Evidence of Rubus Yellow Net Virus Integration into the Red Raspberry Genome.

Rubus yellow net virus (RYNV) infects Rubus spp., causing a severe decline when present in mixed infections with other viruses. RYNV belongs to the family Caulimoviridae, also known as plant pararetroviruses, which can exist as episomal or integrated elements (endogenous). Most of integrated pararetroviruses are noninfectious; however, a few cases have been reported where they excised from the plant genome and formed infectious particles. Graft transmission onto indicator plants R. occidentalis "Munger" has been the standard test method for RYNV detection in certification programs. Previously, it was noticed that some RYNV PCR-positive plants did not induce symptoms on "Munger", suggesting an integration event. In this study, bio-indexing and different molecular techniques were employed to differentiate between integrated and episomal RYNV sequences. Reverse transcription-PCR using RYNV-specific oligonucleotides after DNase treatment generated positive results for the virus in graft transmissible isolates (episomal) only. To confirm these results, rolling circle amplification on DNA preparations from the same samples resulted in amplicons identified as RYNV only from plants with graft transmissible RYNV. High-throughput sequencing was used to identify the RYNV-like sequences present in the host DNA. These results indicate the integration of RYNV into the red raspberry genome and highlight the necessity to recognize this phenomenon (integration) in future Rubus quarantine and certification programs.

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018.

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.

A variant of Rubus yellow net virus with altered genomic organization.

Rubus yellow net virus (RYNV) is a member of the genus Badnavirus (family: Caulimoviridae). RYNV infects Rubus species causing chlorosis of the tissue along the leaf veins, giving an unevenly distributed netted symptom in some cultivars of red and black raspberry. Recently, a strain of RYNV was sequenced from a Rubus idaeus plant in Alberta, Canada, exhibiting such symptoms. The viral genome contained seven open reading frames (ORFs) with five of them in the sense-strand, including a large polyprotein. Here we describe a graft-transmissible strain of RYNV from Europe infecting cultivar 'Baumforth's Seedling A' (named RYNV-BS), which was sequenced using rolling circle amplification, enzymatic digestion, cloning and primer walking, and it was resequenced at a 5X coverage. This sequence was then compared with the RYNV-Ca genome and significant differences were observed. Genomic analysis identified differences in the arrangement of coding regions, promoter elements, and presence of motifs. The genomic organization of RYNV-BS consisted of five ORFs (four ORFs in the sense-strand and one ORF in the antisense-strand). ORFs 1, 2, and 3 showed a high degree of homology to RYNV-Ca, while ORFs 4 and 6 of RYNV-BS were quite distinct. Also, the predicted ORFs 5 and 7 in the RYNV-Ca were absent in the RYNV-BS sequence. These differences may account for the lack of aphid transmissibility of RYNV-BS.

A discovery 70 years in the making: characterization of the Rose rosette virus.

Rose rosette was first described in the early 1940s and it has emerged as one of the most devastating diseases of roses. Although it has been 70 years since the disease description, the rosette agent is yet to be characterized. In this communication, we identify and characterize the putative causal agent of the disease, a negative-sense RNA virus and new member of the genus Emaravirus. The virus was detected in 84/84 rose rosette-affected plants collected from the eastern half of the USA, but not in any of 30 symptomless plants tested. The strong correlation between virus and disease is a good indication that the virus, provisionally named Rose rosette virus, is the causal agent of the disease. Diversity studies using two virus proteins, p3 and p4, demonstrated that the virus has low diversity between isolates as they share nucleotide identities ranging from 97 to 99%.

A New Potyvirus sp. Infects Verbena Exhibiting Leaf Mottling Symptoms.

Verbena 'Taylortown Red' plants showed virus-like mottling symptoms. Virus purifications disclosed the presence of elongated and spherical particles, evidence of mixed virus infections, whereas double-stranded RNA analysis revealed the presence of several bands absent in healthy plants. After shotgun cloning, three viruses were identified in 'Taylortown Red': Broad bean wilt virus-1, Coleus vein necrosis virus, and a previously undescribed potyvirus. Given the importance of verbena to the ornamental industry, we studied the viruses found in 'Taylortown Red' and, in this article, we present our findings on the new potyvirus, provisionally named Verbena virus Y (VVY). VVY belongs to the Potato virus Y subgroup in the genus Potyvirus, has solanaceous plants, including potato, as alternative hosts, and can be transmitted by a ubiquitous pest in the ornamental industry, the green peach aphid.

A new, widespread emaravirus discovered in blackberry.

A new virus was identified in blackberry plants exhibiting leaf mottling, chlorotic ringspots and curved midribs, symptoms associated with blackberry yellow vein disease (BYVD). The genome of the new virus, provisionally named blackberry leaf mottle-associated virus (BLMaV), was characterized and phylogenetic analysis revealed its close relationship to recognized members of the genus Emaravirus. BLMaV was transmitted by a yet to be described eriophyid mite species, further reinforcing its placement in the genus. Detection protocols were developed and used to determine the presence of the virus in plants collected from several areas in the U.S.A. The incidence of BLMaV was greater than 40% in BYVD-affected material.

Evidence of sympatric speciation of elderberry carlaviruses.

Five new carlaviruses infecting elderberry were characterized and tentatively named as elderberry virus A-E (ElVA-ElVE). Their genome organization is similar to that of other carlaviruses with size ranging from 8540 to 8628 nucleotides, excluding the polyadenylated tails. ElVA, ElVB and ElVD share a common ancestor as do ElVC and ElVE, indicating that speciation may be sympatric with all viruses having emerged in elderberry. Analyses of the carlavirus conserved domains indicate that the 2-oxoglutarate and Fe(II)-dependent oxygenase motifs are reliable indicators of virus phylogenetic classification with recombination playing a significant role in the evolution of the genus. A universal RT-PCR assay that detects all the elderberry carlaviruses and potentially other members of the genus has been developed. This tool can be used for research and regulatory purposes as elderberry cultivation is rapidly expanding to new areas where the viruses may be absent.

A novel emaravirus is associated with redbud yellow ringspot disease.

Yellow ringspot is the only virus-like disease reported in redbud (Cercis spp.) with symptoms including vein clearing, chlorotic ringspots and oak-leaf pattern. A putative new emaravirus was present in all trees displaying typical yellow ringspot symptoms and the name redbud yellow ringspot associated virus is proposed. The virus genome is composed of at least five RNA segments. Two coding regions were studied to determine isolate diversity with results pointing to a homogeneous virus population. Host range was evaluated using graft transmission and by testing species found in close proximity to infected trees. Mite transmission with Aculops cercidis, the predominant species found in redbud trees in the epicenter of the disease, was evaluated but was not found to be a vector of the virus. Based on this study and the accumulated knowledge on emaravirus evolution we propose that speciation is allopatric, with vectors being a major component of the process.

Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates.

The population structure of blueberry mosaic associated virus (BlMaV), a putative member of the family Ophioviridae, was examined using 61 isolates collected from North America and Slovenia. The studied isolates displayed low diversity in the movement and nucleocapsid proteins and low ratios of non-synonymous to synonymous nucleotide substitutions, indicative of strong purifying selection. Phylogenetic analyses revealed grouping primarily based on geography with some isolates deviating from this rule. Phylogenetic incongruence in the two regions, coupled with detection of reassortment events, indicated the possible role of genetic exchange in the evolution of BlMaV.

Identification and Detection of a Virus Associated with Strawberry Pallidosis Disease.

The etiology of pallidosis, a disease of strawberry identified more than 45 years ago, remains unknown. We report a putative agent of the disease, a virus belonging to the Crinivirus genus of the Closterovirideae family. A sensitive reverse transcription-polymerase chain reaction (RTPCR) test has been developed. Polyclonal antibodies that can be used to detect the virus in petiole tissue blots were developed using a recombinant virus coat protein. The nucleotide sequences of regions of the viral genome that encode the heat shock protein 70 homolog and the major coat protein were obtained. Alignments of the major coat protein show that the virus isolated from strawberry plants positive for pallidosis is most closely related to Cucumber yellows virus (syn. Beet pseudo-yellows virus) and Cucurbit yellow stunt disorder virus, members of the Crinivirus genus.

A new ophiovirus is associated with blueberry mosaic disease.

Blueberry mosaic disease (BMD) was first described more than 60 years ago and is caused by a yet unidentified graft transmissible agent. A combination of traditional methods and next generation sequencing disclosed the presence of a new ophiovirus in symptomatic plants. The virus was detected in all BMD samples collected from several production areas of North America and was thus named blueberry mosaic associated virus. Phylogenetic analysis, supported by high bootstrap values, places the virus within the family Ophioviridae. The genome organization resembles that of citrus psorosis virus, the type member of the genus Ophiovirus. The implications of this discovery in BMD control and blueberry virus certification schemes are also discussed.

Molecular characterization and population structure of blackberry vein banding associated virus, new Ampelovirus associated with yellow vein disease.

Blackberry yellow vein disease is the most important viral disease of blackberry in the United States. Experiments were conducted to characterize a new virus identified in symptomatic plants. Molecular analysis revealed a genome organization resembling Grapevine leafroll-associated virus 3, the type species of the genus Ampelovirus in the family Closteroviridae. The genome of the virus, provisionally named blackberry vein banding associated virus (BVBaV), consists of 18,643 nucleotides and contains 10 open reading frames (ORFs). These ORFs encode closterovirid signature replication-associated and quintuple gene block proteins, as well as four additional proteins of unknown function. Phylogenetic analyses of taxonomically relevant products consistently placed BVBaV in the same cluster with GLRaV-3 and other members of the subgroup I of the genus Ampelovirus. The virus population structure in the U.S. was studied using the replication associated polyprotein 1a, heat shock 70 homolog and minor coat proteins of 25 isolates. This study revealed significant intra-species variation without any clustering among isolates based on their geographic origin. Further analyses indicated that these proteins are under stringent purifying selections. High genetic variability and incongruent clustering of isolates suggested the possible involvement of recombination in the evolution of BVBaV.