1a.Objectives (from AD-416):
1. Sequence whole genomes for at least 3 PVY-N:O strains that do not cause tobacco vein necrosis, and analyze their sequences against at least 3 other PVY-N:O strains that do.
2. Sequence whole genomes for at least 3 PVY isolates that cause tuber necrosis in potato and analyze their sequences against at least 3 other PVY strains that do not.
3. Express PVYO and O5 capsid proteins in bacteria and correlate the monoclonal 1F5 reactivity with the point mutation in the PVY-O capsid protein.
1b.Approach (from AD-416):
Potato virus Y isolates collected by State Potato Seed Certification agencies and partially characterized by either State laboratories or the ARS laboratory at Ithaca, NY will be sent to the University of Idaho. Virus genomes will be amplified using a RT-PCR protocol and the products sequenced. Sequence information will be catalogued in a bioinformatics database that will be made available to all collaborators on the project. Virus protein fragments will be expressed in bacteria and used to characterize epitopes that recognize monoclonal antibodies.
The tuber necrotic strain of Potato virus Y that emerged recently in the United States has the potential to cause significant yield and quality losses to both the seed and commercial potato industries. It is important that seed lots infected with this virus be identified and eliminated from seed stocks before they can be planted. The detection of the tuber necrotic virus strain is usually accomplished using several commercially available antibodies, however there are reports that different results can be obtained depending on the antibody used. Researchers investigated the specificity of each of the commercial antibodies by identifying the exact amino acid sequence of the virus coat protein that reacts with each of the antibodies. This allowed the researchers to identify if there was a difference among the antibodies and if the virus coat protein was variable in these regions and could be responsible for varying results. Three of the four antibodies were mapped to a linear segment of the coat protein; the fourth antibody binds to a nonlinear, conformational segment of the protein and could not be mapped. Of the three antibodies whose binding site was mapped, two recognize the same piece of the coat protein but that piece of the coat protein is variable in its sequence so these antibodies do not recognize some of the tuber necrotic isolates of the virus. The third antibody binds to a different piece of the protein and consistently recognized all of the tuber necrotic viruses. This is valuable information for testing laboratories when making selections of antibodies for diagnostics testing purposes.