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Cucumbers are susceptible to infections with many characterised virus species. In some cases, mixed virus infections occur and produce novel symptoms. In Greece, routine screening is carried out when virus infection is suspected, or novel symptoms are observed in the field. To identify the viruses associated with distinct symptoms observed in samples from commercial cucumber production areas on the island of Crete, Greece, we carried out high‐throughput sequencing (HTS) from a pool of six samples. Following assembly and BLAST analysis, we identified at least seven viruses based on similarity to published sequences. Two of these sequences represented novel, near‐complete genomes of a putative new nepovirus and of zucchini yellow fleck virus (ZYFV). To confirm the HTS results, the six samples were screened for all identified viruses, and their presence was confirmed through Sanger sequencing of PCR products. The full‐length genomes of both the nepovirus and ZYFV were amplified by PCR and confirmed by Sanger sequencing. We have generated the complete genome of a novel nepovirus from cucumber as well as the first complete genome sequence of a cucumber‐infecting ZYFV isolate from Crete. The nepovirus was mechanically transmissible to Nicotiana benthamiana and induced typical cytopathological modifications consistent with virus infection, as revealed by TEM studies. We propose to name this new virus Cucumber nepovirus A (CuNVA). [ABSTRACT FROM AUTHOR]

Tomato black ring virus (TBRV) and beet ringspot virus (BRSV) are closely related but distinct members of subgroup B of the genus Nepovirus. Both viruses have broad host ranges and are transmitted by seed, pollen, and ectoparasitic nematodes. Although 13 TBRV and 3 BRSV genome sequences were already available, no attempt has been made to link sequence data from these recent sequences with those of historical isolates studied in the pre‐sequencing era. High‐throughput sequencing was used to generate eight new TBRV and BRSV genome sequences from three historical >60‐year‐old and two >30‐year‐old isolates, and three more recent isolates. These eight isolates were from the Czech Republic, Germany, and the UK. We compared these with all genomes sequenced previously. Intraspecies recombination (three of four TBRV and two of four BRSV isolates) was frequent amongst the eight new genomes. Interspecies recombination was also present within the RNA1 of TBRV isolates BRSV‐3393 SG GB and BRSV‐9888 ST GB. No satellite RNAs were associated with the eight new genomes. Two commercial enzyme‐linked immunosorbent assay (ELISA) kits used to detect TBRV during routine testing differed in that one detected only TBRV and the other only BRSV, so they are likely to provide incorrect but potentially complementary virus occurrence information. We suggest both ELISA kits, or appropriate molecular tests, be used by biosecurity authorities to avoid this problem. This study illustrates the value of sequencing historical isolates preserved from the pre‐sequencing era. [ABSTRACT FROM AUTHOR]

In 2015 and 2016, lettuce (Lactuca sativa) plants showing yellow ringspots and line patterns were observed in Argolida (Peloponnese, Greece). Symptoms resembled those caused by nepoviruses, especially artichoke yellow ringspot virus (AYRSV), an endemic virus in artichoke (Cynara scolymus) and onion (Allium cepa var. cepa) crops in this area. The aim of this work was to identify and characterize the viral agent associated with the observed disease. We carried out mechanical inoculations onto Nicotiana benthamiana and lettuce plants and also virus‐specific reverse transcription (RT)‐PCR. Results confirmed the presence of AYRSV in all lettuce plants with symptoms tested, and the natural and experimental host range of the virus was studied. The species Heliotropium europaeum, Solanum nigrum, Origanum vulgare, Lactuca seriola, and Sinapis arvensis, are recorded as hosts of AYRSV for the first time. Also, AYRSV was detected by RT‐PCR in seeds and in the produced seedlings of lettuce, N. benthamiana, N. glutinosa, N. tabacum, and S. nigrum. Sequencing of a partial fragment of the gene encoding the RNA‐dependent RNA polymerase was performed for 29 isolates of AYRSV originating from different hosts (lettuce, artichoke, onion, and arable weeds). Phylogenetic analysis of AYRSV isolates classified them in diverse clusters that exhibited no host grouping. All groups included isolates from cultivated and wild plant species. In this study, AYRSV was associated with a new lettuce disease for the first time, thus increasing the known natural host range of the virus. [ABSTRACT FROM AUTHOR]

Summary: Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs. [ABSTRACT FROM AUTHOR]

The stone fruit genus Prunus, within the family Rosaceae, comprises more than 230 species, some of which have great importance or value as ornamental or fruit crops. Prunus are affected by numerous viruses and viroids linked to the vegetative propagation practices in many of the cultivated species. To date, 44 viruses and three viroids have been described in the 9 main cultivated Prunus species. Seven of these viruses and one viroid have been identified in Prunus hosts within the last 5 years. This work addresses recent advances and prospects in the study of viruses and viroids affecting Prunus species, mostly concerning the detection and characterisation of the agents involved, pathogenesis analysis and the search for new control tools. New sequencing technologies are quickly reshaping the way we can identify and characterise new plant viruses and isolates. Specific efforts aimed at virus identification or data mining of high-throughput sequencing data generated for plant genomics-oriented purposes can efficiently reveal the presence of known or novel viruses. These technologies have also been used to gain a deeper knowledge of the pathogenesis mechanisms at the gene and miRNA expression level that underlie the interactions between Prunus spp. and their main viruses and viroids. New biotechnological control tools include the transfer of resistance by grafting, the use of new sources of resistance and the development of gene silencing strategies using genetic transformation. In addition, the application of next generation sequencing and genome editing techniques will contribute to improving our knowledge of virus-host interactions and the mechanisms of resistance. This should be of great interest in the search to obtain new Prunus cultivars capable of dealing both with known viruses and viroids and with those that are yet to be discovered in the uncertain scenario of climate change. [ABSTRACT FROM AUTHOR]

Virus-like particles ( VLPs) derived from nonenveloped viruses result from the self-assembly of capsid proteins ( CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self-assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus ( GFLV). We found that the N- and C-terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant-based production of nucleic acid-free VLPs. Remarkably, expression of N- or C-terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules. [ABSTRACT FROM AUTHOR]

Grapevine fanleaf virus ( GFLV) is the major causal agent of the grapevine degeneration disease. To characterize the genomic RNA2 segment from Iranian isolates of GFLV, leaf samples were collected from infected vineyards in different locations with a long history of vine cultivation. Four isolates were selected for cloning and sequencing on the basis of the restriction profiles of RT- PCR products. The sequencing data revealed that the RNA2 of the Iranian GFLV isolates were the shortest compared with that of all previously described GFLV isolates. The sizes were 3730 nucleotides (nt) for Shir- Amin and Urmia isolates and 3749 nt for Takestan and Bonab isolates (excluding the poly ( A) tail), due to deletion events in both 5′ and 3′ non-coding regions. In the phylogenetic tree based on the full-length nucleotide sequences of GFLV RNA2, all the GFLV isolates clustered into two groups with the exception of the Hungarian isolate ( GHu). The Iranian isolates grouped as a distinct cluster. Recombination analyses showed that GFLV- NW (Germany), GFLV-F13 (reference isolate), GFLV isolate Shir- Amin ( Iran) and Arabis mosaic virus isolate Lv were recombinant isolates and one of their parents belonged to the same lineage as the Iranian isolates. These findings suggest that these isolates originated from a common ancestor. [ABSTRACT FROM AUTHOR]

Pollen is traded internationally as a source of germplasm and for pollination. Thirty-nine viruses and five viroids are known to be pollen transmitted. We investigated whether reverse transcription-polymerase chain reaction (RT-PCR) could be used to detect viruses reliably in pollen. Four extraction methods yielded nucleic acid in appropriate quantity and quality from Tobacco ringspot virus (TRSV)-infected pollen for RT-PCR amplification. One method, the RNeasy Plant Mini Kit was used subsequently to extract nucleic acid of amplifiable quality from nine plant species, and pollen infected with three ilarvirus and two nepovirus species. A real-time TaqMan™ RT-PCR for the detection of TRSV was reliable and specific using 167 extracts of pollen from plants of Nicotiana glutinosa. The assay was highly sensitive, with extracts testing positive to a 10 dilution, equivalent to a single pollen grain. This demonstrated that RT-PCR methods can detect virus-infected pollen reliably, sensitively and specifically. The possible application of these RT-PCR methods to replace current quarantine procedures without compromising biosecurity is discussed. [ABSTRACT FROM AUTHOR]

A plant of black currant cv. Karlštejnský dlouhohrozen showing symptoms of the severe Russian (R) form of the blackcurrant reversion disease (BCRD) was shown to contain phytoplasma bodies measuring 530–750 nm. Phytoplasma infection was confirmed by polymerase chain reaction (PCR) with the universal primer pair R16F1/R16R0, followed by PCR with the primer pair fU5/rU3. A comparison of the sequence of an amplification product of approximately 880 bp with sequences available in the GenBank confirmed the classification of the phytoplasma in the 16SrI (Aster yellows group). This is the first evidence of the natural occurrence of phytoplasma infection in black currant.Blackcurrant reversion virus(BRV), the cause of BCRD, was confirmed in the plant by RT-PCR. A 481 nt cDNA fragment of BRV was sequenced and compared with sequences in GenBank. Rhabdovirus-like particles were also observed in the plant by electron microscopy. [ABSTRACT FROM AUTHOR]