Two new assays address the challenge of reliably detecting the genetically diverse variants of Grapevine leafroll-associated virus 3
Grapevine leafroll-associated virus 3 (GLRaV-3) is the main etiological agent of grapevine leafroll disease, one of the most important virus diseases of grapevine which is distributed worldwide. The long-distance spread of GLRaV-3, caused by the movement of infected vines, can be controlled effectively if GLRaV-3 is accurately identified and virus-tested clean stock is made available to growers. In turn, accurate GLRaV-3 identification and the production of a large amount of tested planting stock require a high throughput testing method that is sensitive and specific for GLRaV-3. As the source of all California Registered or Certified grapevines, Foundation Plant Services (FPS), has met the need for reliably detecting GLRaV-3 in large sample numbers by focusing on the development of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays.
RT-qPCR assays detect viruses by amplifying a small section of the virus genome to levels that can be easily detected. Amplification is achieved by identifying regions of the genome that are unique to any given virus yet are conserved among all the genetic variants of that particular virus. While identifying conserved regions across genetic variants can be difficult with any RNA virus, due to its relatively high mutation rate, GLRaV-3 has an exceptional number of highly diverse variants. Recent studies based on genome-wide phylogenetic analysis demonstrated that GLRaV-3 variants can be divided into eight distinct groups, six of which have been identified in California. This level of genetic diversity makes it almost impossible to identify a conserved region common to all isolates for design of a single qPCR-based assay. Up to now, FPS has dealt with this problem by designing variant-specific assays, six to date. However, it isn’t feasible to test large numbers of vines using six different assays. In addition, the California Department of Food and Agriculture (CDFA), which works closely with FPS on the grapevine certification process, recently adopted RT-qPCR assays. CDFA tests thousands of grapevine from nursery increase blocks every year, highlighting the need for a reliable single GLRaV-3 assay.
In 2017, Dr. Maher Al Rwahnih, Diagnostic and Research Laboratory Director, at FPS obtained funds from the Pierce’s Disease and Glassy-winged sharpshooter Research Board to conduct the research project, “Survey and analysis of grapevine leafroll-associated virus-3 genetic variants and application towards improved RT-qPCR assay design”. As part of this study, FPS reconstructed the near complete genomes of four new GLRaV-3 variants using high-throughput sequencing (HTS) and incorporated new genetic data into a more complete characterization of genetic variation across GLRaV-3 variants. A small but highly conserved region was identified that was then used to construct a single RT-qPCR assay for detecting all GLRaV-3 variants characterized to date.
The work behind designing and validating the new assay, called FPST, is described below:
- The FPST assay was designed using publicly available GLRaV-3 sequences as well as our own divergent GLRaV-3 variants that were sequenced at FPS. Once a large number of sequences from different genetic variants had been obtained, multiple alignments were used to identify regions with low sequence diversity that were suitable for assay design. In this case, FPST targeted the 3’ terminal region of the virus genome, a highly conserved region that was now supported by the large amount of new sequence data.
- The FPST assay has been empirically tested and validated using single isolate positive controls, representing all the GLRaV-3 groups. When compared with previous GLRaV-3 assays, FPST was the one RT-qPCR assay that detected ALL variants obtained to date (Table 1).
- To further test the new assay, 1,872 samples showing grapevine leafroll disease symptoms were collected from grapevine populations with a historically high incidence of GLRaV-3. These populations included the USDA National Clonal Germplasm Repository in Winters, CA, the Davis Virus Collection at UC-Davis, the FPS pipeline of foreign and domestic introductions, selected vineyards in the main grape-growing areas of California, and samples provided by international collaborators.
- Of the 1,872 samples, 1,148 (61%) samples tested positive for GLRaV-3 using the FPST RT-qPCR assay. These samples corresponded to domestic selections or international plants originating from Israel, Croatia, Portugal, Italy, Hungary, Canada, Japan, Turkmenistan, Spain, South Korea, France or Greece. The large number of samples analyzed resulted in a very rich geographic representation of GLRaV-3 variants.
- Further verification of the FPST assay was obtained by testing the above population with a new GLRaV-3 Enzyme-linked immunosorbent assay (ELISA) kit that was developed using funds from the Fruit Tree, Nut Tree, and Grapevine Improvement Advisory Board, managed by CDFA. This new ELISA kit has detected all known GLRaV-3 variants characterized to date. The side-by-side comparison indicated that all samples testing positive by the FPST RT-qPCR assay also tested positive by the new ELISA kit. The 100% match between the results suggests that both assays have similar efficiencies.
Moving forward, we will continue to test more samples and challenge both the ELISA kit and the FPST RT-qPCR assay against possible new divergent variants of GLRaV-3. Additionally, we will characterize more GLRaV-3 variants using HTS. The availability of more complete GLRaV-3 genome sequences will aid in further characterizing the genetic diversity covered by the assay which will be updated upon finding any new divergent variants. We cannot predict if the FPST assay will detect all the GLRaV-3 variants found in the future, however, the improved assay detects all known variants to date. Finally, FPS is committed to sharing these new detection tools with stakeholders, thus benefiting growers, researchers, and diagnostic labs involved in the grapevine industry in the US and around the world.
Table 1. Test results of divergent GLRaV-3 variants from FPST and individual GLRaV-3 RT-qPCR assays. The results demonstrate the challenge of designing a RT-qPCR assay to a genetically diverse genome. Until FPST was designed, variant-specific assays led to false negative test results.