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

Abstract

Plum bark necrosis stem pitting-associated virus (PBNSPaV), the causal agent of plum bark necrosis stem pitting disease, belongs to the genus Ampelovirus in the family Closteroviridae. The complete genome sequence of PBNSPaV isolates from four Prunus sources was determined by pyrosequencing. All isolates showed the same genomic organization as the PBNSPaV reference isolate. The least divergent isolate, found in a peach tree from China, showed an overall 91.8% of nucleotide identity with the type isolate. Two closely related isolates, defining a second cluster of diversity, were found in two Japanese plum lines from France and showed only 82.8% identity with the type isolate. On the other hand, they were highly homologous with two recently described PBNSPaV divergent isolates from China. The fourth and most divergent isolate, from a Chinese peach, showed only 71.2% identity to other PBNSPaV isolates and was not detected by currently available PBNSPaV reverse-transcription polymerase chain reaction detection assays. Complete sequencing of the divergent isolates allowed the development of a more broad-spectrum detection test targeting a conserved region in the P61 gene. Taken together, these results indicate a much broader diversity of PBNSPaV than previously thought and provide for a more robust detection of this still poorly characterized pathogen.

Characterization of New Isolates of Apricot vein clearing-associated virus and of a New Prunus-Infecting Virus: Evidence for Recombination as a Driving Force in Betaflexiviridae Evolution

“…Primers were designed from the contig sequences and amplifications using Aze204 source TNA were performed in order to complete and validate the sequence of this tentative scaffold. As in previous efforts [19,20,29], essentially no differences were observed between the initially assembled scaffold and the resequenced regions, thus validating both the initial contigs and the tentative scaffolding. The assembled sequence was deposited under accession number KM507061 in the GenBank database.…”

“…These results further illustrate the power of the NGS (Next Generation Sequencing) based approaches to identify and characterize known or novel fruit tree viruses [12,19–21,29,32,33]. …”

“…These sequences were then treated as previously described [19]. Blast comparisons following de novo assembly of contigs showed that in the Pair source three viruses were present in co-infection, namely PBNSPaV (two isolates, collectively representing 60.9% of the reads, [29]), Prunus necrotic ring spot virus (PNRSV, 1.3% of the reads), and an isolate of AVCaV, representing 10.8% of the reads [29]. For the latter virus, a long contig of 8,120 nucleotides (nt) was obtained, spanning the AVCaV genome from nt 205 to nt 8,325.…”

“…A two-step RT-PCR assay was applied. TNA (5 μl) were submitted to a reverse transcription following the protocol described by Marais et al [29]. The cDNA (5 μl) was then amplified in a 50-μl volume containing 10 mM Tris-HCl (pH8.8), 1.5 mM MgCl 2 , 50 mM KCl, 250 μM dNTPs, primers at 1 μM each, and 1 U of DyNAzyme II DNA polymerase (Finnzymes/Fisher Scientific, Illkirch, France) and 40 cycles were applied, each of 95°C for 30 sec, 48°C (NB-F1i/R1) or 42°C (Pol-F1i/R1) for 30 sec, and 72°C for 30 sec, followed by a final extension step of 10 min at 72°C.…”

Next-Generation Sequencing and Genome Editing in Plant Virology

“…The data obtained may afford a better knowledge of the polyvalence or specificity of existing assays and, if needed, facilitate the design of new detection primers of broader specificity for improving the classical detection assays. For example, the NGS discovery of a non-detectable isolate of plum bark necrotic and stem pitting associated virus led Marais et al (2014) to develop a new PCR assay of broader specificity.…”