Evidence of Grapevine enamovirus 1 Infecting Grapevine ( L.) in Canada.

Grapevine enamovirus 1 (GEV1) belongs to the genus Enamovirus, in the family Solemoviridae. It has been reported from several countries infecting grapevines including Brazil (Silva et al. 2017), China (Ren et al. 2021) and France (Hily et al. 2022). To assess the prevalence and diversity of economically important grapevine viruses in nine Canadian vineyards, total RNA and double-stranded RNA (dsRNA) (Fall et al. 2020) were extracted from 30 and 100 composite samples respectively, with each consisting of five vines of the same cultivars. The cultivars included in this study are Frontenac noir (n=34), Vidal (n=32), Marquette (n=33), Riesling (n=31), and Pinot noir (n=31). The total RNA and dsRNA samples were subsequently multiplexed and diagnosed by high-throughput sequencing (HTS) on NovaSeq (600 S4 PE100) and MiSeq (2 × 250 cycle PE) respectively. From NovaSeq and MiSeq sequencing, an average of 410,000 to 1.3 million reads/sample were obtained, respectively, with mapped viral reads representing 10.92% to 12.48% of the total reads. After sequence quality was verified using Trimmomatic v.0.40 (Bolger et al. 2014), the clean sequences were screened against all possible viruses in the databases using the Virtool (Rott et al. 2017) and VirFind virus detection pipelines (Ho and Tzanetakis 2014). GEV1 was detected in clean sequences from two, three, and two leaf samples of cultivars 'Marquette' 'Riesling' and 'Frontenac noir' respectively. Six of the seven HTS-assembled GEV1 genomes were partial, ranging from 4,523 to 6,000 nucleotide (nt) with genome coverage varying from 71% to 89%. Only one 6,314 nt long assembled contig (Accession No. OR021829), represented a nearly complete genome, being only 53 and 3 nt shorter than Sd-CG (MT536978) at 5' and 3' untranslated regions (UTR), respectively. Isolate 3- Riesling-CAN (OR021829) shares 90.56 to 94.19% nt identities with several GEV1isolates at 96-99% of query coverage. Phylogenetically, OR021829 is closer to GEV1 isolates from France and China (Figure S1). To validate the HTS results, the developed primer pair SetF and Set1R (Silva et al., 2017) was used for RT-PCR detection. The amplicons from all seven HTS-positive samples were sequenced using Sanger sequencing, confirming the presence of GEV-1 in three studied grape cultivars in Canadian vineyards. Symptoms associated with the specific GEV1-infected vines could not be explained as composite samples were used. Each of the combined samples HTS library also tested positive for at least one of the known grape virus/viroids, namely grapevine leafroll associated-virus -3, grapevine pinot gris virus, grapevine rupestris stem pitting-associated virus, Marafivirus syrahense grapevine Syrah virus-1 and hop stunt viroid. To our knowledge, this is the first report of GEV1 being detected in grapevines in Canada, or in any North American vineyard. GEV1 is a relatively new virus, and its biology remains largely unknown. Based on this sequence new GEV1 primers can be developed to know the genetic variability among GEV-1 and improve the detection of this virus in vineyards.