Analysis of Grapevine rupestris stem pitting-associated virus in Slovakia Reveals Differences in Intra-Host Population Diversity and Naturally Occurring Recombination Events

Abstract

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a worldwide-distributed pathogen in grapevines with a high genetic variability. Our study revealed differences in the complexity of GRSPaV population in a single host. A single-variant GRSPaV infection was detected from the SK30 grapevine plant. On the contrary, SK704 grapevine was infected by three different GRSPaV variants. Variant-specific RT-PCR detection protocols have been developed in this work to study distribution of the three different variants in the same plant during the season. This study showed their randomized distribution in the infected SK704 grapevine plant. Comparative analysis of fulllength genome sequences of four Slovak GRSPaV isolates determined in this work and 14 database sequences showed that population of the virus cluster into four major phylogenetic lineages. Moreover, our analyses suggest that genetic recombination along with point mutations could play a significant role in shaping evolutionary history of GRSPaV and contributed to its extant genetic diversification.

Grapevine virus T diversity as revealed by full-length genome sequences assembled from high-throughput sequence data

“…Taken together with the abnormal behavior of isolate SK809 between the REP and the CP phylogenetic tree, these results indicate that, similar to many other Betaflexiviridae members, recombination appears to have played a significant role in the evolution of GVT. This observation was not unexpected in view of the complex mixed infections of GVT isolates reported here and of the known impact of recombination on the evolution of the closely related GRSPaV [ 9 , 17 – 18 ].…”

NGS of Virus-Derived Small RNAs as a Diagnostic Method Used to Determine Viromes of Hungarian Vineyards

“…In a striking contrast, with RT-PCR using diagnostic primers amplifying a very short part of the replicase we obtained a product in 16 of our samples (Figure 2B ). GRSPaV's viral RdRp has a very low proofreading activity and frequent recombination events, because the coexistence of different variants in the same plant led to the evolution of diverse variants (Morelli et al, 2011 ; Glasa et al, 2017 ). To test that if the number of virus-specific reads or coverage will increase if we use a different reference during bioinformatics analysis, we made the direct sRNA BWA using 5 distinct full GRSPaV genomes (AF057136 = NC_001948 _Ref_1_USA, AY881627_BS_Canada, KR054734_JF_China, AY881626_SG1_USA, AY368590_SY_USA) (Supplementary Table 7 ).…”

A complex virome unveiled by deep sequencing analysis of RNAs from a French Pinot Noir grapevine exhibiting strong leafroll symptoms.

“…In addition possible differences in sampling time and the fact that different parts of the sampled plants were used could play a role, probably reflecting a non-homogenous distribution of the various viruses in the P70 plant. Other library preparation strategies are being developed and are now available in order to 'enrich' viral reads by (i) focusing directly on viral genome sequences using the VANA (Viral associated nucleic acid) methodology [11], (ii) via viral small interfering (vsi)RNAs sequencing, focusing on small RNAs resulting from the gene silencing-based machinery being activated as part of the natural defense mechanism of plants [13,17]; or (iii) depleting host ribosomal RNAs from total RNA extracts [18,49,50]. Each of these sequencing strategies have been proven to have their advantages but also some drawbacks [51]; therefore it is possible that no generally optimal strategy may exist.…”