No abstract

Vitivirus are ssRNA(+) viruses in the family Betaflexiviridae (subfamily Trivirinae). There are currently ten ICTV recognized virus species in the genus; nevertheless, the extended use of NGS technologies is rapidly expanding their diversity and six more have been proposed recently. Here, we present the characterization of a novel virus from grapevines, which fits the genomic architecture and evolutionary constraints to be classifiable within the Vitivirus genus. The detected virus sequence is 7,607 nt long, including a typical genome organization of ORFs encoding a replicase (RP), a 22 kDa protein, a movement protein, a coat protein (CP) and a nucleic acid binding protein. Here, we present the characterization of a novel virus from grapevines.Phylogenetic analyses based on the predicted RP and CP protein unequivocally places the new virus within the Vitivirus genus. Multiple independent RNAseq data confirmed the presence of the detected virus in berries at diverse developmental stages. Additionally, we detected, confirmed, and assembled virus sequences from grapevine samples of distinct cultivars from America, Europe, Asia and Oceania, sharing 74.9%-97.9% nt identity, suggesting that the identified virus is widely distributed and diverse. We propose the name grapevine virus L (GVL) to the detected Vitivirus.

Vitivirus is a genus within the family Betaflexiviridae (Tymovirales) of ssRNA(+) viruses. There are currently ten ICTV recognized virus species in the genus; nevertheless, the extended use of NGS technologies is rapidly expanding their diversity and six more have been proposed recently. Here, we present the in silico characterization of a novel virus detected in grapevine (cv Riesling) which fits the genomic architecture and evolutionary constraints to be classifiable within the Vitivirus genus. The detected virus sequence is 7,607 nt long, including a typical genome organization of ORFs encoding a replicase (RP), a 22 kDa protein, a movement protein, a coat protein (CP) and a nucleic acid binding protein. Phylogenetic insights based on the predicted RP and CP, which share a 70.2% and 78.2% identity with its closest neighbour's grapevine virus E proteins, unequivocally clusters the identified virus within the Vitivirus genus. Multiple independent RNAseq data confirmed the presence of the detected virus in berries at diverse developmental stages. We tentatively propose the name grapevine virus L (GVL) to the detected putative Vitivirus. KeywordsVitivirus, Grapevine, cv Riesling, virus discovery, Betaflexiviridae Accession numberGrapevine virus L sequence (GVL-rs) has been deposited in GenBank under accession number MH248020. CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/314674 doi: bioRxiv preprint first posted online May. 4, 2018; ReportVitiviruses present flexuous, non-enveloped, filamentous virus particles of 725-785 nm and 12 nm length and diameter. Their nucleocapsid is cross-banded and striated diagonally. They have a linear ssRNA(+) genome (~7.3-7.6 kb), with a methylated nucleotide cap at the 5' end and a 3' poly (A) tail [1]. There are ten species of vitiviruses recognized by the ICTV, nevertheless, just in the last six months, six new species have been proposed, five of them infecting grapevine [2][3][4][5][6]. Grapevine is their most prevalent natural host but they have also been found to infect several crops such as Mint (Mentha x glaciaris), arracacha (Arracacia xanthorrhiza) blue agave (Agave tequilana) and kiwi (Actinidia chinense) [7][8][9].Vitiviruses appear to be latent in Vitis vinífera cultivars, and so far, only Grapevine virus A and Grapevine virus B had been consistently associated to grapevine diseases of the rugose wood complex (Grapevine vitiviruses) or Shiraz disease (reviewed by Minafra [10]). The etiological role of the recently described vitiviruses should be assessed in order to establish its relationships with known or unknown viral diseases. The synergistic effects between vitiviruses and other grapevine viruses appears to be significant [11]. The availability of complete sequences of these viruses could allow the development of full-length infectious clones, and ...

Apricot vein clearing-associated virus is the type species of genus Prunevirus, family Betaflexiviridae. The virus was first discovered from an Italian apricot tree (Prunus armeniaca) showing leaf vein clearing and mottling symptoms (Elbeaino et al. 2014). Since then, apricot vein clearing-associated virus (AVCaV) has been reported in symptomatic and asymptomatic plants from other countries (Marais et al. 2015; Kinoti et al. 2017; Kubaa et al. 2014). In 2018, a domestic selection of a flowering apricot (P. mume cv. Peggy Clarke) (PC01) with no discernible foliar virus-like symptoms was received for inclusion in the Foundation Plant Services (UC-Davis) collection. The plant originated from a private Prunus collection located in California. Total nucleic acids (TNA) were isolated from PC01 leaves using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific). The TNA were analyzed for a panel of 15 Prunus-infecting viruses by reverse-transcription quantitative PCR (RT-qPCR) (Diaz-Lara et al. 2020). In addition, to screen for sap-transmissible viruses, young leaves of PC01 were homogenized in inoculation buffer and were rubbed onto leaves of herbaceous indicator plants, Chenopodium amaranticolor, C. quinoa, Cucumis sativus, and Nicotiana clevelandii (Rowhani et al. 2005). The source PC01 tested negative for the 15 screened viruses. Interestingly, vein clearing symptoms were observed on leaves of C. quinoa and C. amaranticolor plants (Figure S1). These results suggested the presence of a mechanically transmissible virus in PC01. To determine the identity of mechanically transmissible viral agent, symptomatic C. quinoa and PC01 plant were advanced for high throughput sequencing analysis. Aliquots of TNA from PC01 and C. quinoa were rRNA-depleted and used for cDNA library preparation with TruSeq Stranded Total RNA kit (Illumina). The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). A total of 47,261,138 and 8,812,296 single-end reads were obtained from cDNA libraries of PC01 and C. quinoa, respectively. The de novo assembly generated near-complete contigs resembling AVCaV genome ) from both PC01 and C. quinoa, which were 99.8% identical at the nucleotide level. The longest contig (8,342 nucleotides, 73.5x coverage depth) obtained from PC01 was further completed using SMARTer RACE 5’/3’ kit (Takara Bio). The complete genome sequence of AVCaV-PC01 is 8,364 nucleotides long (GenBank: MK170158). The full-length virus genome was compared with GenBank database using BLASTn, which the best hit corresponded to KY132099 with 98% identity. Additionally, AVCaV infection was confirmed in both PC01 selection and the symptomatic C. quinoa by RT-PCR as previously described (Marais et al. 2015). Lastly, symptomatic leaves of C. quinoa were used in leaf dip method to visualize virus particles by transmission electron microscope. As a result, flexuous rod-shaped virions were observed from leaf dips of symptomatic C. quinoa plants (Figure S2). Therefore, our results represent the first report of AVCaV in California, USA. Furthermore, mechanical transmission of an AVCaV isolate infecting flowering apricot to herbaceous hosts was confirmed. Field surveys and biological studies are underway to determine the prevalence of AVCaV in commercial orchards and assess its effect on tree performance.