Abstract：
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.
摘要：
葡萄enamovirus1(GEV1)属于enamovirus属,在Solemoviridae家族中。据报道，包括巴西在内的几个国家感染了葡萄藤（Silva等人。2017),中国(任等人。2021年)和法国(Hily等人。2022年）。为了评估九个加拿大葡萄园中经济上重要的葡萄病毒的流行和多样性，总RNA和双链RNA(dsRNA)(Fall等人。2020)分别从30个和100个复合样品中提取，每个葡萄藤由相同品种的五个葡萄藤组成。本研究包括的品种是Frontenacnoir(n=34)，Vidal(n=32),马奎特（n=33），雷司令(n=31),和黑皮诺（n=31）。随后将总RNA和dsRNA样品多重化,并分别在NovaSeq(600S4PE100)和MiSeq(2x250循环PE)上通过高通量测序(HTS)诊断。从NovaSeq和MiSeq测序，平均获得了410,000到130万个读数/样本，分别,映射的病毒读段占总读段的10.92%至12.48%。使用Trimmoaticv.0.40验证序列质量后（Bolger等人。2014),使用Virtool对数据库中所有可能的病毒筛选干净的序列(Rott等人.2017)和VirFind病毒检测管道(Ho和Tzanetakis2014)。从两个干净的序列中检测到GEV1，三,和两个品种\'Marquette\'\'Riesling\'和\'Frontenacnoir\'的叶片样品。七个HTS组装的GEV1基因组中有六个是部分的，范围从4,523到6,000个核苷酸（nt），基因组覆盖率从71％到89％不等。只有一个6,314nt长的组装重叠群（登录号OR021829),代表了一个几乎完整的基因组，在5'和3'非翻译区（UTR）处仅比Sd-CG（MT536978）短53和3nt，分别。Isolate3-Riesling-CAN（OR021829）与多个GEV1分离株共享90.56至94.19％的nt身份，占查询覆盖率的96-99％。系统发育,OR021829更接近于来自法国和中国的GEV1分离株(图S1)。为了验证HTS结果，开发的引物对SetF和Set1R(Silva等人。,2017)用于RT-PCR检测。使用Sanger测序对来自所有七个HTS阳性样品的扩增子进行测序，确认在加拿大葡萄园的三个研究葡萄品种中存在GEV-1。与特定的GEV1感染的葡萄藤相关的症状无法解释，因为使用了复合样品。每个组合样品HTS文库还对至少一种已知的葡萄病毒/类病毒进行了阳性测试，即葡萄卷叶相关病毒-3，葡萄比诺格里斯病毒，葡萄茎点蚀相关病毒，马拉夫病毒西拉葡萄西拉病毒1和啤酒花特技类病毒。据我们所知,这是加拿大在葡萄藤中检测到GEV1的第一份报告，或任何北美葡萄园。GEV1是一种相对较新的病毒，它的生物学在很大程度上仍然未知。基于此序列，可以开发新的GEV1引物，以了解GEV-1之间的遗传变异性，并改善葡萄园中对该病毒的检测。
