Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences.

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.

New food from a potato somatic hybrid: nutritional equivalence and safety assessment by a feeding study on rats.

BACKGROUND: Potato tubers from the STBd somatic hybrid line that exhibited improved tolerance to salinity and resistance to fungal and PVY infections were characterised. They were compared for their chemical composition to the Spunta variety produced by conventional agronomic practices. This study aimed to compare nutritional value and safety by feeding rats with STBd or commercial tubers added to the standard diet (20/80 w/w). RESULTS: The analysis of soluble sugar, fat, fibre and ash content of tubers did not reveal any significant differences between the hybrid line and the control Spunta variety. Small differences were observed in dry matter, starch and protein content of hybrid potatoes in comparison to controls. However, all values were within normal ranges reported in the literature. The feeding study on rats showed that overall health, weight gain, food consumption, morphological aspects and weights of organs were comparable between rat groups fed the STBd hybrid and the Spunta variety. CONCLUSION: Taken together, 28 days of consumption of STBd hybrid potato did not exert any adverse effect on rats compared with commercial Spunta potato. The STBd potato line was therefore considered to be as safe for food utilisation as the commercial variety.

Immune response to a potyvirus with exposed amino groups available for chemical conjugation.

BACKGROUND: The amino terminus of the tobacco etch virus (TEV) capsid protein is located on the external surface of infectious TEV particles, as proposed by previous studies and an in silico model. The epsilon amino groups on the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as antigen carriers. The availability of amino groups on the surfaces of TEV particles was determined and the immune response to TEV evaluated. RESULTS: Using a biotin-tagged molecule that reacts specifically with amino groups, we found that the TEV capsid protein has amino groups on its surface available for coupling to other molecules via crosslinkers. Intraperitoneal TEV was administered to female BALB/c mice, and both their humoral and cellular responses measured. Different IgG isotypes, particularly IgG2a, directed against TEV were induced. In a cell proliferation assay, only spleen cells from vaccinated mice that were stimulated in vitro with TEV showed significant proliferation of CD3+/CD4+ and CD3+/CD8+ subpopulations and secreted significant amounts of interferon γ. CONCLUSIONS: TEV has surface amino groups that are available for chemical coupling. TEV induces both humoral and cellular responses when administered alone intraperitoneally to mice. Therefore, TEV should be evaluated as a vaccine adjuvant when chemically coupled to antigens of choice.

Engineering broad-spectrum resistance against RNA viruses in potato.

RNA silencing technology has become the tool of choice for inducing resistance against viruses in plants. A significant discovery of this technology is that double-stranded RNA (dsRNA), which is diced into small interfering RNAs (siRNAs), is a potent trigger for RNA silencing. By exploiting this phenomenon in transgenic plants, it is possible to confer high level of virus resistance by specific targeting of cognate viral RNA. In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have constructed a chimeric expression vector containing three partial gene sequences derived from the ORF2 gene of Potato virus X, Helper Component Protease gene of Potato virus Y and Coat protein gene of Potato leaf roll virus. Solanum tuberosum cv. Desiree and Kuroda were transformed with this chimeric gene cassette via Agrobacterium tumefaciens-mediated transformation and transgenic status was confirmed by PCR, Southern and double antibody sandwich ELISA detection. Due to simultaneous RNA silencing, as demonstrated by accumulation of specific siRNAs, the expression of partial triple-gene sequence cassette depicted 20% of the transgenic plants are immune against all three viruses. Thus, expression of a single transgene construct can effectively confer resistance to multiple viruses in transgenic plants.

Overexpression of the wild potato eIF4E-1 variant Eva1 elicits Potato virus Y resistance in plants silenced for native eIF4E-1.

Potato virus Y (PVY) is the most important viral pathogen of cultivated potato (Solanum tuberosum) from a commercial perspective, causing severe losses in both tuber quality and yield worldwide. Specific accessions of wild potato species exhibit resistance against PVY but efforts to transfer the trait to cultivated material have not yielded widely adopted varieties. Because amino acid substitutions at specific domains of host factor eIF4E-1 often confer resistance to various crops, we sequenced the associated genes expressed in wild potato plants. A novel eIF4E-1 variant, designated here as Eva1, was identified in S. chacoense, S. demissum, and S. etuberosum. The protein contains amino acid substitutions at ten different positions when compared to its cultivated potato (S. tuberosum) homolog. In the yeast two-hybrid system, Eva1 failed to bind VPg, a viral protein required for infectivity. Overexpression of the associated cDNA conferred PVY resistance to transgenic potato plants silenced for the native eIF4E-1 gene. Because the gene sources of Eva1 are sexually compatible with potato, the molecular strategies described can be employed to develop 'intragenic' potato cultivars.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.

A test of taxonomic and biogeographic predictivity: resistance to Potato virus Y in wild relatives of the cultivated potato.

A major justification for taxonomic research is its assumed ability to predict the presence of traits in a group for which the trait has been observed in a representative subset of the group. Similarly, populations in similar environments are expected to be more alike than populations in divergent environments. Consequently, it is logical to assume that taxonomic relationships and biogeographical data have the power to predict the distribution of disease resistance phenotypes among plant species. The objective of this study was to test predictivity in a group of widely distributed wild potato species, based on hypotheses that closely related organisms (taxonomy) or organisms from similar environments (biogeography) share resistance to a simply inherited trait (Potato virus Y [PVY]). We found that wild potato species with an endosperm balance number (EBN) of 1 (a measure of cross compatibility) shared resistances to PVY more than species with different EBN values. However, a large amount of variation was found for resistance to PVY among and within species. We also found that populations from low elevations were more resistant than those from high elevations. Because PVY is vectored by aphids, we speculate that the distribution of aphids may determine the level of selection pressure for PVY resistance.

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.

Cellular localisation of calcium ions during potato hypersensitive response to Potato virus Y.

Ca(2+) is one of the most universal and versatile signalling molecules and is involved in almost every aspect of cellular processes. Accumulating evidence suggests that Ca(2+) serves as a messenger in many growth and developmental processes and in plant responses to biotic and abiotic stresses. Numerous signals have been shown to induce transient elevation of cytoplasmic [Ca(2+)](cyt) in plants. The calcium free ions were detected cytochemically in Solanum tuberosum cv. Rywal tissues as a hypersensitive response (HR) from 10h to 5 days after a Potato virus Y (PVY) infection. Calcium was detected in vivo by its reaction with Alizarin S Red, producing an intense red staining in contact with calcium free ions. Calcium was found in the necrotic area of the epidermal and mesophyll cells 3 days after the PVY infection (when morphological symptoms on potato leaves appeared). Calcium ions were detected cytochemically in HR also by its reaction with potassium pyroantimonate. Inoculation with PVY(NTN) and also PVY(N) Wi induced a rapid hypersensitive response during which highly localised increased accumulation of electron-dense deposits of calcium pyroantimonate were detected. Calcium deposition was observed in necrotic and non-necrotic areas, starting from 10h after PVY infection. The deposits were present along ER cisternae, chloroplasts and mitochondria envelopes connected with PVY particles. The precipitates of calcium antimonate were detected near the nuclear envelope, inside karyolymph and along tracheary elements, especially when virus particles were present inside.

Production of polyclonal antibodies to a recombinant coat protein of potato virus Y.

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Omega. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.

Multiplex microsphere immuno-detection of potato virus Y, X and PLRV.

To monitor seed potatoes for potato virus X, Y and PLRV, a multiplex microsphere immunoassay (MIA) was developed based on the Luminex xMAP technology, as an alternative to ELISA. The xMAP technology allowed detection of a number of antigens simultaneously whereas ELISA only allowed simplex detection of antigens. The use of paramagnetic beads in the MIA procedure allowed efficient removal of excess sample compounds and reagents. This resulted in lower background values and a higher specificity than a non-wash MIA procedure using conventional beads. In a simplex MIA detection, levels for PVY and PLRV in potato leaf extracts were 10 times lower than ELISA but for PVX 10 timers higher, whereas the specificity was similar. Results of a multiplex assay performed on viruses added to potato leaf extracts were largely similar to those of ELISA for individual viruses. Results of samples infected naturally with PVX, PVY or PLRV were comparable with ELISA.

A point mutation changes the serotype of a potato virus Y isolate; genomic determination of the serotype of PVY strains.

A Syrian isolate of Potato virus Y (PVY), named PVY-12, reacted to two monoclonal antibodies that are specific to PVY(O,C) and PVY(N) strains, although its coat protein (CP) belongs to the PVY(N) strain. Analysis of the CP of PVY-12 revealed that a point mutation in its N terminus switched it from PVY(N)-like to PVY(O)-like at this position. This mutation changed the second nucleotide of the codon that encodes the 29th amino acid of the CP of PVY-12 from A to G, which resulted in one amino acid substitution from Glu(29 )to Gly(29). The role of Gly(29) in the binding of PVY-12 to PVY(O,C)-specific monoclonal antibody was confirmed by gene expression in Escherichia coli. The N terminus of the CP gene of PVY-12 and another PVY isolate of the N serotype with identical CP to PVY-12 except for one amino acid substitution from Gly(29 )to Glu(29) was cloned and expressed in E. coli using a pUC18 vector. Resulting antigens showed similar reactivity to the relevant antibodies as same as the native CPs of these two isolates. Further analysis of the CP of PVY isolates showed that Gly(29) was conserved in the CP of PVY(O), PVY(C), PVY(N)W, and non-potato isolates of PVY while Gln(17) and Glu(31 )were conserved in the CP of PVY(N/NTN). Therefore, these amino acids are characteristic of the CP for these strain groups and subgroups in agreement with the serotype and phylogenetic relationships previously determined.

Cloning and expression of functional single-chain Fv antibodies directed against NIa and coat proteins of potato virus Y.

Three single-chain variable fragment (scFv) antibodies recognizing the nuclear inclusion a (NIa) and capsid proteins of potato virus Y were obtained from two mouse derived hybridoma clones secreting, respectively, an anti-NIa (22-1) and an anti-coat protein (136-13) monoclonal antibodies. The first monoclonal antibody was able to inhibit in vitro the PVY polyprotein cleavage by blocking the NIa protease activity. The amplified scFv cDNAs were first inserted into the TOPO vector and then sequenced. Several recombinant E. coli clones carrying the accurate scFv sequences were selected and the corresponding cDNAs were subcloned in pHEN phagemid and transferred in E. coli strain. The expressed scFv fragments showed an antibody activity that recognized the viral target proteins in infected tissues. Their activity was comparable to the parental monoclonal antibodies.

New indicator plants for potato virus X.

For over 2 years (2002-2004); a sever virus like disease on leaves in Pisum sativum was observed from Tehran in Iran. The identity of which was established by the following host reactions and serological and molecular assays. Collected samples were tested for the presence of the virus using the DAS-ELISA (direct double antibody sandwich-ELISA). Leaf sap of each test samples was diluted 1:10 in ELISA sample buffer. The samples reacted in ELISA with antibody against Potato virus (x)(PVX). The samples were passed through three single chlorotic local lesion transfers on Gompherena globosa, which showed chlorosis 5 to 7 days after mechanical inoculation. Finally one local was homogenized in phosphate buffer (0.05 M, pH 7.2) and rubbing the inoculums on carbourundum--dusted leaves of Nicotiana glutinosa. For host range studies extracts prepared from infected N. glutinosa leave were inoculated to the 35 species. The virus induces systemic mosaic symptoms on Petunia hybrida, Physalis floridana, Nicandera physaloides and Solanum nigrum. This has not been yet record as assay hosts for PVX. Total RNA extracted from symptomatic, plants and RNA extracted from purified virus preparations were tested using reverse transcription polymerase chain reaction (RT-PCR) with specific primers designed to amplify a fragment of the RNA-dependent RNA-polymerase gen. Back inoculation from different symptomatic plants, had been done to Pisum sativum and confirmed by DAS-ELISA. This is the first report of Petunia hybrida, Physalis floridana, Nicandera physaloides and Solanum nigrum as new indicator plants for PVX.

Mapping of viral genomic regions important in cross-protection between strains of a potyvirus.

Cross-protection was tested between potato and tobacco strains of Potato virus A, a member of the genus Potyvirus (PVA), in tobacco plants. Cross-protection was effective only at the initiation of infection. The potato strains provided only weak cross-protection against the tobacco strain, whereas the tobacco strain provided strong cross-protection against potato strains. The tamarillo strain (TamMV) showed cross-protection phenotypes mostly resembling those of the potato strains. Chimera of the PVA strains were utilized to map viral genomic regions important for cross-protection. The coat protein (CP) encoding region and the helper component proteinase (HCpro) affected cross-protection and virus accumulation. An amino acid substitution at the CP N-terminus reduced virus accumulation and the ability to overcome cross-protection, whereas amino acid substitutions introduced to the HCpro increased virus accumulation and the ability to overcome cross-protection. Closer sequence relatedness between the protector and challenger isolate, as determined by the CP-encoding sequence, was correlated with an increased cross-protection ability. Cross-protection was not overcome by inoculation with nonencapsidated viral RNA. Thus, the differences in cross-protection abilities between PVA strains and chimera were not explained with the "re-encapsidation model" described for strains of Tobacco mosaic tobamovirus but may be associated with a virus infection-induced RNA silencing mechanism.

Polyclonal antibodies to a recombinant coat protein of Potato virus A.

Specific mouse antibodies against a recombinant coat protein (CP) of Potato virus A (PVA) were produced. The PVA CP gene was cloned in an expression vector pMPM4omega. After expression in Escherichia coli the presence of the expressed CP was proved by Western blot analysis using polyclonal and monoclonal antibodies (MAbs). The expressed CP was purified by centrifugation in CsCl density gradient or on a sucrose cushion. The production of virus-like particles (VLPs) was proved by electron microscopy. The purified CP was used for preparation of a mouse antiserum which had a titer of 1:1024 in ELISA and reacted specifically in Western blot analysis and indirect plate-trapped antigen ELISA (PTA-ELISA).

Phenotypic recombination between bean common mosaic and bean common mosaic necrosis potyviruses in vivo.

The US-5 strain of bean common mosaic virus (BCMV) and the NL-8 strain of bean common mosaic necrosis virus (BCMNV) are both seedborne potyviruses in common bean (Phaseolus vulgaris L). They have contrasting and highly stable biological characteristics which are genetically controlled. BCMV strain US-5 belongs to pathogenicity group IV. BCMNV strain NL-8 belongs to pathogenicity group III. The two strains have contrasting serological characteristics: NL-8 is serotype A; US-5 is serotype B. When these two strains were maintained separately or as a mixture for more than three years (39 serial transfers) or in more than 100 plants of either of two susceptible hosts, we were unable to isolate a single virus strain that exhibited mutant-like or recombinant-like characteristics. However, within 28 days (during the 1st passage) after these 2 strains were inoculated to opposite primary leaves of bean plants that were susceptible to one virus and resistant to the other, we were able to recover 17 strains that clearly possessed recombinations of various phenotypic characteristics from each of the two "parental" viruses. Three types of phenotypic characteristics were recombined singly or in combination during a single passage in vivo: 1) Biological characteristics known to be controlled by genes for pathogenicity; 2) Serotype; and 3) Temperature-induced hypersensitive vascular necrosis. Each of the phenotypic recombinant strains contained only pathogenicity genes or serological characteristics found in one or both parents. In no case did we isolate a strain that could be described as a random mutation or one that contained pathogenicity or serological characteristics which were not found in at least one parent strain. This is the first known demonstration of phenotypic recombinations between distinct potyviruses in vivo. Implications for the evolution of new virus strains through the use of resistant cultivars and its impact on breeding programs and bean seed production are discussed.

Detection of different strains of Potato virus Y and their mixed infections using competitive fluorescent RT-PCR.

A competitive fluorescent RT-PCR assay (CF RT-PCR) was developed for the rapid and reliable detection and discrimination of the two most common strains of Potato virus Y (PVY) found in potato (necrotic and ordinary). The assay incorporates two strain specific primers labelled with fluorescent labels, used in conjunction with a universal PVY primer. The strain specific primers compete for the same annealing site which further increases specificity. Discrimination is conferred by the fluorescent labels; green PCR products for PVY(O) and red for PVY(N), whilst mixed infections are detected as orange PCR products without the need for staining agarose gels. The assay can be scaled up for the processing of 96 samples simultaneously, with the detection of PCR products directly using a fluorescent microtitre plate reader. The assay successfully discriminated between 20 isolates of PVY tested, and could be used for the direct detection of PVY in potato tubers.

Effect of pH, ions, bovine serum albumin and heterologous antisera on the stability of immunosorbed flexuous potato viruses.

Electron microscopic studies on the stability of immunosorbed (trapped) virions of potato viruses X, S and Y0 (PVX, PVS and PVY0) revealed disintegration and dislodging of PVY0 virions upon incubation with (1) antisera to PVX, PVS, or both diluted in saline, (2) 0.86% NaCl (saline) or 0.1 mol/l CaCl2 but not with 0.1 mol/l CaSO4 or 0.1 mol/l MgSO4. PVX virions, on the other hand, showed partial dislodging upon incubation with an antiserum to PVS diluted in saline, but complete disintegration and dislodging with saline. 0.1 mol/l CaCl2 caused partial dislodging while MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) had no apparent adverse effect. PVS virions were not affected by saline, CaCl2, MgCl2, CaSO4 or MgSO4 (all 0.1 mol/l) and were only partially dislodged by antisera to PVX or PVY0. Disintegration and/or dislodging of the PVX and PVY0 virions was prevented when (1) they were fixed with glutaraldehyde prior to incubation or (2) the virus extract contained bovine serum albumin (BSA) or (3) heterologous antisera were diluted in 0.1 mol/l phosphate buffer (PB) before use except the PVS antiserum which still caused disintegration and dislodging of PVY0 virions. Prior fixation of virions prevented their disruption and dislodging by saline only in the case of PVY0 but not PVX. On the other hand, BSA reverted the adverse effect of saline but not that of the PVS antiserum on PVY0 virions. The results presented here suggest (1) a disruptive effect of Cl' on PVX and PVY0 virions particularly when it was associated with Na+ and (2) an interaction between the immunosorbed virions of PVX or PVY0 and the antiserum to PVS.

Compositional analysis of tubers from insect and virus resistant potato plants.

Genetically modified potato plants that are resistant to the Colorado potato beetle, plus either the potato leaf roll virus or potato virus Y, have recently been commercialized. As part of the safety assessment for plants produced by modern biotechnology, the composition of the food/feed must be compared to that of the food/feed produced by an equivalent plant variety from a conventional source. The composition of important nutritional and antinutritional factors in tubers produced by virus- and insect-resistant potato plants were compared to tubers produced by conventional potato plants. Key nutritional, quality, and antinutritional components measured were total solids, vitamin C, dextrose, sucrose, soluble protein, and glycoalkaloids. Proximate analyses included fat, ash, calories, total protein, and crude fiber. Minor nutrients measured were vitamin B6, niacin, copper, magnesium, potassium, and amino acids. The results from these analyses confirm that tubers produced by insect- and virus-protected varieties are substantially equivalent to tubers produced by conventional potato varieties.