Progress 12/15/08 to 12/14/13

Outputs
Target Audience: Plant pathologists working on management of PVY, plant virologists interested in virus evolution. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One MS student was involved in the work on this project, K.J. Evans, who is preparing for her thesis defense in May 2014. One visiting MS student from University of Chihuahua (Mexico) received training in methods of whole-genome sequencing and sequence analysis using PVY as a model. How have the results been disseminated to communities of interest? Results have been published as peer-reviewed papers and presented at professional meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Potato virus Y (PVY) exists as a complex of strains, including a growing number of recombinants. Evolution of PVY proceeds through accumulation of mutations and more rapidly through recombination, combining large sections of parental genomes, which leads to adaptation of the virus to multiple potato cultivars and a wider range of environmental conditions. The role of recombination in PVY evolution and origin of the common PVY recombinants, such as PVYN:O, PVYN-Wi, and PVYNTN, were studied here through whole genome sequencing of PVY genomes and subsequent recombination and phylogenetic analysis. A collection of 76 newly sequenced PVY isolates and 148 PVY genomes from the GenBank database was subjected to phylogenetic analysis, focusing on large genome sections commonly involved in recombination. A new recombinant between PVYO and PVYC genomes was sequenced and identified. A substantial diversity was revealed within non-recombinant parental strain types PVYO and PVYEu-N, with several distinct lineages identified. This diversity in the parental sequences allowed us to trace the origins and evolution of all recombinant types of PVY, which also show considerable diversity in most cases. From the clade placement for different genome sections, it was elucidated that certain recombinant types are formed from different parental sequences and hence likely to have some selective advantages.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Karasev, A.V. and Gray, S.M. (2013) Genetic diversity of Potato virus Y complex. American Journal of Potato Research 90: 7-13. Quintero-Ferrer, A. and Karasev, A.V. (2013) First report of Potato virus Y in potato in Jalisco, Mexico. Plant Disease 97: 430-430. Karasev, A.V. and Gray, S.M. (2013) Continuous and emerging challenges of Potato virus Y in potato. Annual Review of Phytopathology 51: 571-586. Chikh Ali, M., Karasev, A.V., Furutani, N., Taniguchi, M., Kano, Y., Sato, M., Natsuaki, T., and Maoka, T. (2013) Occurrence of Potato virus Y strain PVYNTN in foundation seed potatoes in Japan, and screening for symptoms in Japanese potato cultivars. Plant Pathology 62: 1157-1165. Chikh Ali, M., Gray, S.M., and Karasev, A.V. (2013) An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y. Plant Disease 97: 1370-1374. Quintero-Ferrer, A., Robles-Hernandez, L., Gonzalez-Franco A.C., Kerlan, C., and Karasev, A.V. (2014) Molecular and biological characterization of a recombinant isolate of Potato virus Y from Mexico. Archives of Virology, published on-line January 9, 2014 (DOI 10.1007/s00705-013-1968-0).

Progress 12/15/11 to 12/14/12

Outputs
OUTPUTS: Potato virus Y (PVY) infects four major solanaceous crops, potato, tomato, pepper, and tobacco, and exists as a group of strains and variants which differ in host specificity, symptom expression, and genome structure. In potato, five genetic groups have been identified based on hypersensitive resistance (HR) response in potato indicators bearing different N genes. In addition, multiple recombinants have been sequenced, in particular, PVYNTN strain, associated with tuber necrosis in potato, with the genome composed of four segments from two parental strains, PVYO and PVYN, and having 3 recombinant junctions. Since neither PVYO nor PVYN genomes are normally associated with tuber necrosis, the origin and evolution of the PVYNTN strain causing necrosis in potato tubers are unclear. Location of a specific virus determinant responsible for the tuber necrosis induction is also unknown at the moment. We subjected individual segments of PVYO and PVYN specific sequences from PVYNTN isolates to phylogenetic analysis along with respective sequences from parental, non-recombinant genomes, and also with other types of PVY recombinants, like PVYN:O and PVYN-Wi. These recombinant and non-recombinant sequences were generated in the course of a PVY whole genome sequencing project conducted on the North American collection of PVY isolates from 16 U.S. states. Phylogenetic analysis suggested that the PVYNTN strain may have polyphyletic origin, with only one segment in the recombinant genome coming from a common PVYN parent for all PVYNTN isolates sequenced. PARTICIPANTS: Alexander Karasev, Associate Professor; Celeste Brown, Professor, Kelsie Evans, Graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
A panel of 36 PVY isolates, randomly collected in Brazil from potato between 1985 and 2009, was subjected to a systematic molecular and serological typing using RT-PCR and a series of PVYO and PVYN-specific monoclonal antibodies. The data collected were combined with biological characterization of the same isolates in tobacco. Of the 36 isolates tested, three were typed as PVYO, ten PVYN:O/N-Wi, twenty-one PVYNTN, and two "unusual" or inconclusive. Of the ten isolates from the recombinant PVYN:O/N-Wi strain group, one isolate, MAF-VOY, was found to have an unusual serological profile identical to the non-recombinant PVYO-O5 strain group. The twenty one tested PVYNTN isolates included one isolate that did not induce vein necrosis in tobacco and two isolates with an unusual serological profile, i.e. displaying negative reactivity to one commercial PVYN-specific monoclonal antibody. Whole genome sequences were determined for four PVY isolates from Brazil, representing PVYO, PVYNTN, and PVYN-Wi strains. The genome of the MAF-VOY isolate was found recombinant having characteristic N-Wi structure with two recombinant junctions, and carrying a single mutation in the capsid protein at position 98 which lead to an unusual O5 serological reactivity. Taken together, the data obtained suggest that the two recombinant strains, PVYNTN and PVYN:O/N-Wi, now are apparently dominant in Brazilian potato crop. The data also suggest that recombinant isolates in Brazil often have unusual serological reactivity which may hamper their correct identification by conventional typing based on ELISA.

Publications

• Galvino-Costa, S.B., Figueira, A., Camargos, V.V., Geraldino, P.S., Hu, X., Nikolaeva, O.V., Kerlan, C., and Karasev, A.V. (2012) A novel type of Potato virus Y recombinant genome, determined for the genetic strain PVYE. Plant Pathology 61: 388-398.
• Nikolaeva, O.V., Roop, D., Galvino-Costa, S.F.B., Figueira, A.R., Gray, S.M., and Karasev, A.V. (2012) Epitope mapping for monoclonal antibodies recognizing tuber necrotic strains of Potato virus Y. American Journal of Potato Research 89: 121-128.
• Galvino-Costa, S.B.F., Figueira, A.R., Rabelo-Filho, F.A.C., Moraes, F. H. R., Nikolaeva, O.V., and Karasev, A.V. (2012) Molecular typing of Potato virus Y isolates from Brazil reveals a diverse set of recombinant strains. Plant Disease 96: 1451-1458.

Progress 12/15/10 to 12/14/11

Outputs
OUTPUTS: During characterization of recombinant Potato virus Y (PVY) isolates collected in Brazil, two isolates, PVY-AGA and PVY-MON, were identified with a novel type of PVYNTN recombination pattern. Whole genome sequence analysis revealed that PVY-AGA and PVY-MON represented recombinants between two novel parent genomes, PVYNTN and PVY-NE11. Specifically, new recombinants had an ordinary PVYNTN genome structure for approximately 6.7-kb from the 5'- end of the genome, while the 3'-terminal 3.0-kb segment had two fragments of NE-11 sequence separated by another small NTN fragment. Only PVY-AGA induced vein necrotic reaction in tobacco. Both PVY-AGA and PVY-MON isolates did not induce hypersensitive resistance (HR) in potato cultivars carrying Ny, Nc, or (putative) Nz genes. An ordinary PVYNTN isolate PVY-AST induced systemic HR in cultivar Maris Bard carrying a putative Nz gene. All three isolates, PVY-AGA, PVY-MON, and PVY-AST, induced typical potato tuber necrotic ringspot disease in a susceptible potato cv Yukon Gold under greenhouse conditions. In a standard multiplex RT-PCR assay, PVY-AST, PVY-AGA, and PVY-MON were all typed as ordinary PVYNTN isolates, consistent with the presence of two prominent recombinant junctions in their genome, characteristic of an ordinary PVYNTN strain. Ability of these new PVY recombinants to overcome resistance genes in potato producing mild or no foliar symptoms presents a significant threat posed by these isolates to seed potato production areas. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Potato virus Y (PVY) is an important viral pathogen of potato responsible for reducing tuber yield and quality across the globe. The PVYN and PVYNTN strains, the latter of which induces potato tuber necrotic ringspot disease (PTNRD), are regulated for international potato trade, and have been routinely detected using monoclonal antibodies (MAbs) that discriminate between PVYN and PVYO serotypes. Here, we identify the distinct binding sites in the capsid protein of PVY for three of the four main PVYN-specific MAbs, Bioreba-N, SASA-N, and Neogen-N, available commercially. These binding domains were mapped through a combination of TAS-ELISA testing of MAbs on multiple reference isolates of PVY, sequence analysis, heterologous expression of capsid protein fragments, and synthetic peptide binding experiments. All three MAbs were found to bind linear epitopes located within the first 31 N-terminal amino acids of the capsid protein. Bioreba-N MAb epitope spanned aa 1-17 and included three positions, aa 1, aa 11, and aa 17, which differ between PVYN and PVYO serotypes. Both SASA-N and Neogen-N epitopes spanned aa 22-30, and included two positions, aa 24 and aa 29, which differ between PVYN and PVYO serotypes. Epitopes for SASA-N and Neogen-N MAbs are likely to be identical or overlapping. Examination of available sequences for tuber necrotic isolates of PVY that do not react with PVYN-specific MAbs SASA-N and Neogen-N indicated possible selection for substitutions in corresponding epitopes leading to the loss of reactivity towards these antibodies. The data obtained suggested that testing with more than one PVYN serotype-specific MAb could assure a reliable serological identification of a PVYN or PVYNTN isolate.

Publications

• Karasev, A.V., Hu, X., Brown, C.J., Kerlan, C., Nikolaeva, O.V., Crosslin, J.M., and Gray, S.M. (2011) Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains. Phytopathology 101: 778-785.
• Kerlan, C., Nikolaeva, O.V., Hu, X., Meacham, T., Gray, S.M.,and Karasev, A.V. (2011) Identification of the molecular make-up of the Potato virus Y strain PVYZ: Genetic typing of PVYZ-NTN. Phytopathology 101: 1052-1060.

Progress 12/15/09 to 12/14/10

Outputs
OUTPUTS: The ordinary strain of Potato virus Y (PVY), PVYO, causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel sub-strain of PVYO was recently reported, PVYO-O5, which is spreading in the U.S. and is distinguished from other PVYO isolates serologically, i.e. reacting to the otherwise PVYN-specific monoclonal antibody 1F5. To characterize this new PVYO-O5 sub-group, and address possible reasons for its continued spread, we conducted a molecular study of PVYO and PVYO-O5 isolates from a North American collection of PVY through whole genome sequencing and phylogenetic analysis. Forty-four PVYO isolates were sequenced, including 31 from the previously defined PVYO-O5 group, and subjected to whole genome analysis. PVYO-O5 isolates formed a separate lineage within the PVYO genome cluster in the whole genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVYO sequences separated into at least two distinct lineages on the whole genome phylogenetic tree. To shed light on the origin of the three most common PVY recombinants, a more detailed phylogenetic analysis of a sequence fragment, nt 2,406-5,821, that is present in all recombinant and non-recombinant PVYO genomes was conducted. The analysis revealed that PVYN:O and PVYN-Wi recombinants acquired their PVYO segments from two separate PVYO lineages, while the PVYNTN recombinant acquired its PVYO segment from the same lineage as PVYN:O. These data suggest that PVYN:O and PVYN-Wi recombinants originated from two separate recombination events involving two different PVYO parental genomes, while the PVYNTN recombinants likely originated from the PVYN:O genome via additional recombination events. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In order to characterize molecular and biological features of the new PVYO-O5 variant that may be responsible for its spread in the U.S., 44 isolates from the survey collection were subjected to whole genome sequencing and subsequent phylogenetic analysis. A majority of these isolates were PVYO-O5 variants, however, 13 additional PVYO isolates were included to provide better reference between the two PVYO sub-groups. We determined that PVYO-O5 represents a phylogenetically distinct lineage of isolates within a broader PVYO clade, which demonstrated considerable diversity. Phylogenetic analyses of the PVYO sequences common between non-recombinant PVYO and recombinant PVYNTN, PVYN-Wi, and PVYN:O genomes allowed an insight into the origin of recombinant PVY strains.

Publications

• Gray, S.M., DeBoer, S.H., Lorenzen, J., Karasev, A.V., Whitworth, J., Nolte, P., Singh, R.P., Boucher, A., and Xu, H. (2010) Potato virus Y: a significant and evolving threat to potato crops in the United States and Canada. Feature Article. Plant Disease 94, 1384-1397.
• Karasev, A.V., Nikolaeva, O.V., Hu, X., Sielaff, Z., Whitworth, J., Lorenzen, J.H., and Gray, S.M. (2010). Serological properties of ordinary and necrotic isolates of Potato virus Y: a case study of PVYN misidentification. Amer. J. Potato Res. 87, 1-9.
• Hu, X., Karasev, A.V., Brown, C.J., and Lorenzen, J.H. (2009). Sequence characteristics of Potato virus Y recombinants. J. Gen. Virol. 90, 3033-3041.
• Kerlan, C., Marhadour, S., and Karasev, A.V. (2010) A novel source of hypersensitive resistance against the PVYN strain of Potato virus Y (PVY) in an unidentified potato cultivar. Abstracts of the 94th Annual Meeting of the Potato Association of America, August 15-19, 2010; Corvallis, OR.
• Kerlan, C., Nikolaeva, O.V., Hu, X., Meacham, T., Gray, S.M., and Karasev, A.V. (2010) Identification of the molecular make-up of the Potato virus Y strain PVYZ. Abstracts of the Annual Meeting of the American Phytopathological Society, August 7-11, 2010; Charlotte, NC.

Progress 12/15/08 to 12/14/09

Outputs
OUTPUTS: A novel PVY isolate, L26, recovered from a Frontier potato line was initially typed as a PVYNTN strain using multiplex RT-PCR and serological assays. L26 induced mosaic and mild vein clearing symptoms in tobacco rather than vein necrosis characteristic of the PVY NTN strain. The whole genome sequence was determined for L26 and two other PVYNTN isolates, HR1 and N4, from Idaho that did induce vein necrosis in tobacco. The sequence of all three isolates was similar to typical European PVYNTN isolates that contain three recombination junctions in their genome. The sequence of the L26 genome was nearly identical to the genomes HR1, N4, and to a previously characterized PVYNTN isolate, 423-3, differing by only five nucleotides in the entire ca. 9.7-kb genome, only one resulting in a corresponding amino acid change, D-205 to G-205 in the central region of HC-Pro. Two "signature" amino acid residues, thought involved in induction of the vein necrosis syndrome in tobacco, K-400 and E-419, were present in the C-terminal region of HC-Pro of all three isolates. Multiple alignment of the whole genome sequences of L26 and other PVYNTN isolates whose phenotype in tobacco has been reported, suggests that a single nucleotide change (A-1,627 to G-1,627) resulting in the single amino acid change (D-205 to G-205) in the HC-Pro cistron of L26 correlates with the loss of the vein necrosis phenotype in tobacco. Taken together, these data suggest that the vein necrosis genetic determinant of PVY in tobacco is complex and includes other element(s), in addition to the C-terminal fragment of HC-Pro. In the course of a multi-year survey of PVY incidence and diversity in seed potato certification systems in North America, an unusual sub-set of PVY isolates was identified. These isolates, collectively named PVYO5, exhibited N-specific immunoreactivity when subjected to a standard, NAPPO-approved testing in TAS-ELISA. Consequently, potato lots with these PVY isolates would have triggered a quarantine action and not allowed for cross-border shipments and trade. We demonstrated that PVYO5 isolates belong to a common group of PVYO isolates, by a variety of methods. The unusual immunoreactivity in the NAPPO-approved testing protocol is due to a single amino acid substitution in the PVYO5 capsid protein, R-98 to Q-98. We demonstrated that this amino acid substitution affects a specific epitope recognized by the monoclonal antibody 1F5 which is used in the NAPPO test. We provided evidence that if another N-specific monoclonal antibody, is used in place of 1F5, this misidentification does not occur, and PVYO5 isolates are identified correctly by TAS-ELISA. PVY survey has demonstrated that PVYO5 sub-set is wide-spread in North America, and in some states represents a substantial proportion among PVY-positive potato samples resulting in frequent misidentification of PVYO5 as PVYN/NTN with clear quarantine implications for export shipments of potato. We propose to replace 1F5 antibody in PVY typing for quarantine purposes, to avoid possible unwarranted misidentification of PVYO5 strains as PVYN type. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Through analysis of the sequence of the novel L26 isolate of PVY and its biological properties, we proved that the tobacco vein necrosis determinant of PVY is distinct from the tuber necrosis-inducing determinant, resulting in potato tuber necrosis ringspot disease (PTNRD). This has a substantial value for the development of diagnostic reagents to identify PTNRD inducing strains of the virus. Through analysis of the epitope specificity of several monoclonal antibodies used for PVY quarantine purposes, we identified an epitope for the 1F1 monoclonal approved by NAPPO. We demonstrated that this antibody mistakenly identifies a sub-set of ordinary PVY strains (PVY-O) as belonging to the necrotic PVY strains (PVY-N), and thus directly affects potato trade between Canada, Mexico, and the U.S. This subset is named PVY-O5, and we demonstrated that it can be correctly identified as PVY-O group by another monoclonal antibody with a different epitope specificity.

Publications

• Hu, X., Meacham, T., Ewing, L., Gray, S.M., and Karasev, A.V. A novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco. Virus Res. 143: 68-76, 2009.
• Karasev, A.V., Nikolaeva, O.V., Hu, X., Sielaff, Z., Whitworth, J., Lorenzen, J., and Gray, S.M. Serological properties of ordinary and necrotic isolates of Potato virus Y: a case study of PVYN misidentification. Amer. J. Potato Res. 86, 2009 (in press), published on-line on September 3, 2009 as doi: 10.1007/s12230-009-9110-2.
• Karasev, A.V., Hu, X., and Gray, S.M. (2009) Sequencing the North American collection of Potato virus Y isolates: applications to identification of disease determinants and strain differentiation. Abstracts of the 93rd Annual Meeting of the Potato Association of America, August 9-14, 2009; Fredericton, NB, #62.
• Hu, X., Meacham, T., Ewing, L., Gray, S.M., and Karasev, A.V. (2009) A novel recombinant strain of Potato virus Y suggests a new viral genetic determinant of vein necrosis in tobacco. Abstracts of the 2009 Annual Meeting of the American Society for Virology, July 11-15, 2009; Vancouver, BC, W16-12.