High prevalence and low genetic diversity of grapevine yellow speckle viroid 1 in Slovakia: down the rabbit hole of RNA secondary structure and phylogeny

Grapevine yellow speckle viroid 1 (GYSVd-1) is a widespread subviral pathogen affecting grapevine ( Vitis vinifera L.), with potential implications for viticulture. This study investigates the prevalence, genetic variability, intrahost population polymorphism and secondary RNA structure of GYSVd-1 in Slovakia, where its molecular characteristics have been largely unexplored. Leaf samples from 49 grapevine plants across western and southern Slovakia were analyzed using RT-PCR, followed by sequencing and phylogenetic analyses. GYSVd-1 was detected in 73% of the samples, demonstrating high prevalence rates across vineyards in Slovakia. Sequence analysis revealed a high degree of genetic similarity among Slovak isolates, with nucleotide identity ranging from 96.2% to 100%, consistent with low variability reported globally. Phylogenetic analyses showed no specific grouping of Slovak isolates. Intrahost variability, assessed via subcloning of RT-PCR products, demonstrated the presence of random mutations among viroid clones, consistent with quasispecies nature of viroids in their hosts. These findings suggest that both interhost and intrahost viroid populations evolve under similar pressures to maintain structural stability essential for viroid function. Secondary RNA structure analysis of Slovak GYSVd-1 isolates revealed that all unambiguous sequences exhibited the characteristic rod-like conformation. In contrast, a significant proportion of sequences from mixed populations failed to form proper rod-like structures, suggesting that these variants may lack biological viability. Overall, this study unveils the prevalence of GYSVd-1 in Slovak vineyards and provides valuable insights to guide future phylogenetic studies and structural analyses of viroids.