Alexander V. Karasev

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.

Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus

The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.

Potato virus Y: An Evolving Concern for Potato Crops in the United States and Canada

North American potato production differs from other geographical regions such as Europe in that it is essentially a closed system, i.e., seed potatoes are not imported and production is dominated by only a few cultivars. The lack of significant seed imports provides a mechanism for seed certification to be extremely effective at minimizing virus levels in seed lots, especially if the changes in seed laws, postharvest testing, and tolerance limits discussed above are adopted. This is an opportunity to effectively manage PVY at levels that are at or below detection and well below economic significance. Aiding the seed certification programs in the adoption of the Canada/US-Management Plan for Potato Viruses that Cause Tuber Necrosis has and continues to build consensus and cooperation within the industry to reform and modernize seed certification practices and, as importantly, modernize best management practices that growers can implement so that their production meets or exceeds virus tolerances set within the seed certification standards. Seed inspectors could also benefit from continually updated information from the research community to help them better recognize the spectrum of symptoms caused by the various strains and variants of PVY in all the different cultivars now being grown in their states and provinces. They could also benefit from improved field diagnostics that will assist them and the growers in identifying problem plants that should be rogued. If PVY levels in seed can be minimized and on-farm management strategies can be optimized, then PVY incidence in the potato crop will be marginalized. The restricted distribution of the tuber necrotic strains also offers an opportunity to prevent these strains from becoming economically significant if appropriate testing of seed lots in those areas could prevent them from being planted. Shipping point inspections of tubers will also help in identifying and eliminating tuber necrotic viruses. The dominance of a few cultivars has been eroding in recent years. Russet Burbank, a cultivar introduced over 100 years ago, still accounts for 40 to 50% of the U.S. acreage, but acreage in the Northwestern United States has been declining steadily as other russet cultivars come on the market and gain acceptance. Potato cultivar has had a significant impact on the PVY problem, as with the release and widespread acceptance of Shepody, Russet Norkotah, and other asymptomatic carriers of PVY (http://oregonstate. edu/potatoes/latenttoPVYlist.htm), which in 2008 comprised more than 15 and 12% of the total U.S. and Canadian seed acreage, respectively. These cultivars have certainly contributed to the overall increase in PVY in the seed potato crop and by extension the commercial potato crop. The increased diversity of potato cultivars grown in both countries has also introduced a wider spectrum of PVY symptoms, most notably the milder symptoms that are characteristic of the PVYN/NTN and PVYN-Wi strains on many cultivars. Since the success of seed certification is dependent upon visual assessment of the crop, mild or absent symptoms means that many more infected plants go unnoticed. The more symptomatic PVYO strains are observed and removed, but the other strains remain in the crop and are passed along in the seed, contributing to an overall increase in PVY incidence and more importantly to a shift in PVY strain composition. The U.S. and Canadian potato industry stakeholders are increasingly aware of the PVY-associated challenges and have been moving rapidly to work with researchers and all aspects of the industry to implement plans to suppress PVY incidence. Continued education of growers, seed certification officials, and researchers alike, coupled with the development and adoption of new or revised best management practices and diagnostic tools, and the renewed inter est of breeders to develop virus resistant cultivars, will be the keys to success in bringing PVY incidence under control and in minimizing tuber necrotic strains.

Continuous and Emerging Challenges of Potato virus Y in Potato

Potato virus Y (PVY) is one of the oldest known plant viruses, and yet in the past 20 years it emerged in the United States as a relatively new and very serious problem in potato. The virus exists as a complex of strains that induce a wide variety of foliar and tuber symptoms in potato, leading to yield reduction and loss of tuber quality. PVY has displayed a distinct ability to evolve through accumulation of mutations and more rapidly through recombination between different strains, adapting to new potato cultivars across different environments. Factors behind PVY emergence as a serious potato threat are not clear at the moment, and here an attempt is made to analyze various properties of the virus and its interactions with potato resistance genes and with aphid vectors to explain this recent PVY spread in potato production areas. Recent advances in PVY resistance identification and mapping of corresponding genes are described. An updated classification is proposed for PVY strains that takes into account the most current information on virus molecular genetics, serology, and host reactivity.

PUTATIVE 65 KDA PROTEIN OF BEET YELLOWS CLOSTEROVIRUS IS A HOMOLOGUE OF HSP70 HEAT SHOCK PROTEINS

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.

Genetic diversity of the ordinary strain of Potato virus Y (PVY) and origin of recombinant PVY strains.

The ordinary strain of Potato virus Y (PVY), PVY(O), causes mild mosaic in tobacco and induces necrosis and severe stunting in potato cultivars carrying the Ny gene. A novel substrain of PVY(O) was recently reported, PVY(O)-O5, which is spreading in the United States and is distinguished from other PVY(O) isolates serologically (i.e., reacting to the otherwise PVY(N)-specific monoclonal antibody 1F5). To characterize this new PVY(O)-O5 subgroup and address possible reasons for its continued spread, we conducted a molecular study of PVY(O) and PVY(O)-O5 isolates from a North American collection of PVY through whole-genome sequencing and phylogenetic analysis. In all, 44 PVY(O) isolates were sequenced, including 31 from the previously defined PVY(O)-O5 group, and subjected to whole-genome analysis. PVY(O)-O5 isolates formed a separate lineage within the PVY(O) genome cluster in the whole-genome phylogenetic tree and represented a novel evolutionary lineage of PVY from potato. On the other hand, the PVY(O) 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, nucleotides 2,406 to 5,821, that is present in all recombinant and nonrecombinant PVY(O) genomes was conducted. The analysis revealed that PVY(N:O) and PVY(N-Wi) recombinants acquired their PVY(O) segments from two separate PVY(O) lineages, whereas the PVY(NTN) recombinant acquired its PVY(O) segment from the same lineage as PVY(N:O). These data suggest that PVY(N:O) and PVY(N-Wi) recombinants originated from two separate recombination events involving two different PVY(O) parental genomes, whereas the PVY(NTN) recombinants likely originated from the PVY(N:O) genome via additional recombination events.

The complete nucleotide sequence and genome organization of tomato chlorosis virus

Summary.The crinivirus tomato chlorosis virus (ToCV) was discovered initially in diseased tomato and has since been identified as a serious problem for tomato production in many parts of the world, particularly in the United States, Europe and Southeast Asia. The complete nucleotide sequence of ToCV was determined and compared with related crinivirus species. RNA 1 is organized into four open reading frames (ORFs), and encodes proteins involved in replication, based on homology to other viral replication factors. RNA 2 is composed of nine ORFs including genes that encode a HSP70 homolog and two proteins involved in encapsidation of viral RNA, referred to as the coat protein and minor coat protein. Sequence homology between ToCV and other criniviruses varies throughout the viral genome. The minor coat protein (CPm) of ToCV, which forms part of the “rattlesnake tail” of virions and may be involved in determining the unique, broad vector transmissibility of ToCV, is larger than the CPm of lettuce infectious yellows virus (LIYV) by 217 amino acids. Among sequenced criniviruses, considerable variability exists in the size of some viral proteins. Analysis of these differences with respect to biological function may provide insight into the role crinivirus proteins play in virus infection and transmission.

HSP70-related 65 kDa protein of beet yellows closterovirus is a microtubule-binding protein

Beet yellows virus (BYV) genome encodes a 65 kDa protein homologous to the HSP70 family of cellular heat‐shock proteins (Agranovsky, A.A., Boyko, V.P., Karasev, A.V., Koonin, E.V. and Dolja, V.V. (1991) J. Mol. Biol. 217, 603–610). The respective gene was cloned and expressed in vitro yielding a product of the expected size (p65). This product was found to bind to the purified microtubules with a binding constant of 4 × 10−7 M. The binding of p65 was stimulated if ATP presented in the translation mixture was hydrolyzed by apyrase. Removal of the short C‐terminal domains of α‐ and β‐tubulin by subtilisin digestion abolished the binding, demonstrating its specificity. The possible role of p65 association with microtubules in the movement of virus within and/or between plant cells is proposed.

Serological properties of ordinary and necrotic isolates of Potato virus Y: a case study of PVYN misidentification.

In the course of a multi-year survey of Potato virus Y (PVY) incidence and diversity in the U.S. seed potato crop, an unusual PVY variant was identified in low but significant levels in multiple states. This variant, PVYO-O5, was initially detected by a commercially available PVYN-specific monoclonal antibody, 1F5. This antibody is widely used by U.S. Seed Certification programs to test for PVYN and is one of two antibodies designated by the North American Plant Protection Organization (NAPPO) for pre-shipment testing of tuber lots that are to be transported between countries. Consequently, PVYN positives identified by the 1F5 antibody have triggered quarantine actions, prevented cross-border shipments and impacted trade. Here, we demonstrate by a variety of methods that the PVYO-O5 is a variant within the ordinary PVY strain (PVYO). Specifically, the PVYO-O5 variant likely arose due to a single amino acid substitution within the capsid protein. This variant does not induce vein necrosis in tobacco or tuber necrosis in susceptible varieties of potato. Furthermore, it is identified by RT-PCR based diagnostics as PVYO and it has a typical PVYO genome sequence. We demonstrate that another PVYN specific monoclonal antibody, SASA-N, recognizes an epitope distinct from that recognized by 1F5, and correctly identifies the PVYO-O5 variants as belonging to the PVYO serotype. Since the PVYO-O5 variant is present in many seed producing states and misidentification of PVYO-O5 as PVYN/NTN has clear quarantine implications for export shipments of potato, the limitations of the commercially available monoclonal antibodies should be considered in any certification or phytosanitary testing program.ResumenA lo largo del estudio de varios años sobre la incidencia y diversidad del virus Y de la papa (PVY) en los cultivos de papa para semilla en los Estados Unidos (EU), se identificó a una variante inusual a niveles bajos pero significativos en múltiples estados. Esta variante, PVYO-O5, se detectó inicialmente con un anticuerpo monoclonal comercialmente disponible específico para PVYN, el 1F5. Este anticuerpo es ampliamente usado por los Programas de Certificación de Semilla en los EU para PVYN, y es uno de los dos anticuerpos designados por la Organización Norteamericana de Protección de Plantas (NAPPO) para pruebas de pre-envío de lotes de tubérculos que serán transportados entre países. Consecuentemente, los PVYN positivos identificados con el anticuerpo 1F5 han disparado acciones cuarentenarias, evitando envíos trans-fronteras y han impactado al comercio. Aquí, nosotros demostramos con diversos métodos que PVYO-O5 es una variante del PVY ordinario (PVYO). Específicamente, la variante PVYO-O5 es probable que haya surgido debido a una substitución de un aminoácido dentro de la proteína de la cápside. Esta variante no induce necrosis de las venas en tabaco o necrosis del tubérculo en variedades susceptibles de papa. Aún mas, se le identifica como PVYO mediante RT-PCR y tiene la típica secuencia genómica del PVYO. Demostramos que otro anticuerpo monoclonal específico para PVYN, el SASA-N, reconoce un epítope distinto al reconocido por 1F5, e identifica correctamente a las variantes PVYO-O5 como pertenecientes al serotipo PVYO. Tomando en cuenta que la variante PVYO-O5 esta presente en muchos estados que producen semilla, y que la identificación equivocada de PVYO-O5 como PVYN/NTN tiene claras implicaciones cuarentenarias para envíos de exportación de papa, se deberían de considerar las limitaciones de los anticuerpos monoclonales disponibles comercialmente en cualquier programa de pruebas para certificación o fitosanidad.

Expression strategy of the potato virus X triple gene block.

The mode of expression of the overlapping genes of the triple block positioned internally in potato virus X (PVX) RNA was examined. The results of In vitro translation of synthetic RNA transcripts and natural PVX-specific methylmercuric hydroxide-denatured dsRNAs suggest that the 25K protein is expressed as a single translation product of the 2.1 kb subgenomic (sg) RNA and that both the 12K and 8K proteins are expressed from the same 1.4 kb sgRNA.