Cucumber mosaic virus (CMV; Cucumovirus, Bromoviridae) is an omnipresent virus characterized by a large host range and high genetic variability. Using high-throughput sequencing, we have characterized near complete genomes of 14 Slovak CMV variants from different plant hosts. Of these, three variants originated from the Papaveraceae species (oilseed poppy, common poppy and great celandine), previously poorly described as CMV natural hosts. Based on a BLAST search and phylogenetic analysis, the Slovak CMV isolates can be divided into two genetically different Groups, Ia and II, respectively. The SL50V variant, characterized by a divergent RNA2 sequence, potentially represents a reassortant variant. In four samples (T101, SL50V, CP2, MVU2-21), the presence of satellite CMV RNA was identified along with CMV. Although mechanically transmitted to experimental cucumber plants, the role of satellite RNA in the symptomatology observed could not be established due to a complex infection of original hosts with different viruses.

The hop stunt viroid (HSVd) is a widespread subviral pathogen infecting a broad spectrum of plant hosts including grapevine (Vitis vinifera L.). Despite its omnipresence in virtually all grapevine growing areas around the world, molecular data characterizing HSVd populations are missing from Slovakia. Analysis of the complete nucleotide sequences of 19 grapevine variants revealed the existence of two genetic HSVd groups in Slovakia (internally named the “6A” and “7A” groups based on the particular stretch of adenines at nucleotide positions 39–44/45, respectively). Despite their sampling at different times in various unrelated vineyards, the 6A and 7A groups are characterized by low intra-group divergence (~0.3 and 0.2%, respectively). On the other hand, inter-group divergence reached 2.2% due to several mutations, seven of which were found to be group-specific and mainly (except for one) located in the region of the pathogenic domain. Interestingly, in addition to their frequent co-existence within the same geographical location, the mixed infection of the 6A and 7A type sequence variants was also unequivocally and repeatedly proven within single grapevine plants. The RNA secondary structure analysis of representative isolates from each of these two genetic groups indicated a potential compensatory explanation of such mutations. These group-specific sites could be pointing towards the evolutionary selection linked to the necessity of the viroid to retain its structural conformational integrity, crucial for its functional biochemical ability to interact with specific grapevine cellular host factors required for HSVd propagation.

Plant viral vectors have become a promising tool for the rapid and cost-effective production of recombinant proteins in plants. Among the numerous genera of viruses that have been used for heterologous expression, potyviruses offer several advantages, such as polyprotein expression strategy or a broad host range. In our work, the expression vectors pAD/pAD-agro based on the plum pox virus (PPV) genome were used for the heterologous expression of different foreign polypeptides: alfalfa mosaic virus capsid protein (AMV CP), zucchini yellow mosaic virus capsid protein (ZYMV CP), the small heat-shock protein of Cronobacter sakazakii fused with hexahistidine (sHSP-his), a fragment of influenza A virus hemagglutinin (HA2-2), influenza A virus protein PB1-F2, SARS-CoV-2 nucleocapsid protein (CoN2-his), and its N- and C-terminal fragments (CoN-1-his and CoN3-his, respectively), each fused with a hexahistidine anchor. Particular proteins differed in their accumulation, tissue localization, stability, and solubility. The accumulation rate of produced polypeptides varied from low (N, hemagglutinin fragment) to relatively high (plant viral CPs, N-terminal fragment of N, PB1-F2). Some proteins preferentially accumulated in roots (sHSP, hemagglutinin fragment, PB1-F2), showing signs of proteolytic degradation in leaf tissues. Thus, each expression requires an individual approach and optimization. Here, we summarize our several-year experiments and discuss the usefulness of the pAD/pADep vector system.

Analysis of the viromes of three symptomatic Fabaceae plants, i.e., red clover (Trifolium pratense L.), pea (Pisum sativum L.), and common bean (Phaseolus vulgaris L.), using high-throughput sequencing revealed complex infections and enabled the acquisition of complete genomes of a potyvirus, bean yellow mosaic virus (BYMV). Based on phylogenetic analysis, the Slovak BYMV isolates belong to two distinct molecular groups, i.e., VI (isolate FA40) and XI (isolates DAT, PS2). Five commercial pea genotypes (Alderman, Ambrosia, Gloriosa, Herkules, Senator) were successfully infected with the BYMV-PS2 inoculum and displayed similar systemic chlorotic mottling symptoms. Relative comparison of optical density values using semi-quantitative DAS-ELISA revealed significant differences among virus titers in one of the infected pea genotypes (Ambrosia) when upper fully developed leaves were tested. Immunoblot analysis of systemically infected Alderman plants showed rather uneven virus accumulation in different plant parts. The lowest virus accumulation was repeatedly detected in the roots, while the highest was in the upper part of the plant stem.

Over the past two decades, plant viral vectors have emerged as a powerful tool for the production of recombinant proteins in plants. Among the different plant viruses engineered to carry foreign genes of interest in their genomes, potyviruses have gained attention due to their polyprotein expression strategy and broad host range. To date, at least eleven different species belonging to the genus Potyvirus have been used for heterologous gene expression in both their natural and experimental hosts. This review article provides an overview of the current state of potyvirus-based plant viral vectors, discussing the advantages and limitations of these systems. We also discuss the future challenges and potential applications of potyvirus-based expression vectors, including the production of vaccines, nanoparticles, therapeutics, and metabolic engineering. Overall, we highlight the potential of potyvirus-based vectors as a versatile tool for recombinant protein production in plants.

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.