Evaluation of the RNA Silencing Suppression Activity of Three Cherry Virus F-Encoded Proteins.

Cherry virus F (CVF) is a newly emerged sweet cherry virus. CVF has been identified in a small number of countries and it has not been associated with discrete symptomatology. RNA silencing is a natural defense mechanism of plants against invaders that degrades viral RNA in a sequence-specific manner. As a counter-defense, plant viruses encode one or more RNA silencing suppressors (RSSs) interfering with the silencing pathway via several mechanisms. To identify putative RSSs, the three proteins (MP, CPL, CPS) encoded by the RNA2 of CVF were selected and separately cloned into the binary vector pART27. The clones were used for transient expression experiments in Nicotiana benthamiana leaves, using co-agroinfiltration with a GFP-expressing vector. In both CPL and CPS, a rapid decrease in fluorescence was recorded, comparable to the negative control, whereas the MP of CVF retained the GFP's fluorescence for a few days longer even though this was observed in a small number of infiltrated leaves. Further experiments have shown that the protein was not able to inhibit the cell-to-cell spread of the silencing signal; however, a putative interference with systemic silencing was recorded especially when the induction was carried out with double-stranded GFP RNA. Overall, our results indicate that the MP of CVF is putatively implicated in the suppression of RNA silencing, though further experimentation is needed to unveil the exact mode of action.

Identification of divergent isolates of cherry latent virus 1 in Greek sweet cherry orchards.

In this study, samples collected from eight sweet cherry trees in northern Greece were analyzed by high-throughput sequencing for the presence of viruses. Bioinformatic analysis revealed the presence of divergent isolates of cherry latent virus 1 (CLV-1), a recently identified trichovirus in a sweet cherry accession imported into the USA from the Republic of Georgia. The complete genome sequences of seven CLV-1 isolates were determined, and phylogenetic analysis indicated that they belonged to a separate clade from the previously characterized Georgian isolate. A small-scale survey confirmed the presence of CLV-1 in 47 out of 151 sweet cherry samples tested, and partial sequencing of 15 isolates showed a high degree of nucleotide sequence similarity among them.

Identification and complete genome sequencing of a divergent olive virus T isolate and an olive leaf yellowing-associated virus isolate naturally infecting olive trees in Greece.

Several new full genome sequences of olive viruses came to light recently via high-throughput sequencing (HTS) analysis. In this study, total RNA HTS analysis of two Greek olive trees revealed the presence of an olive virus T (OlVT) isolate and an olive leaf yellowing-associated virus (OLYaV) isolate. The full viral genome of OlVT isolate (50Ch) is composed of 6862 nucleotides encoding for three proteins (replicase, movement protein, and capsid protein) with typical betaflexiviruses' genomic features. However, both sequence and phylogenetic data analysis exhibited high levels of variability between 50Ch and the previously characterized OlVT isolates. In addition, the almost full genome of the Greek OLYaV isolate (OL2) was obtained, which is composed of 16,693 nucleotides encoding for 11 open reading frames (ORFs) and shares common genomic features with the recently characterized OLYaV isolates from Spain and Brazil. Sequence and phylogenetic analysis revealed high similarity between these three isolates. Due to problems encountered with the detection of both viruses, new nested RT-PCR assays were developed and applied. In addition, recombination events were observed in OlVT isolates (50Ch GR-168), thus highlighting the potential role of this mechanism in the evolution of the virus. This study is adding further knowledge to the limited information available about these recently characterized olive infecting viral pathogens and highlights their widespread distribution in Greece, one of the most important olive producing countries of the world.

Interplay of Cucurbit Yellow Stunting Disorder Virus With Cucurbit Chlorotic Yellows Virus and Transmission Dynamics by Bemisia tabaci MED.

Cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) are two closely related criniviruses that often coinfect cucurbits and are associated with cucurbit yellows disease. Both viruses are distributed worldwide and are transmitted in a semipersistent manner by the whitefly vectors Bemisia tabaci MED or MEAM1. The major goal of this study was to provide insight into the interaction of CCYV and CYSDV in cucumber and to study the effect on transmission by B. tabaci MED. The titers of both viruses were estimated in single- and dually infected cucumber plants via reverse transcription PCR assays. In mixed infections, the accumulation of both viruses was significantly decreased. When B. tabaci MED adults were placed on cucumber infected with both viruses, their simultaneous transmission efficiency was significantly higher, whereas transmission efficiency of each individual virus was low. Moreover, nonviruliferous whiteflies preferentially settled on crinivirus-infected cucumber plants, whereas viruliferous whiteflies were attracted by healthy cucumber plants. Finally, the titer of both viruses was calculated in five commercial cucumber hybrids, followed by subsequent transmission experiments. Our results show that although the titers of CYSDV and CCYV were significantly lower in mixed infections in cucumbers, their simultaneous transmission increased.

The population structure of Rose rosette virus in the USA.

Rose rosette virus (RRV) (genus Emaravirus) is the causal agent of the homonymous disease, the most destructive malady of roses in the USA. Although the importance of the disease is recognized, little sequence information and no full genomes are available for RRV, a multi-segmented RNA virus. To better understand the population structure of the virus we implemented a Hi-Plex PCR amplicon high-throughput sequencing approach to sequence all 7 segments and to quantify polymorphisms in 91 RRV isolates collected from 16 states in the USA. Analysis revealed insertion/deletion (indel) polymorphisms primarily in the 5' and 3' non-coding, but also within coding regions, including some resulting in changes of protein length. Phylogenetic analysis showed little geographical structuring, suggesting that topography does not have a strong influence on virus evolution. Overall, the virus populations were homogeneous, possibly because of regular movement of plants, the recent emergence of RRV and/or because the virus is under strong purification selection to preserve its integrity and biological functions.

High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics.

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing.

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species.

Genome sequence and detection of peach rosette mosaic virus.

Peach rosette mosaic disease was first described in the 1940s affecting peach and plum. It was later determined that peach rosette mosaic virus (PRMV) is the causal agent of the disease. PRMV, a member of the genus Nepovirus, infects several perennial crops including stone fruit, grape and blueberry as well as several weed species found in orchards around the world. The molecular characterization of the virus is limited to partial genome sequences making it difficult to develop reliable and sensitive molecular detection tests; the reason that detection is routinely performed using ELISA with antibodies risen against a single virus isolate. Given the potential economic impact of the virus and the modes of transmission which, in addition to nematodes, include seed we studied PRMV in more depth using a modified dsRNA extraction protocol to obtain the virus genome. We determined the full nucleotide sequence and developed a protocol that detects conserved regions present in RNA 1 and RNA 2, making it an excellent alternative to the detection protocols used today.

Development of a Real-Time RT-PCR for the Universal Detection of LChV1 and Study of the Seasonal Fluctuation of the Viral Titer in Sweet Cherry Cultivars.

Little cherry virus 1 (LChV1) is a sweet cherry pathogen which has lately been reported in other Prunus spp. LChV1 variability makes reliable detection a challenging undertaking. The objective of this work was to develop a rapid, sensitive, and reliable one-tube, real-time reverse-transcription polymerase chain reaction (RT-PCR) for the detection and quantification of LChV1. Primers and a TaqMan probe were designed, using conserved regions of the capsid protein gene. Detection range was evaluated using several divergent viral isolates. The amplification efficiency of the method was estimated at 96.7%, whereas the detection limit was about 100 RNA copies. The protocol was applied in the study of virus fluctuation within leaves and phloem tissue throughout the year and the best periods to test and plant tissues to sample were determined. Comparative analysis of this method with a previously published nested RT-PCR revealed the higher analytical and diagnostic sensitivity of the new test, making it a reliable tool that can be used in routine testing and certification programs.