Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design

Authors: DIAZ-LARA, ALFREDO - University Of California; KLAASSEN, VICKI - University Of California; STEVENS, KRISTIAN - University Of California; Sudarshana, Mysore; ROWHANI, ADIB - University Of California; MAREE, HANS - Stellenbosch University; CHOOI, KAR - Auckland University Of Technology; BLOUIN, ARNAUD - Auckland University Of Technology; HABILI, NUREDIN - University Of Adelaide; SONG, YASHU - University Of Guelph; ARAM, KAMYAR - University Of California; ARNOLD, KARI - University Of California Agriculture And Natural Resources (UCANR); COOPER, MONICA - University Of California Agriculture And Natural Resources (UCANR); WUNDERLICH, LYNN - University Of California Agriculture And Natural Resources (UCANR); BATTANY, MARK - University Of California Agriculture And Natural Resources (UCANR); BETTIGA, LARRY - University Of California Agriculture And Natural Resources (UCANR); SMITH, RHONDA - University Of California Agriculture And Natural Resources (UCANR); BESTER, RACHELLE - Stellenbosch University; XIAO, HUOGEN - University Of Guelph; MENG, BAOZHONG - University Of Guelph; Preece, John; GOLINO, DEBORAH - University Of California; ALRWAHNIH, MAHER - University Of California

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2018
Publication Date: 12/12/2018
Citation: Diaz-Lara, A., Klaassen, V., Stevens, K., Sudarshana, M.R., Rowhani, A., Maree, H.J., Chooi, K.M., Blouin, A.G., Habili, N., Song, Y., Aram, K., Arnold, K., Cooper, M.L., Wunderlich, L., Battany, M.C., Bettiga, L.J., Smith, R.J., Bester, R., Xiao, H., Meng, B., Preece, J.E., Golino, D., Alrwahnih, M. 2018. Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design. PLoS One. 13(12):e0208862. https://doi.org/10.1371/journal.pone.0208862.
DOI: https://doi.org/10.1371/journal.pone.0208862

Interpretive Summary: Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank or generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small fragment at the 3’ end of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that detects all known GLRaV-3 variants characterized to date. Additionally, four new GLRaV-3 divergent variants were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but considerably similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on phylogenetic analysis a new GLRaV-3 subclade is proposed and named as group X.

Technical Abstract: Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank or generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small fragment at the 3’ end of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that detects all known GLRaV-3 variants characterized to date. Additionally, four new GLRaV-3 divergent variants were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but considerably similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on phylogenetic analysis a new GLRaV-3 subclade is proposed and named as group X.