Analysis of Virome by High-Throughput Sequencing Revealed Multiple Infection and Intra-Virus Diversity in a Single Grapevine Plant

The ribosomal-depleted total RNA from white-berry grapevine (Vitis vinifera, SK933) plant showing severe chlorosis and downrolling of leaves was used for the high-throughput sequencing (HTS) analysis in order to unravel the potential contribution of the viral pathogens to the symptomatology observed. The combination of de novo assembly and mapping of ca. 1.1 millions of HTS reads enabled to identify and characterise a complex viral/viroid infection involving Grapevine leafroll-associated virus-2 (GLRaV-2), Grapevine leafroll-associated virus-3 (GLRaV-3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine rupestris vein feathering virus (GRVFV), Grapevine Syrah virus-1 (GSyV-1) and Hop stunt viroid (HSVd). The determined nearly complete genomes of GLRaV-2 SK933 showed its high genetic divergence from previously characterised isolates. In case of GRSPaV, two variants representing different evolutionary lineages have been identified in the plant. The results further pinpoint the complexity of grapevine viral diseases and show that mixed virus infection of grapevine is rather a rule than an exception

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

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

Molecular and Biological Characterisation of Turnip mosaic virus Isolates Infecting Poppy (Papaver somniferum and P. rhoeas) in Slovakia

In recent years, the accumulated molecular data of Turnip mosaic virus (TuMV) isolates from various hosts originating from different parts of the world considerably helped to understand the genetic complexity and evolutionary history of the virus. In this work, four complete TuMV genomes (HC9, PK1, MS04, MS15) were characterised from naturally infected cultivated and wild-growing Papaver spp., hosts from which only very scarce data were available previously. Phylogenetic analyses showed the affiliation of Slovak Papaver isolates to the world-B and basal-B groups. The PK1 isolate showed a novel intra-lineage recombination pattern, further confirming the important role of recombination in the shaping of TuMV genetic diversity. Biological assays indicated that the intensity of symptoms in experimentally inoculated oilseed poppy are correlated to TuMV accumulation level in leaves. This is the first report of TuMV in poppy plants in Slovakia

Experimental Infection of Different Tomato Genotypes with Tomato mosaic virus Led to a Low Viral Population Heterogeneity in the Capsid Protein Encoding Region

The complete genome sequence of a Slovak SL-1 isolate of Tomato mosaic virus (ToMV) was determined from the next generation sequencing (NGS) data, further confirming a limited sequence divergence in this tobamovirus species. Tomato genotypes Monalbo, Mobaci and Moperou, respectively carrying the susceptible tm-2 allele or the Tm-1 and Tm-2 resistant alleles, were tested for their susceptibility to ToMV SL-1. Although the three tomato genotypes accumulated ToMV SL-1 to similar amounts as judged by semi-quantitative DAS-ELISA, they showed variations in the rate of infection and symptomatology. Possible differences in the intra-isolate variability and polymorphism between viral populations propagating in these tomato genotypes were evaluated by analysis of the capsid protein (CP) encoding region. Irrespective of genotype infected, the intra-isolate haplotype structure showed the presence of the same highly dominant CP sequence and the low level of population diversity (0.08–0.19%). Our results suggest that ToMV CP encoding sequence is relatively stable in the viral population during its replication in vivo and provides further demonstration that RNA viruses may show high sequence stability, probably as a result of purifying selection

Molecular characterization of Prune dwarf virus cherry isolates from Slovakia shows their substantial variability and reveals recombination events in PDV RNA3

Molecular characterization of Prune dwarf virus cherry isolates from Slovakia shows their substantial variability and reveals recombination events in PDV RNA