Association of Little cherry virus 1 (LChV1) with the Shirofugen Stunt Disease and Characterization of the Genome of a Divergent LChV1 Isolate

Abstract

Double-stranded RNAs purified from the V2356 ('Successa') sour cherry source of the Shirofugen stunt disease (SSD) were sequenced using a 454 pyrosequencing multiplex approach. The 15,646 reads obtained were assembled into 279 contigs, 5 of which, totaling almost 16.9 kbp and 5,332 reads (34% of sample reads), showed high Blast scores and homology to Little cherry virus 1 (LChV1). The five contigs were further assembled manually into three supercontigs spanning the full LChV1 genome with only two small gaps (17 and 55 bases). Completion of the sequencing of the viral genome was performed using targeted polymerase chain reaction and primers designed from the contigs. No evidence for the presence of other viral agents in the V2356 source could be obtained in the remaining contigs or singletons. The V2356 LChV1 isolate is only ≈76% identical with the reference complete LChV1 sequences and, in particular, with the ITMAR isolate associated with the Kwanzan stunting syndrome. However, it is highly homologous (97 to 100% identity) in two short genome regions with divergent LChV1 from North America, providing the first complete sequence for such divergent isolates. Although not providing a definite proof, the failure to detect any other viral agent in the V2356 SSD source and the identification of LChV1 in a second, independent, source of the disease suggests that LChV1 isolates could be responsible for the SSD syndrome.

Characterization by Deep Sequencing of Divergent Plum bark necrosis stem pitting-associated virus (PBNSPaV) Isolates and Development of a Broad-Spectrum PBNSPaV Detection Assay

“…or Nanjing Prunus sources and submitted to 454 pyrosequencing. The sequences were treated as described in a previous work (6). In the PR258 source, 38 contigs were annotated as belonging to two distinct PBNSPaV isolates and represented 52.5% of the reads.…”

“…Double-stranded RNAs (dsRNAs) extracted from leaves of GF305 grafted with the various PBNSPaV sources (Ta Tao 25, PR258, Pair, and Nanjing) were analyzed by 454 pyrosequencing, using the strategy described by Candresse et al (6). Following assembly, PBNSPaV contigs were identified by identity to the PBNSPaV reference genome (PL186 isolate, GenBank accession number EF546442).…”

Insights into the genetic diversity and evolution of Little cherry virus 1

“…Distinct fruit symptoms involving a dramatic reduction of fruit size, colour and taste occur in susceptible sweet cherry cultivars, whereas other cultivars show only moderate fruit deformations or are tolerant. Different isolates of LChV-1 have also been associated with various other disorders such as Kwanzan stunting syndrome and Shirofugen stunt disease (Matic et al, 2009;Candresse et al, 2013). Recently, LChV-1 was reported in other Prunus species such as almond, peach and plum, although without inducing any obvious symptoms (Matic et al, 2007(Matic et al, , 2009.…”

“…The complete genome sequences of three LChV-1 isolates have been determined (Keim-Konrad & Jelkmann, 1996;Jelkmann et al, 1997;Matic et al, 2009;Candresse et al, 2013). The RNA genome of the virus, c. 17 kb, is organized in eight open reading frames (ORFs) that potentially encode the conserved replication domains of papain-like protease, methyltransferase and helicase (ORF1a), the RNA-dependent RNA polymerase (RdRp; ORF1b), a small hydrophobic protein (p4; ORF2), a 70 kDa heat-shock protein homologue (HSP70 h; ORF3), a polypeptide of 61 kDa (p61; ORF4), the coat protein (CP; ORF5) and CP minor (CPm; ORF6) proteins and two polypeptides with currently unknown function of 21 kDa (p21; ORF7) and 27 kDa (p27; ORF8) .…”

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

“…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).…”