Department of Plant Pathology, University of California-Davis, Davis, California, United States of America.
Foundation Plant Services, University of California-Davis, Davis, California, United States of America.
PLoS One. 2020 Sep 22;15(9):e0239522. doi: 10.1371/journal.pone.0239522. eCollection 2020.
The genus Vitivirus in the family Betaflexiviridae includes eleven viruses known to infect grapevine: grapevine vitiviruses A, B, D, E, F, G, H, I, J, L and M (GVA-GVM). Three of these viruses, GVA, GVB and GVD, have been associated with the etiology of rugose wood disease in grapevine and cause agronomically significant losses. The other vitiviruses were more recently discovered and their effects on grapevine are undetermined. To certify grape material for propagation as virus tested, an updated reverse transcription PCR (RT-PCR) assay to detect all known vitiviruses is desirable. To accomplish this, multiple grapevine vitivirus sequences were aligned at the amino acid level to search for conserved motifs. Two highly conserved motifs were found at an ideal distance for RT-PCR detection in the RNA-dependent RNA polymerase region of the replicase protein. The amino acid motifs were back translated to create degenerate primers and used to successfully amplify all eleven grapevine vitiviruses. The RT-PCR primers were used to test a panel of vitivirus-infected vines for inclusivity as well as vines infected with closely related viruses in the Betaflexiviridae family (i.e. grapevine pinot gris virus and grapevine rupestris stem pitting-associated virus) for exclusivity. Broader use of these primers to detect vitiviruses in other plant hosts was investigated. In summary, an end-point RT-PCR assay that detects all the known grapevine vitiviruses and potentially other members of the genus Vitivirus has been developed. The universal assay represents an alternative to individual assays to reduce the work associated with the diagnosis of vitiviruses, including for regulatory purposes.
贝塔柔膜病毒科的葡萄病毒属包括 11 种已知感染葡萄的病毒：葡萄病毒 A、B、D、E、F、G、H、I、J、L 和 M（GVA-GVM）。其中 3 种病毒，GVA、GVB 和 GVD，与葡萄皱木病的病因有关，会造成严重的农业损失。其他的葡萄病毒是最近才发现的，其对葡萄的影响尚未确定。为了对用于繁殖的葡萄材料进行病毒检测认证，需要一种更新的逆转录聚合酶链反应（RT-PCR）检测方法来检测所有已知的葡萄病毒。为了实现这一目标，对多个葡萄病毒序列进行了氨基酸水平的比对，以寻找保守的基序。在复制酶蛋白的 RNA 依赖性 RNA 聚合酶区域发现了两个高度保守的基序，其距离非常适合 RT-PCR 检测。将氨基酸基序回译以创建简并引物，并成功扩增了所有 11 种葡萄病毒。使用 RT-PCR 引物对一组感染了葡萄病毒的葡萄藤进行了包容性检测，以及对感染了贝塔柔膜病毒科（如葡萄皮尔斯灰葡萄孢病毒和葡萄岩斑病毒）的葡萄藤进行了排他性检测。还研究了这些引物在其他植物宿主中检测葡萄病毒的更广泛应用。总之，已经开发出一种终点 RT-PCR 检测方法，可以检测所有已知的葡萄病毒和该属的其他潜在成员。通用检测方法代表了一种替代个别检测方法的方法，可减少与葡萄病毒诊断相关的工作量，包括法规目的。
