In this study a CIM monolith based procedure was developed for the fast purification from plant tissue of the filamentous Potato virus Y (PVY) (virion size, 740 nm × 11 nm), which is one of the most important plant viruses causing great economical losses in potato production. The optimized procedure involves initial clarification steps, followed by chromatography using CIM quaternary amine (QA) monolithic disk column. In comparison to classical purification procedure involving ultracentrifugation through sucrose and caesium chloride, the developed CIM-QA purification achieved comparable yield, concentration and purity. Plant nucleic acids were successfully removed. Purification showed good reproducibility and moreover it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where a filamentous virus was purified using CIM monolithic supports. The advantages of this new purification procedure make it an attractive method in serological diagnostic tool production, which requires purified viruses for the immunization step. Moreover, the outcome of this study could serve as starting point for the improvement of the purification methods of other important filamentous viruses.
COBISS.SI-ID: 21244646
Two state of the art characterization tools based on detecting molecular markers – RT-qPCR (Kogovsek et al., 2008) and SNaPshot (Rolland et al., 2008) – were assessed for their ability to assign PVY accurately to the correct group. The results were validated by bioassay, ELISA and in silico sequence analysis. The spectrum of PVY strain groups distinguished by SNaPshot is broader than that by RT-qPCR. However, the latter was more reliable in discriminating the PVYNTN group members, known for their ability to induce PTNRD on selected potato cultivars. The difference in discrimination precision was due to different molecular markers being targeted by RT-qPCR and SNaPshot. Both tools use genotypic markers for detecting PVYNTN strain groups. Future development, however, should be focused on identifying the genomic determinants of the tuber necrosis property. Until then, the RT-qPCR and SNaPshot methods remain the most powerful diagnostic tools for detecting the PVY subgroup isolates found in Europe.
COBISS.SI-ID: 2745423
The availability of the potato genome sequence opens up the possibility of advancing the understanding of the physiology of potato response to virus infection. Interaction between potato plants and Potato virus Y (PVY), the most devastating potato virus, will be described, starting with the biological variability of PVY, available detection methods, viral movement, and its accumulation in plants. The response of potato plants to PVYinfection will be described, first the symptomsʼ development from differentpoints of view, from macroscopical to cytological, and second the main metabolic pathways involved in response to infection. Alterations in photosynthesis, hormonal pathways, defense and signaling mechanisms, and otherpathways will be demonstrated. The most important methods enabling disclosure of potato response mechanisms and pathways will be described.
COBISS.SI-ID: 2662735
Differences in the early responses of two potato cultivars, Igor and Nadine, to two isolates of Potato virus Y (PVY), the aggressive PVYNTN and the mild PVYN, were monitored for the first time. Microarray and quantitative real-time PCR analyses were carried out to identify differentially expressed genes after inoculation with each virus isolate.
COBISS.SI-ID: 2254415
This chapter in the book entitled “Monolithic chromatography and its modern applications” is an overview of applications with monolithic columns for purification and separation of large bio-molecules. As it is described in this publication methacrylate monoliths were already in the past proven to be very efficient in purification and concentration of different viruses.
COBISS.SI-ID: 3825016