Development of RT-PCR degenerate primers to overcome the high genetic diversity of grapevine virus T

Authors: DIAZ-LARA, ALFREDO - University Of California; GOLINO, DEBORAH - University Of California; Preece, John; RWAHNIH, MAHER AL - University Of California

Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/8/2020
Publication Date: 5/16/2020
Citation: Diaz-Lara, A., Golino, D., Preece, J.E., Rwahnih, M. 2020. Development of RT-PCR degenerate primers to overcome the high genetic diversity of grapevine virus T. Journal of Virological Methods. 282. Article 113883. https://doi.org/10.1016/j.jviromet.2020.113883.
DOI: https://doi.org/10.1016/j.jviromet.2020.113883

Interpretive Summary: Grapevine virus T (GVT) is a new member of the genus Foveavirus, which has been reported in several European countries infecting grapevine. In 2017, GVT was detected for the first time in California infecting a domestic selection of wine grape, cv. Lambrusca di Alessandria, via high throughput sequencing (HTS). To further investigate the presence of GVT in other grapevine plants, a two-step reverse transcription (RT)-PCR assay involving degenerate primers was developed. Aiming to cover the high genetic diversity of GVT, available sequenced isolates were aligned identifying a conserved region in the coat protein that was suitable for use in the assay. As a result, GVT was found in three additional grapevine selections using RT-PCR and later confirmed by HTS. The four characterized GVT isolates were divergent, sharing an overall 81% pairwise identity, which suggests that the new RT-PCR assay is reliable and can detect a broad-range of GVT variants. The RT-PCR detection method developed in this study will be useful for routine virus testing.

Technical Abstract: Grapevine virus T (GVT) is a new member of the genus Foveavirus, which has been reported in several European countries infecting grapevine. In 2017, GVT was detected for the first time in California infecting a domestic selection of wine grape, cv. Lambrusca di Alessandria, via high throughput sequencing (HTS). To further investigate the presence of GVT in other grapevine plants, a two-step reverse transcription (RT)-PCR assay involving degenerate primers was developed. Aiming to cover the high genetic diversity of GVT, available sequenced isolates were aligned identifying a conserved region in the coat protein that was suitable for use in the assay. As a result, GVT was found in three additional grapevine selections using RT-PCR and later confirmed by HTS. The four characterized GVT isolates were divergent, sharing an overall 81% pairwise identity, which suggests that the new RT-PCR assay is reliable and can detect a broad-range of GVT variants. The RT-PCR detection method developed in this study will be useful for routine virus testing.