Plant Virus Nanoparticles for Vaccine Applications.

In the rapidly evolving field of nanotechnology, plant virus nanoparticles (pVNPs) are emerging as powerful tools in diverse applications ranging from biomedicine to materials science. The proteinaceous structure of plant viruses allows the capsid structure to be modified by genetic engineering and/or chemical conjugation with nanoscale precision. This means that pVNPs can be engineered to display peptides and proteins on their external surface, including immunodominant peptides derived from pathogens allowing pVNPs to be used for active immunization. In this context, pVNPs are safer than VNPs derived from mammalian viruses because there is no risk of infection or reversion to pathogenicity. Furthermore, pVNPs can be produced rapidly and inexpensively in natural host plants or heterologous production platforms. In this review, we discuss the use of pVNPs for the delivery of peptide antigens to the host immune in pre-clinical studies with the final aim of promoting systemic immunity against the corresponding pathogens. Furthermore, we described the versatility of plant viruses, with innate immunostimulatory properties, in providing a huge natural resource of carriers that can be used to develop the next generation of sustainable vaccines.

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.

Alarm lateral flow immunoassay for detection of the total infection caused by the five viruses.

This study presents new type of the lateral flow immunoassay (LFIA) for multi-target analysis. A test, named alarm-LFIA, has an essentially new function that consists in notice (signaling the danger) about the presence at least one target from the controlled list without identification. The design of the alarm-LFIA assumes one test zone, which contains a mixture of antibodies, and multi-specific conjugate that binds the several targets. The alarm test is based on the novel conjugate with broaden specificity due to the immobilisation of a mix of antibodies, specific to several structurally different targets, on the surface of gold nanoparticles. For proof of concept, multi-specific conjugate to five important potato viruses (potato virus X, -M, -S, -Y and potato leaf roll virus) was fabricated using five antibodies with different specificity. The alarm-LFIA was developed for rapid detection of the total infection caused by up to five viruses. Detection limits of the viruses in potato leaf extracts are from 10 to 30 ng/mL. The alarm-LFIA was successfully used for viruses' detection in potato leaves; results were confirmed by enzyme-linked immunosorbent assay. The proposed approach of alarm-LFIA shows great potential for the various cases when different targets of interest can occur simultaneously or separately in samples.

Detection of potato mop-top virus in soils and potato tubers using bait-plant bioassay, ELISA and RT-PCR.

The hilly region of Northwest of Pakistan is leading seed potato producing areas of the country. Soil and plant samples were collected from the region and tested for PMTV using both conventional and molecular techniques. The bait plants exhibited PMTV-characteristic v-shaped yellow leaf markings in Nicotiana debneyi plants grown in putative viruliferious soils from 20/26 locations. The results were confirmed by back inoculation of sap from both roots and leaves of bait plant on indicator hosts (N. debneyi, Nicotiana benthamiana). The root samples of bait plants grown in soils of 25 locations and leaves of 24 locations reproduced systemic infection on indicator hosts upon back inoculation. The virus was identified in bait plants grown in soils from 25/26 locations using double antibody sandwich-enzyme linked immunosorbent assay (DAS)-ELISA and reverse transcription and polymerase chain reaction (RT-PCR) methods. The products of the 566bp were amplified from coat protein region of PMTV RNA 3 in both root and leaf samples of baited plants. The virus was detected in 10 potato cultivars commercially grown in the region using DAS-ELISA and RT-PCR. The virus was also detected in zoospores of Spongospora subterranea derived from the peels of selected scabby tubers using triple antibody sandwich (TAS)-ELISA. The results indicate that a bait plant bioassay, infectivity assay, ELISA and RT-PCR can detect PMTV in roots and leaves of baited plants, field samples, zoospores of S. subterranea and tubers of 10 potato cultivars commercially grown in the region.

Dot immunobinding assay method with chlorophyll removal for the detection of southern rice black-streaked dwarf virus.

Southern rice black-streaked dwarf virus (SRBSDV), a new virus from Fiji, has seriously damaged rice crops in southern China and northern Vietnam in recent years. This virus is difficult to diagnose in the early stages of infection, and is very destructive at the late stage. In the present study, a dot immunobinding assay (DIBA) that has a high sensitivity for diagnosing SRBSDV was developed. Two kinds of treatment for the DIBA were evaluated to determine the most effective one for removing chlorophyll interferences via rice extraction. The first included several reagents to remove chlorophyll, namely, the alkaline reagents like magnesium oxide and alumina oxide, the adsorbent reagents like activated carbon and bentonite, as well as the extraction agent acetone. The second and third treatments, which were used to remove chlorophyll in blot membrane-nitrocellulose and polyvinylidene fluoride (PVDF), included several organic solvents containing methanol, ethanol, acetone, ethyl acetate, and diethyl ether. The results showed that activated carbon and methanol yielded the best contrasting purple color for the infected samples by decreasing the chlorophyll content.

Analysis of the resistance-breaking ability of different beet necrotic yellow vein virus isolates loaded into a single Polymyxa betae population in soil.

The genome of most Beet necrotic yellow vein virus (BNYVV) isolates is comprised of four RNAs. The ability of certain isolates to overcome Rz1-mediated resistance in sugar beet grown in the United States and Europe is associated with point mutations in the pathogenicity factor P25. When the virus is inoculated mechanically into sugar beet roots at high density, the ability depends on an alanine to valine substitution at P25 position 67. Increased aggressiveness is shown by BNYVV P type isolates, which carry an additional RNA species that encodes a second pathogenicity factor, P26. Direct comparison of aggressive isolates transmitted by the vector, Polymyxa betae, has been impossible due to varying population densities of the vector and other soilborne pathogens that interfere with BNYVV infection. Mechanical root inoculation and subsequent cultivation in soil that carried a virus-free P. betae population was used to load P. betae with three BNYVV isolates: a European A type isolate, an American A type isolate, and a P type isolate. Resistance tests demonstrated that changes in viral aggressiveness towards Rz1 cultivars were independent of the vector population. This method can be applied to the study of the synergism of BNYVV with other P. betae-transmitted viruses.

[One-step cytometric bead array for rapid detection of Potato Virus A].

OBJECTIVE: Based on immunoassay, we developed a method to detect Potato Virus A (PVA) using one-step cytometric bead array (CBA). METHODS: Using fluorescent beads as carriers, we formed the complexes of bead-capture antibody-PVA-fluoresceinisothiocyanate (FITC) labeled detection antibody by immunoreaction of double antibody sandwich on bead surface. We measured the fluorescent signals produced from beads and from FITC with a multiparameter flow cytometer. RESULTS: By optimization test, capture antibody and detection antibody (from PVA detection kit, Agdia, USA) were finally diluted at 1:50 (4 ug/mL) and 1:25 to be used in one step CBA, respectively. A total CBA reaction time of 2 h was needed. No cross reactivity with other plant virus similar to potato virus Y, lettuce mosaic virus and tomato ring spot virus were found. Detection sensitivity for positive control sample in one-step CBA was 4 folds higher than that in normal plate double antibodies sandwich enzyme-linked immunosorbent assay (DAS-ELISA). CONCLUSION: A one-step CBA method for rapid and sensitive detection of PVA is developed.

Production of recombinant Potato mop-top virus coat protein in Escherichia coli and generation of antisera recognising native virus protein.

Potato mop-top virus (PMTV, Pomovirus) is difficult to detect because it is unevenly distributed and present at low concentration in infected tissues. The production of PMTV-free seed relies on sensitive and specific detection methods of virus detection, including serological methods. The possibility of using a PMTV recombinant coat protein (CP) as an antigen for antiserum production was investigated. The region encoding the PMTV CP was inserted into pET3A, expressed in Escherichia coli, and the recombinant PMTV CP produced was used to raise antibodies in rabbits. Three antisera were produced. All recognised efficiently the recombinant CP in Western blot analysis and the most sensitive antiserum (H5003) detected native CP on Western blots and in ELISA. Thus, recombinant CP can be used as an alternative to purified virus for the production of specific antibodies against PMTV.

The Beet soilborne pomovirus in Belgium and relationship with rhizomania.

The objective of this study was to determine the extend of the Beet soilborne pomovirus (BSBV) and the Beet virus Q in sugar beet fields in Belgium. During the 2000 sugar beet growing season, more than 80 fields located in Belgium were investigated for the presence of the Beet necrotic yellow vein benyvirus (BNYVV), the BSBV and Polymyxa betae, the plasmodiophorid vector of both viruses. Fields investigated were identified either using previous recorded data or by visual identification of yellow leaves on sugar beets or root symptoms. Sampling position (longitude-latitude) was recorded using the global positioning system (G.P.S.) with the view to follow-up infested fields in the following years. Three different techniques were used to evidence the aforementioned biological agents: enzyme-linked immunosorbent assay (ELISA), a RT-PCR assay to detect the viruses and direct coloration of Polymyxa betae in plant root tissues, using lactophenol-aniline blue. ELISA allowed the detection of 43 BSBV-infested soils, largely distributed in all Belgian sugar beet growing areas. These results were largely confirmed by RT-PCR using two different primers pairs targeting respectively a 400 bp fragment of the 145K ORF located on virus RNA-1 and a 970 bp fragment of the conserved 3' end of the viral genome. Five other primer's pairs have also been evaluated for BSBV identification. The detection of BSBV-infested soils without BNYVV, as well as BNYVV-infested soils without BSBV allowed the design of a competition assay between both viruses. Among the samples, 21 were selected randomly and tested for the presence of Beet virus Q by RT-PCR. Here also, six fields were detected positive for this virus. Sequence data reveal a clonal population of BSBV isolates in Belgium though a high level of diversity is observed for the coat protein region. Compared to BSBV, BVQ isolates show less diversity at sequence level.

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.

An onion isolate of tobacco rattle virus: reactivity with an antiserum to Hypochoeris mosaic virus, a putative furovirus, and molecular analysis of its RNA 2.

A tubular virus from onion was found to react with an antiserum to Hypochoeris mosaic virus (HyMV), a putative furovirus. Sequence analysis of its genomic RNAs and further serological tests, however, indicated it to be a tobravirus rather than a furovirus. The reactivity of the HyMV antiserum with several isolates of tobacco rattle virus (TRV) suggests that HyMV itself may be a tobravirus. The deduced amino acid sequences of the putative proteins encoded on RNA 2 of the onion virus isolate (ON) suggest close evolutionary relationships to the TRV isolate TCM from tulip. However, RNA 2 of the ON isolate contains a shorter RNA 1-like sequence on its 3'-end and an additional small ORF upstream of its RNA 1-like part. The sequence of its 315 5'-terminal nucleotides is more similar to that of RNA 2 of the PLB isolate from potato than to that of TCM RNA 2.

Monoclonal antibodies against potato virus A -- immunoblot analysis.

Monoclonal antibodies (MoAbs) against potato virus A (PVA) were examined in their reactivity with PVA and its denatured capsid protein (PVA-CP) bound to the nitrocellulose membrane. Five MoAbs reacted with native PVA, three of them also with PVA-CP. One MoAb gave no reaction in dot-blot test. In polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) PVA-CP migrated as two major bands. In immunoblot analysis, two MoAbs reacted only with the slower band, one only with the faster one. We presume that those bands do not correspond to the intact CP but they do to truncated N- and C-terminal CP molecules, respectively, and that the corresponding epitopes reacting with MoAbs are localized near to both termini of CP molecules. After mild trypsinolysis of PVA particles no MoAb reacted with resulting "core" CP.

Genetic modification of plants: significant issues and hurdles to success.

Transformation and regeneration is routine for many crop plants. A genetically engineered tomato with a longer shelf life at full ripeness was introduced in the United States in 1994, and other soon-to-be-released products, both foods and fibers, incorporate genes for resistance to pests, diseases, and environmentally benign herbicides. Other possibilities are altered plant fats and oils, methionine- and lysine-enhanced grain and legume proteins, plant foods that can deliver immunizing antigens, and other ways of controlling fruit ripening. Food safety concerns include the inadvertent production of toxicants and allergens. Foreign DNA can be introduced into plants by bacterial vectors, direct uptake by protoplasts, and mechanical introduction on metal particles or other materials. Limitations include little or no control of copy number or site of integration of the introduced DNA, dependence on selectable markers for recovery of traits, and inadequate knowledge of how to control key metabolic steps to maximize desirable traits. Directed genetic change still requires conventional crop breeding to deliver benefits to farmers and consumers.

Optimum conditions for the storage of potato virus S.

The effect of storage conditions on the serological activity of two strains of potato virus S (PVS), Andean and the ordinary, was studied by ELISA. Virus purificates, infected leaves and their homogenates, stored in lyophilized, frozen and dissolved form at various temperatures were tested. Virus purificates were most stable in lyophilized form, their activity decreased after 9 months only by 20-30%. Also non-purified virus was most stable as a lyophilized leaf homogenate, its activity decreased after 12 months by 30%. When lyophilized leaves were stored, the virus activity dropped after 12 months by 45%. Both the Andean and the ordinary strain of PVS behaved similarly during storage under the conditions tested.

Antigenic structure of the coat protein of potato mop-top furovirus.

The antigenic structure of the coat protein (CP) of potato mop-top furovirus (PMTV) was studied by electron microscopy of virus particles labeled with gold-conjugated monoclonal antibodies (MAbs) and by the reactions of MAbs with overlapping octapeptides (Pepscan) representing the complete amino acid sequence of the CP. A total of seven epitopes were identified in the CP. MAb SCR 69 detected a continuous epitope, which was located at the extreme N-terminus of the CP, was exposed at the surface along the sides of PMTV particles, and was removed by treating them with trypsin. MAb SCR 68 detected a discontinuous epitope found at the concave end of PMTV particles. Five other epitopes, which were detected by Pepscan tests, were located internally in, and at intervals along, the CP amino acid sequence. A tentative model of the PMTV CP subunit was produced, based on computer-aided prediction of its secondary structure and apparent similarities with the CP of tobacco mosaic virus. In this model, four of the epitopes occur at high radius in each of the pairs of parallel and anti-parallel alpha-helices in the CP subunit. The fifth is at low radius in the putative left radial alpha-helix. The epitope detected by MAb SCR 77, although amenable to study by Pepscan, contains three reactive elements, separated by short runs of nonessential residues, in a sequence of 13 amino acids. In intact virus particles, the CPs of beet necrotic yellow vein furovirus and PMTV apparently differ in the accessibility of their N- and C-termini.

Characterization and subcellular localization of tomato ringspot nepovirus putative movement protein.

Sequence comparison of the tomato ringspot nepovirus (TomRSV) genome with related viruses suggested that the region of the RNA-2-encoded polyprotein immediately upstream of the coat protein may be involved in the cell-to-cell movement of the virus (Rott et al., 1991, J. Gen. Virol. 72, 1505-1514). To further study the role of this portion of the genome, monoclonal antibodies against the putative movement protein were raised. Western blots of plant extracts allowed the detection of a viral nonstructural protein of M(r) 45K present only in TomRSV-infected tissues. Immunogold-labeling studies revealed that in Nicotiana clevelandii the putative movement protein was found only in infected cells immediately adjacent to the necrotic tissue, and that it was associated with tubular structures containing virus-like particles present in or near the cell wall. This provides further evidence that this protein is involved in the cell-to-cell movement of the virus and that this movement might take place via the formation of tubular structures.

Purification of PVYo and its soluble antigen by physico chemical means.

Potato virus Yo was purified by centrifugation of infected and minced plant tissue in the virus extraction rotor. As the initial seeding material was heavily contaminated with tobacco mosaic virus (TMV) this virus was isolated and antibody was elicited in chickens. The chicken antibody (IgY) against TMV was used for removing this extraneous virus from the original PVYo seeding material prior to propagating PVYo in tobacco plants, CV Glutinosa.

Production and characteristics of strain common antibodies against a synthetic polypeptide corresponding to the C-terminal region of potato virus Y coat protein.

Comparing the predicted amino acid sequence between two Japanese potato virus Y (PVY) strains, necrotic strain and ordinary strain, it was found that the C-terminal regions (H2N-HTTEDVSPSMHTLLGVKNM-COOH) of the coat proteins in the two strains were completely conserved. The conserved amino acid sequence was also found in the C-terminal region coat protein of PVY-36, a strain which did not react with monoclonal antibodies specific to the necrotic and the ordinary strain respectively. Antibodies were produced against a synthetic polypeptide PVY-C19 consisting of 19 amino acids, which correspond to the C-terminal region of the coat protein, using 4 coupling combinations of polypeptide PVY-C19 to protein carriers. Carrier-free polypeptides and those coupled to ovalbumin with ECDI (ethyl-dimethylaminopropyl carbodiimide) produced high titer of antibodies and detected PVY strains from PVY-infected plants by Western blot analysis and by ELISA.

Analysis of epitopes on potato leafroll virus capsid protein.

Pepscan hexapeptides prepared to the capsid protein amino acid sequence of potato leafroll luteovirus (PLRV) were tested against both polyclonal and monoclonal antibodies. Twelve continuous epitopes were identified: 11 were detected by two different PLRV polyclonal antisera, but only 4 were detected by both antisera. The 12th epitope reacted with monoclonal antibody BG3. The location of most of the epitopes did not correlate well with antigenic areas predicted by computer algorithms. Comparison of the amino acid sequences of PLRV and southern bean mosaic virus capsid proteins allowed a preliminary assignment of epitopes 4-12 to different regions of the putative S domain of the PLRV subunit. Five out of 14 monoclonal antibodies and both of the polyclonal antisera reacted with epitope 1 at the N-terminus. ELISA data indicated that even though the N-terminus is hydrophobic, it is exposed at the surface of the particles.

Cross-reactive potential of monoclonal antibodies raised against proteolysed tobacco etch virus.

Monoclonal antibodies (mAb) capable of reacting with different potyviruses were obtained by immunizing mice with proteolysed tobacco etch virus. The mAb were not equally effective in all ELISA formats and some were specific for different conformational states of the viral coat protein. The mAb also detected antigenic differences between purified virus particles and viral antigen in infected plant sap. In an ELISA format using antigen-coated plates, 5 different potyviruses (out of 7 viruses tested) could be detected in plant sap by one mAb. Different combinations of mAb and polyclonal antiserum could also be used for detecting several potyviruses by ELISA.