APPLICATION OF NEW DIAGNOSTIC REAGENTS FOR IDENTIFICATION OF DIVERSE STRAINS OF POTATO VIRUS Y
Vegetable and Forage Crops Production Research
2009 Annual Report
1a.Objectives (from AD-416)
Develop new monoclonal antibodies that can be used for rapid detection and identification of Potato virus Y (PVY) strains that can be utilized on a large scale by certification agencies, researchers, and diagnostic facilities.
1b.Approach (from AD-416)
Nucleoproteins of diverse strains of PVY will be purified using standard methods. These will be used to immunize mice for production of monoclonal antibodies (MAbs) by hybridoma technology. The MAbs will be tested for virus strain specificity using a large panel of characterized PVY isolates available to the cooperator. Documents SCA with University of Idaho. Formerly 5354-21220-002-19S (5/08).
Potato virus Y (PVY) is a type member of the genus Potyvirus in the family Potyviridae. PVY has a filamentous particle, ca. 740 nm long and 11 nm in diameter, which is composed of a single 9.7-kb positive strand RNA and multiple copies of ca. 30-kDa capsid protein (CP). A single copy of the VPg protein is covalently attached to the 5’-terminus of the PVY genomic RNA and is also present in the assembled PVY particle. PVY is a major pathogen in potato, reducing tuber yield and quality. Different pathotypes of PVY, in particular PVYO and PVYN, have long been differentiated based on their reactivities towards various monoclonal antibodies (MAbs). With the wide spread of recombinant variants of PVY, inconsistencies emerged in specificity and reliability of available MAbs, and prompted this study on antigenic structure and immunoreactivity of PVY. We tested a panel of commercially available and newly produced MAbs against representative isolates in our PVY strain collection from North America (more than 2,500 isolates) which included several distinct strain groups. Tests were conducted on PVY antigens in solution – by ELISA in various formats, and after immobilization on solid supports – by Western blots or direct ELISA. Using immunodetection data in conjunction with multiple sequence alignments, we classified and mapped epitopes for widely used commercial MAbs. Based on the data obtained we propose amendments to existing PVY differentiation protocols which involve MAbs. This project was monitored via phone calls and emails with the lead researcher.