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Among foods protected by the European Union with the PGI (Protected Geographical Indication) mark, several ecotypes of “Fagioli di Sarconi” common beans, typical legumes of Basilicata Region, are included. This work aimed to conduct a survey of seed-borne pathogens isolated from “Ciuoto” and “Cannellino rosso”, ecotypes of “Fagioli di Sarconi” common beans, in two years and cultivation areas of the National Park of the Agri Valley, for identifying resistant and climatic changes well-adapted genotypes. Three validated methods were used for the seed-borne pathogens screening. Eighteen fungi were differently found for ecotype and year of observation by the washing test. Saprophyte contaminants pathogens isolated and detected by the blotter test were strongly reduced by 1% sodium hypochlorite treatment. Using the between paper test, specific for detecting Colletotrichum lindemuthianum, the presence of this pathogen for both ecotypes, years and cultivation areas, and also some bacteria were individuated. Therefore, area-, environment- and ecotype-dependent differences were revealed, probably also caused by a different polyphenolic content and thickness of integument of two ecotypes. This study represents a baseline information for further studies, development of forecasting models and management of seed-borne diseases associated with common beans.

The yield and fruit quality of processing tomatoes (Solanum lycopersicum L.) have increased markedly over the past decades. The aim of this work was to assess the effects of the organic (OFS) and conventional farming systems (CFS) on the main agronomic parameters involved in processing tomato yield components and fruit quality traits of heirloom and modern genotypes. Marketable yield increased from heirloom to modern genotypes, both in OFS and in CFS, showing a difference of ≈20 t per hectare in favor of CFS. Total fruit yield (TY) was not improved from heirloom to modern assessed genotypes, and a difference of ≈35 t per hectare was observed in favor of CFS. In both farming systems, the highest marketable yield of modern genotypes was due to a higher number of fruits per plant, harvest index, nitrogen agronomic efficiency (NAE), and fruit water productivity. Moreover, the main growth parameters involved in the yield differences between OFS and CFS were the number of leaves per plant, the average fruit weight, the normalized difference vegetation index (NDVI), and NAE. It is noteworthy that fruit quality improvement in terms of color and brix per hectare was paralleled by a decrease of tomato pH in both farming systems. According to our results, we conclude that to reduce the current yield gap between OFS and CFS, agronomic and breeding efforts should be undertaken to increase leaf area index, fruit number per plant, and NAE for better genotype adaptation to organic farming systems.

Currently, vegetable production systems have been changed to improve yield and quality, leading to an improvement in sustainability. In horticultural cultivation, one of the most important sectors is the seedling production in nurseries. In the past, horticultural seedlings were produced directly by farmers. Nowadays, industrialized nurseries provide seedlings characterized by a uniform growth and an early and contemporaneous development. In addition, consumer concern about the impact of food production on the environment is driving an increased demand for organic vegetables with a consequent increase of agricultural land cultivated by organic methods. Hence, there is a need to produce high-quality seedlings suitable to be cultivated in the organic farming system. Root zone management, for improving seedling quality of organic horticultural crops, remains largely unexploited, such that this review highlights some of the current research and future development priorities, providing useful information to nursery growers. In particular, we reviewed all the scientific and modern knowledge on the production of organic, healthy and vigorous seedlings including the use of: (1) compost and compost tea; (2) agroindustrial byproducts; (3) microbial and non-microbial plant biostimulants; (4) beneficial microorganisms.

This study evaluated, for the first time, the suitability of on-farm green compost (GC) from the fourth range sector for the production, in a controlled environment, of six baby leaf species (radish, lettuce, rocket, chard, sorrel and dill), characterized by short cultivation cycles. The pH and electrical conductivity of potting soil-compost mixtures were affected by the different amounts of GC added to the potting soil. Germination tests on Lactuca sativa seeds indicated no phytotoxic effects when GC was used to prepare aqueous extracts diluted at 50% and 25% (v/v). In the pot experiment, GC was used at four different dosages in the growing medium, inducing different impacts on growth parameters, depending on the species and applied dose. The greatest plant growth responses were determined by using less than 3.5 g of GC in 100 g of potting soil. Significant positive correlations between the dry weights of root and shoot (r = 0.77 **), seedling height (r = 0.51 **) and the number of true leaves number (r = 0.56 **) confirmed the close relationship between the root system biomass and the growth parameters of aboveground organs. The study highlights that on-farm green compost, used as partial growing substrate, provides a good opportunity to obtain baby leaf species with well-developed root systems.

Common bean (Phaseolus vulgaris L.) is a major grain legume consumed worldwide with high nutritional value. Seed-borne pathogens represent a serious problem for seeds, causing losses of yield and quality reduction. Basilicata Region (southern Italy) is a traditional, vocational area of bean production, in particular for "Fagioli di Sarconi" common beans, including different ecotypes protected by the European Union with the mark PGI (Protected Geographical Indication). The study aimed to determine the presence of seed-borne pathogens isolated from seeds of two ecotypes of "Fagioli di Sarconi" common beans, "Ciuoto" and "Cannellino rosso", collected from three different cultivation areas of Basilicata Region during the years 2018 and 2019, for identifying genotypes resistant to seed-borne pathogens and well adapted to climatic changes. The seeds were preliminary evaluated for their purity and germinability and treated or not with sodium hypochlorite for seed-borne pathogens screening, by using three validated seed health testing methods, according to the 2020 International Rules for seed testing: washing, blotter and between paper tests. The washing test identified 17 fungal pathogens, different for ecotype and year of observation. Furthermore, the 1% sodium hypochlorite treatment strongly reduced the contaminants maintaining the Rhizoctonia solani, Colletotrichum lindemuthianum, Fusarium oxysporum, Fusarium solani pathogens; instead, using the blotter test, Aspergillus spp., Cladosporium spp., Botrytis spp., Mucor spp., Rhizophus spp. saprophyte pathogens resulted. Finally, between paper test, specific for detecting the C. lindemuthianum, revealed the presence of this pathogen for the both ecotypes and years, in all areas. This assay individuated some bacteria, too.

A screening strategy using micropropagation glass tubes with a gradient of distances between germinating seeds and a metal-contaminated medium was used for studying alterations in root architecture and morphology of Arabidopsis thaliana treated with cadmium (Cd) and zinc (Zn) at the concentration of 10-20μM and 100-200μM, respectively. Metal concentrations in plant shoots and roots were measured by quadrupole inductively coupled plasma mass spectrometry. After 21days from germination, all plants in the tubes were scanned at high resolution and the root systems analyzed. The localization of indole-3-acetic acid (IAA) in the primary root and lateral root apices was monitored using DR5:GUS, LAX3:GUS and AUX1:GUS Arabidopsis transgenic lines. Total phenol content in leaves was measured spectrophotometrically. Shoot and root dry weight and leaf area did not change in Zn-exposed plants and significantly decreased in Cd-exposed plants, compared to control plants. Cadmium induced a reduction of root length, of mean number of roots and of total root surface. Both Cd- and Zn-exposed plants showed a reduced specific root length. This morphological behavior, together with an observed increase in root diameter in metal-exposed plants could be interpreted as compensatory growth, and the observed thicker roots could act as a barrier to protect root from the metals. In comparison with the apical localization of the IAA signal in the control plants, Zn generally reinforced the intensity of IAA signal, without affecting its localization. In Cd-exposed plants, IAA localization remained apical but weaker compared to control plants. Total phenols decreased in plants exposed to Zn and Cd. Therefore, we propose that the remodelling of the root architecture and the production of some secondary metabolites, such as IAA and phenols could be two responses of plants subjected to metal stress. This knowledge can open the way to future phytoremediation strategies of contaminated sites.

In most countries the historical-cultural heritage also refers to the foods traditionally consumed. Southern Italy maintains a strong tradition of dishes based on wild plants, considered an important dietary foundation. Despite this, the nutritional properties and nutraceutical value of some of these plants are little known and, therefore, need to be investigated. Sixteen spontaneous species, traditionally used as food plants, were collected from their natural habitat in four different areas of Campania Region, and their phenolic content and nutritional value were determined according to European model of food labelling. The species analysed resulted quite homogeneous regarding the nutritional value, as demonstrated by cluster analyses. Energy values were always low. Minerals content (especially calcium and potassium) and phenols differed among the species, the sodium-potassium ratio was almost always

In the last few years, the aromatic plant market for fresh consumption has been growing, especially packaged herbs for Mass Market Retailers. Pruning and selection of aromatic plants during packaging leads to the accumulation of a large amounts of plant residues with consequent disposal costs. In a circular economy perspective, the aim of this study was to recover residues of three aromatic plants (Ocimum basilicum L., Rosmarinus officinalis L. and Salvia officinalis L.) by extracting essential oil and aromatic water and, subsequently, reusing oil-free biomasses for composting. In fact, these by-products are currently considered very interesting and sustainable. The essential oils are natural products that have a wide range of biological activities useful for pharmaceutical, medical, veterinary and agriculture innovative purposes. Hydrolates have a much softer scent than the corresponding essential oils. The compost can be successfully applied for the restoration and maintenance of soil fertility. Results of this study showed that yields of essential oils obtained from plant residues, were sufficiently higher, especially for sage and rosemary, also if they were collected well far from plant balsamic period. Analysis of composition of essential oils confirmed the presence of characteristic compounds for each species. The aromatic waters were found to be an easily usable product due to the favourable physicochemical characteristics and, in particular, the aromatic waters of basil showed high antioxidant activity. About compost, the main physicochemical (humidity, pH, electrical conductivity) and biological (basal respiration and hydrolase activity) properties, were considered. Phytotoxicity tests indicated that composts derived from the three species can be used in agriculture. This research demonstrates that it is possible to eliminate completely plant residues and recover new products from aromatic species, supporting the effectiveness of an eco-friendly model to recover and reuse all fresh aromatic plant residues.

Hydrogen peroxide (H2O2), an important relatively stable non-radical reactive oxygen species (ROS) is produced by normal aerobic metabolism in plants. At low concentrations, H2O2 acts as a signal molecule involved in the regulation of specific biological/physiological processes (photosynthetic functions, cell cycle, growth and development, plant responses to biotic and abiotic stresses). Oxidative stress and eventual cell death in plants can be caused by excess H2O2 accumulation. Since stress factors provoke enhanced production of H2O2 in plants, severe damage to biomolecules can be possible due to elevated and non-metabolized cellular H2O2. Plants are endowed with H2O2-metabolizing enzymes such as catalases (CAT), ascorbate peroxidases (APX), some peroxiredoxins, glutathione/thioredoxin peroxidases, and glutathione sulfo-transferases. However, the most notably distinguished enzymes are CAT and APX since the former mainly occurs in peroxisomes and does not require a reductant for catalyzing a dismutation reaction. In particular, APX has a higher affinity for H2O2 and reduces it to H2O in chloroplasts, cytosol, mitochondria and peroxisomes, as well as in the apoplastic space, utilizing ascorbate as specific electron donor. Based on recent reports, this review highlights the role of H2O2 in plants experiencing water deficit and salinity and synthesizes major outcomes of studies on CAT and APX activity and genetic regulation in drought- and salt-stressed plants.

The study of the biochemical and molecular mechanisms deriving from the host-pathogen-antagonist interaction is essential to understand the dynamics of infectious processes and can be useful for the development of new strategies to control phytopathogens, particularly viruses, against which chemical treatments have no effect. In this work, we demonstrate the ability of the rhizospheric fungus Trichoderma harzianum strain T-22 (T22) to induce defense responses in tomato (Solanum lycopersicum var. cerasiforme) against Cucumber mosaic virus (CMV, family Bromoviridae, genus Cucumovirus) strain Fny. A granule formulation containing T22 was used for treating the plants before, simultaneously or after the CMV inoculation, in order to study the molecular and biochemical aspects of the interaction between T22 and tomato against the virus. Reactive oxygen species (ROS) and the genes encoding for ROS scavenging enzymes were investigated. Histochemical analysis revealed a different increase in the superoxide anion ( $$ {\text{O}}_{2}^{ \bullet - } $$ ) and hydrogen peroxide (H2O2) content in plants infected by CMV alone or in the presence of T22, confirming the involvement of ROS in plant defense responses. Gene expression analysis suggested a definite improvement in oxidative stress when plants were treated with T22 after inoculation with CMV. In conclusion, our data indicate that Trichoderma harzianum T-22 stimulates the induction of tomato defense responses against CMV, an action that implies the involvement of ROS, pointing towards its use as a treatment rather than as a preventive measure.

Arabidopsis thaliana L. is a model plant but little information is available about morphological root changes as part of a phytohormonal common response against both biotic and abiotic stressors. For this purpose, two-week-old Arabidopsis seedlings were treated with 10 mu M CdSO4 or infected with CMV. After 12 days the entire aerial parts and the root system were analyzed, and the presence of CMV or the accumulation of Cd were detected. Microscopic analysis revealed that both CMV and Cd influenced root morphology by a marked development in the length of root hairs and an intense root branching if compared to controls. Among the three treatments, Cd-treated seedlings showed a shorter root axis length and doubled their lateral root diameter, while the lateral roots of CMV-infected seedlings were the longest. The root growth patterns were accompanied by significant changes in the levels of indole-3-acetic acid, trans-zeatin riboside, dihydrozeatin riboside, as a probable consequence of the regulation of some genes involved in their biosynthesis/degradation. The opposite role on root development played by the phythormones studied is discussed in detail. The results obtained could provide insights into novel strategies for plant defense against pathogens and plant protection against pollutants.

The role of apoptosis in the persistence of hepatitis C virus (HCV) infection is controversial. Moreover, conflicting data on the modulation of this process by HCV proteins have been provided. We evaluated the susceptibility of peripheral lymphocytes from patients with chronic hepatitis C to apoptosis both spontaneous and after incubation with a chimeric Cucumber mosaic virus (CMV) carrying 180 copies of the synthetic R9 mimotope obtained from more than 200 hypervariable region-1 sequences of HCV. Resting T lymphocytes were found to be sensitized to apoptosis as a result of chronic HCV infection. The plant virus-derived vector R9-CMV displayed a strong pro-apoptotic effect associated with activation of both caspase-8 and -9, indicating the involvement of both extrinsic and intrinsic apoptotic pathways. A parallel R9-CMV-mediated activation of endoplasmic reticulum-stress was suggested by the significant induction of BiP/GRP78, GADD153 and caspase-12. These data contribute to define the complex HCV/host interaction, and open new prospects for developing a plant-derived antigen-presenting system to strengthen host defences against persistent pathogens.

Nowadays, crop production is at risk due to global warming, especially in Mediterranean areas where the increase of air temperature and/or reduction of precipitation is relevant. Climate changes that are occurring can severely prejudice plant defensive mechanisms during host-pathogen interactions by modifying growth and physiology of the host plant. In particular, viral diseases cause serious economic losses destroying crops and reducing agronomic productivity, and, in some cases such as tomato crops, they become the limiting factor production of both open field and under greenhouse cultivation systems. This is because plant viruses are obligate parasites and require living tissue for their multiplication and spread. Therefore, they are able to interfere with plant metabolism and compete for host plant resources, so determining a decrease of plant growth and productivity. Severe outbreaks of Cucumber mosaic virus (CMV) and other viruses caused disruption of tomato plants in the Mediterranean region and in Southern Italy since the 1970s. In such a scenario, it is necessary to introduce new strategies for controlling plant pathogens and parasites in order to help maintain ecosystems and to boost sustainable agriculture. The aim of this work is to give an up-to-date overview on the recent breakthroughs in the use of microorganisms on plants for improving crop yields, quality and plant tolerance against pathogens. In particular, here we report a case study regarding an innovative strategy to control a viral disease (CMV) in tomato, based on the use of rhizosphere microorganism (Trichoderma harzianum, strain T-22) as an antagonist biocontrol agent (BCA).

A plant bioreactor has enormous capability as a system that supports many biological activities, that is, production of plant bodies, virus-like particles (VLPs), and vaccines. Foreign gene expression is an efficient mechanism for getting protein vaccines against different human viral and nonviral diseases. Plants make it easy to deal with safe, inexpensive, and provide trouble-free storage. The broad spectrum of safe gene promoters is being used to avoid risk assessments. Engineered virus-based vectors have no side effect. The process can be manipulated as follows: (a) retrieve and select gene encoding, use an antigenic protein from GenBank and/or from a viral-genome sequence, (b) design and construct hybrid-virus vectors (viral vector with a gene of interest) eventually flanked by plant-specific genetic regulatory elements for constitutive expression for obtaining chimeric virus, (c) gene transformation and/or transfection, for transient expression, into a plant-host model, that is, tobacco, to get protocols processed positively, and then moving into edible host plants, (d) confirmation of protein expression by bioassay, PCR-associated tests (RT-PCR), Northern and Western blotting analysis, and serological assay (ELISA), (e) expression for adjuvant recombinant protein seeking better antigenicity, (f) extraction and purification of expressed protein for identification and dosing, (g) antigenicity capability evaluated using parental or oral delivery in animal models (mice and/or rabbit immunization), and (h) growing of construct-treated edible crops in protective green houses. Some successful cases of heterologous gene-expressed protein, as edible vaccine, are being discussed, that is, hepatitis C virus (HCV). R9 mimotope, also named hypervariable region 1 (HVR1), was derived from the HVR1 of HCV. It was used as a potential neutralizing epitope of HCV. The mimotope was expressed using cucumber mosaic virus coat protein (CP), alfalfa mosaic virus CP P3/RNA3, and tobacco mosaic virus (TMV) CP-tobacco mild green mosaic virus (TMGMV) CP as expression vectors into tobacco plants. Expressed recombinant protein has not only been confirmed as a therapeutic but also as a diagnostic tool. Herpes simplex virus 2 (HSV-2), HSV-2 gD, and HSV-2 VP16 subunits were transfected into tobacco plants, using TMV CP-TMGMV CP expression vectors.

A plant bioreactor has enormous capability as a system that supports many biological activities, that is, production of plant bodies, virus-like particles (VLPs), and vaccines. Foreign gene expression is an efficient mechanism for getting protein vaccines against different human viral and nonviral diseases. Plants make it easy to deal with safe, inexpensive, and provide trouble-free storage. The broad spectrum of safe gene promoters is being used to avoid risk assessments. Engineered virus-based vectors have no side effect. The process can be manipulated as follows: (a) retrieve and select gene encoding, use an antigenic protein from GenBank and/or from a viral-genome sequence, (b) design and construct hybrid-virus vectors (viral vector with a gene of interest) eventually flanked by plant-specific genetic regulatory elements for constitutive expression for obtaining chimeric virus, (c) gene transformation and/or transfection, for transient expression, into a plant-host model, that is, tobacco, to get protocols processed positively, and then moving into edible host plants, (d) confirmation of protein expression by bioassay, PCR-associated tests (RT-PCR), Northern and Western blotting analysis, and serological assay (ELISA), (e) expression for adjuvant recombinant protein seeking better antigenicity, (f) extraction and purification of expressed protein for identification and dosing, (g) antigenicity capability evaluated using parental or oral delivery in animal models (mice and/or rabbit immunization), and (h) growing of construct-treated edible crops in protective green houses. Some successful cases of heterologous gene-expressed protein, as edible vaccine, are being discussed, that is, hepatitis C virus (HCV). R9 mimotope, also named hypervariable region 1 (HVR1), was derived from the HVR1 of HCV. It was used as a potential neutralizing epitope of HCV. The mimotope was expressed using cucumber mosaic virus coat protein (CP), alfalfa mosaic virus CP P3/RNA3, and tobacco mosaic virus (TMV) CP-tobacco mild green mosaic virus (TMGMV) CP as expression vectors into tobacco plants. Expressed recombinant protein has not only been confirmed as a therapeutic but also as a diagnostic tool. Herpes simplex virus 2 (HSV-2), HSV-2 gD, and HSV-2 VP16 subunits were transfected into tobacco plants, using TMV CP-TMGMV CP expression vectors.

Edible vaccines must survive digestive process and preserve the specific structure of the antigenic peptide to elicit effective immune response. The stability of a protein to digestive process can be predicted by subjecting it to the in vitro assay with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Here, we describe the protocol of producing and using chimeric Cucumber mosaic virus (CMV) displaying Hepatitis C virus (HCV) derived peptide (R9) in double copy as an oral vaccine. Its stability after treatment with SGF and SIF and the preservation of the antigenic properties were verified by SDS-PAGE and immuno western blot techniques.

Edible vaccines must survive digestive process and preserve the specific structure of the antigenic peptide to elicit effective immune response. The stability of a protein to digestive process can be predicted by subjecting it to the in vitro assay with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Here, we describe the protocol of producing and using chimeric Cucumber mosaic virus (CMV) displaying Hepatitis C virus (HCV) derived peptide (R9) in double copy as an oral vaccine. Its stability after treatment with SGF and SIF and the preservation of the antigenic properties were verified by SDS-PAGE and immuno western blot techniques.

The need for new eco-friendly control methods against plant diseases and pests requires that the scientific research is focused on effective tools for a safe environment for humans and animal health. The systemic acquired resistance (SAR) represents a valid opportunity in plant natural protection. Particularly, biocontrol microorganisms (BCMs) can be used as inducers of SAR. At the same time, fungal BCMs are able to promote plant growth and development (so acting as plant growth-promoting microorganisms (PGPMs)) that in turn determine a higher tolerance against abiotic stresses, such as drought and salinity. The ability of BCMs and PGPMs of modulating plant defence mechanisms, like SAR, was demonstrated, but the details of this BCM-plant molecular crosstalk are poorly known and many defensive compounds are likely to exist but remain to be identified. For these reasons, BCMs and PGPMs can be defined as “biostimulant microorganisms”, able to foster plant growth and defence against pathogens throughout the crop life cycle, from seed germination to plant maturity. The aim of this chapter is to give an up-to-date overview on the recent breakthroughs in the use of biostimulant microorganisms on plants for improving crop vigour, yields and quality and for increasing plant tolerance against biotic and abiotic stresses.

To date, almost no information is available in roots and shoots of the model plant Arabidopsis thaliana about the hierarchic relationship between metal accumulation, phytohormone levels, and glutathione/phytochelatin content, and how this relation affects root development. For this purpose, specific concentrations of cadmium, copper and zinc, alone or in triple combination, were supplied for 12 days to in vitro growing seedlings. The accumulation of these metals was measured in roots and shoots, and a significant competition in metal uptake was observed. Microscopic analyses revealed that root morphology was affected by metal exposure, and that the levels of trans-zeatin riboside, dihydrozeatin riboside, indole-3-acetic acid and the auxin/cytokinin ratio varied accordingly. By contrast, under metal treatments, minor modifications in gibberellic acid and abscisic acid levels occurred. Real-time polymerase chain reaction analysis of some genes involved in auxin and cytokinin synthesis (e.g. AtNIT in roots and AtIPT in shoots) showed on average a metal up-regulated transcription. The production of thiol-peptides was induced by all the metals, alone or in combination, and the expression of the genes involved in thiol-peptide synthesis (AtGSH1, AtGSH2, AtPCS1 and AtPCS2) was not stimulated by the metals, suggesting a full post-transcriptional control. Results show that the Cd/Cu/Zn-induced changes in root morphology are caused by a hormonal unbalance, mainly governed by the auxin/cytokinin ratio.

A primary therapeutic goal in Alzheimer’s disease (AD) is to reduce the quantity of amyloid b protein (A b) present in the brain. To develop an effective, safe system for vaccination against Alzheimer’s disease, the plant virus Cucumber mosaic virus (CMV) was engineered genetically to express A b -derived fragments that stimulate mainly humoral immune responses. Six chimeric constructs, bearing the A b 1–15 or the A b 4–15 sequence in positions 248, 392 or 529 of the CMV coat protein (CP) gene, were created. Viral products proved to be able to replicate in their natural host. However, only chimeric A b 1–15-CMVs were detected by A b 1–42 antiserum in Western blot analysis. Experimental evidence of Immunoelectron microscopy revealed a complete decoration of A b 1–15-CMV248 and A b 1–5-CMV392 following incubation with either anti-A b 1–15 or anti-A b 1–42 polyclonal antibodies. These two chimeric CMVs appear to be endowed with features making them possible candidates for vaccination against Alzheimer’s disease.

The Cucumber mosaic virus (CMV) is an isodiametric plant virus with an extremely wide host range, present worldwide. CMV chimeric particles (R9-CMV), engineered to express a 27-aa synthetic peptide derived from Hepatitis C virus (HCV), were demonstrated to be stable under simulated gastric and intestinal conditions. Then the possibility of inducing a humoral immune response in rabbits fed with R9-CMV infected lettuce plants was demonstrated, suggesting that this system could function as a confirming tool of a bioreactor for the production of a stable edible vaccine against HCV.

The Cucumber mosaic virus (CMV) is a three-component isodiametric plant virus with an extremely wide host range, present worldwide. A pseudorecombinant form has been described, deriving from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA 1, 2 components of the CMV-D strain. The CP gene was then engineered to express one or two copies of a synthetic peptide derived from many hypervariable region 1 (HVR1) sequences of the Hepatitis C virus (HCV) envelope protein E2 (the so-called R9 mimotope). Study of the symptoms pattern displayed in tobacco by these chimeric CMV particles, together with determination of their structural characteristics, assessed by circular dichroism (CD) spectroscopy and electron microscopy, revealed a possible relationship between the biological behavior and the structural properties of virus components.

Chimeric plant viruses are emerging as promising vectors for use in innovative vaccination strategies. In this context, cucumber mosaic virus (CMV) has proven to be a suitable carrier of the hepatitis C virus (HCV)-derived R9 mimotope. In the present work, a new chimeric CMV, expressing on its surface the HCV-derived R10 mimotope, was produced but lost the insert after the first passage on tobacco. A comparative analysis between R10- and R9-CMV properties indicated that R9-CMV stability was related to structural features typical of the foreign insert. Thus, in order to combine high virus viability with strong immuno-stimulating activity, we doubled R9 copies on each of the 180 coat protein (CP) subunits of CMV. One of the chimeras produced by this approach (2R9-CMV) was shown to systemically infect the host, stably maintaining both inserts. Notably, it was strongly recognized by sera of HCV-infected patients and, as compared with R9-CMV, displayed an enhanced ability to stimulate lymphocyte IFN-gamma production. The high immunogen levels achievable in plants or fruits infected with 2R9-CMV suggest that this chimeric form of CMV may be useful in the development of oral vaccines against HCV.