La secuenciación profunda de una sola vid reveló un viroma dominado por micovirus (Al Rwahnih et al, 2011).…”

“…Es posible que los métodos de extracción de dsRNAs virales lleguen a utilizarse en la implementación de NGS para la detección e identificación de virus. Esto ha sido demostrado con el uso de dsRNAs para realizar la secuenciación completa de genomas de RNA de plantas y hongos, e identificar elementos que semejan virus en poblaciones microbianas acuáticas utilizando NGS (Nerva et al, 2016), análisis de secuenciación profunda de virus que afectan a la vid (Al Rwahnih et al, 2011;Coetzee et al, 2010), y un estudio metagenómico de muestras ambientales (Roossinck, 2012).…”

Grapevine Biotechnology: Molecular Approaches Underlying Abiotic and Biotic Stress Responses

“…Several techniques have been developed based on PCR variants [83,102], but the use of real-time PCR allows quantification of virus titer [103]. Recently, new generation sequencing (NGS) has been used for rapid identification and sequencing of all putative viruses present in a candidate sample, allowing the identification of new viral agents as well [3,81,99,104]. The use of NGS technologies as diagnostic tool requires no prior knowledge of the pathogens present in the sample, but is still expensive in order to be used as a routine procedure.…”

“…Grapevine leafroll disease (GLD) is one of the most important viral diseases affecting grapevines worldwide [3,[81][82][83]. It is generally accepted that this disease is caused by 11 viral agents, named GLRaV-1 to GLRaV-11 [3], and according to specific genome sequences, their taxonomic classification includes members of the Ampelovirus genus (GLRaV-1, -3, -4, -5, -6 and -9), the Closterovirus genus (GLRaV-2) and the Velarivirus genus (GLRaV-7).…”