1. Please state project objectives and what work was done this quarter to address them:
Objective 1: The development of a antibody based diagnostic assays. We have been consistently successful in using the polyclonal antibody that was made to the sequence of the viral moment protein (MP) to detect CBaPRV MP from protein extracted from plant samples using a dot blot immune assay. We have also been successful in using a Western blot assay to detect the protein. However, some assays using this method were unsuccessful. Attempts to adapt the antibody to an ELISA have been challenging due to the lack of specificity and likely a lack of protein abundance. To overcome these challenges, an antigen concentration method is under testing before an ELISA can become be a viable test. This this is also a necessary step before a usable lateral flow assay test can be considered.
Objective 2: The goal of this objective is to determine how prevalent inserted copies of viral DNA are in commercial citrus. The virus inserts a copy of its DNA into the host DNA as a regular part of the viral life cycle. For objective 2 we are using PCR to screen DNA from 10 citrus varieties (a minimum of 3 trees per variety) for the presence of inserted viral DNA. By the end of the second quarter, we have surpassed the number of varieties by a large number, 42 in total, including 6 different species (orange, lemon, lime, grapefruit, mandarin, tangerine and tangelo) including 13 different varieties of rootstock. All tested citrus varieties have inserted CBaPRV DNA, meaning that they are capable of being infected if the proper triggering conditions occur. We have tested some citrus relatives and found that Murraya species do not have the presence of the inserted virus. Sequencing of one replicate for 8 species of citrus were used to confirm the status of CBaPRV in all species of citrus tested. Sequencing reads matched that of the parent CBAPRV sequence that was previously sequenced in 2016. There were some single point mutations for some nucleotide positions that may be dependent on species, a random mutation within the sequence, or just a sequencing error when the machine read the base.
2. Please state what work is anticipated for next quarter:
Objective 1: To overcome the limit of detection of movement protein, we will use a bead base sample to concentrate the target protein before we use the antibody to detect. We have also begun the process of creating a monoclonal antibody using some of our limited funds to increase the specificity. This will increase our capacity to develop a grower-friendly diagnostic assay to detect CBaPRV.
Objective 2: We need to complete enough representatives for the last few varieties and need to verify the sequencing with three replicates, but at this time we have fulfilled the objectives of the proposal. It is also clear that a CRISPR system is needed for the prevention of viral activation give that it is prevalent in all germplasm tested.
3. Please state budget status (underspend or overspend, and why):
We are on track with our budget for this quarter and have purchased supplies necessary to carry out the objectives of the grant. We are on track for salaries this quarter.
4. Please show all potential commercialization products resulting from this research, and the status of each:
At this time there is no potential commercialization of any products associated with this research.