A virus was isolated from potted plants of an unidentified species of Aeonium (Crassulaceae), a succulent ornamental very common in Southern Italy, showing faint chlorotic spots and rings on both leaf surfaces. The virus was successfully transmitted by sap inoculation to a limited range of hosts, and propagated in Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30 nm in diameter, have a single type of coat protein (CP) subunits 54 kDa in size, that encapsidate single-stranded positive-sense RNA species of 7,549 (RNA1) and 4,010 (RNA2) nucleotides. A third RNA molecule 3,472 nts in size entirely derived from RNA2 was also detected in infected Aeonium plants. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to Tobacco ringspot virus (TRSV). Comparison of each single domain of TRSV and the Aeonium virus polyproteins disclosed a relatively low percentage of identity throughout. Moreover, the Aeonium virus showed to be serologically distinct from TRSV. Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.

The interaction of nanoparticles with proteins has emerged as a key issue in addressing the problem of nanotoxicity. We investigated the interaction of silver nanoparticles (AgNPs), produced by laser ablation with human ubiquitin (Ub), a protein essential for degradative processes in cells. The surface plasmon resonance peak of AgNPs indicates that Ub is rapidly adsorbed on the AgNP surface yielding a protein corona; the Ub-coated AgNPs then evolve into clusters held together by an amyloid form of the protein, as revealed by binding of thioflavin T fluorescent dye. Transthyretin, an inhibitor of amyloid-type aggregation, impedes aggregate formation and disrupts preformed AgNP clusters. In the presence of sodium citrate, a common stabilizer that confers an overall negative charge to the NPs, Ub is still adsorbed on the AgNP surface, but no clustering is observed. Ub mutants bearing a single mutation at one edge ? strand (i.e. Glu16Val) or in loop (Glu18Val) behave in a radically different manner. Human ubiquitin forms amyloids on the surface of silver nanoparticles produced by laser ablation, which induce clustering of the nanoparticles and thioflavin T fluorescence. In the presence of sodium citrate as a stabilizer, ubiquitin only forms a protein corona. A single mutation (Glu16Val) at one edge ? strand of the protein can deeply influence the amyloid transition (see figure). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

We have shown that in tobacco plants Artichoke Italian latent virus (AILV) is unable to interfere with plant defence response based on RNA silencing (RNAi). As a result, plants recover from disease symptoms between 21 and 28 days post inoculation but symptomless leaves still contain infectious virus particles.

Six rhizobacteria isolated from common bean and able to protect bean plants from the common bacterial blight (CBB) causal agent, were in vitro evaluated for their potential antifungal effects toward different plant pathogenic fungi, mostly soil-borne. By dual culture assays, the above bacteria resulted producing diffusible and volatile metabolites which inhibited the growth of the majority of the pathogens under study. In particular, the latter substances highly affected the mycelium growth of Sclerotinia sclerotiorum strains, one of which was selected for further studies either on mycelium or sclerotia. Gas chromatographic analysis of the bacterial volatiles led to the identification of an array of volatile organic compounds (VOCs). Time course studies showed the modification of the VOCs profile along a period of 5 days. In order to evaluate the single detected VOC effects on fungal growth, some of the pure compounds were tested on S. sclerotiorum mycelium and their minimal inhibitory quantities were determined. Similarly, the minimal inhibitory quantities on sclerotia germination were also defined. Moreover, observations by light and transmission electron microscopes highlighted hyphae cytoplasm granulation and ultrastructural alterations at cell organelles, mostly membranes, mitochondria, and endoplasmic reticulum. The membranes appeared one of the primary targets of bacterial volatiles, as confirmed by hemolytic activity observed for the majority of pure VOCs. However, of interest is the alteration observed on mitochondria as well.

A virus was isolated from potted plants of an unidentified species of Aeonium, a succulent ornamental very common in Southern Italy, showing chlorotic spots and rings on both leaf surfaces. It was successfully transmitted by sap inoculation to a limited range of hosts, including Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30nm in diameter, have a single type of coat protein (CP) subunits 54kDa in size, that encapsidate single-stranded positive-sense RNA species of 7,549 (RNA1) and 4,010 (RNA2) nucleotides. A third RNA molecule 3,472 nts in size entirely derived from RNA2 was also found. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to, but serologically distinct from tobacco ringspot virus (TRSV). Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-Fny”2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVYSON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

We have previously developed HIV-1 Pr55gag-based virus-like particles (HIV-VLPs) as presentation and delivery model using a transient Baculovirus expression system. Here we describe the establishment and characterization of stably transfected insect cell line for the constitutive and reproducible production of HIV-VLPs. The persistence of HIV gag coding gene has been verified in clonal resistant insect cells and the protein expression has been confirmed by Western blot analysis. The resulting HIV-VLPs have been evaluated by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. Our results demonstrate that this strategy is highly efficient for constitutive expression of conformational enveloped VLPs which can be employed as presentation and delivery system for pathogen as well as tumor-associated antigens. This represents, to our knowledge, the first example of stably transfected insect cell line for the constitutive production of VLPs.

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.

Viroids are infectious agents identified only in plants so far. In contrast to viruses, the genome of viroids is composed of a tiny circular RNA (250-400 nt) not coding for proteins, but containing in its compact structure all the information needed for parasitizing the transcriptional and RNA trafficking machineries of their hosts. Viroid infections are frequently accompanied by cellular and developmental disorders that ultimately result in macroscopic symptoms. The molecular events linking the structural domains of viroid RNAs with cellular and macroscopic alterations remain largely unexplored, although significant progress has been lately achieved in one specific viroid-host combination, highlighting the ability of viroids to strongly interfere with their host RNA regulatory networks. Cytopathic effects induced by nuclear-replicating viroids, which were investigated since early studies on viroids, consist in irregular proliferations of cell membranes (paramural bodies or plasmalemmasomes), cell wall distortions, and chloroplast malformations. Different alternatives have been proposed regarding how these cytological alterations may influence the onset of macroscopic symptoms. Recently, the cytopathology and histopathology incited by a chloroplast-replicating viroid have been investigated in depth, with defects in chloroplast development having been related to specific molecular events that involve RNA silencing and impairment of chloroplast ribosomal RNA maturation. On this basis, a tentative model connecting specific cytopathologic alterations with symptoms has been put forward. Here, early and more recent studies addressing this issue will be reviewed and reassessed in the light of recent advances in the regulatory roles of small RNAs.

Deep-sequencing technology applied on double stranded RNA recovered from an apricot tree with vein clearing symptoms allowed the identification of a novel virus with a single-stranded RNA genome, for which the provisional name apricot vein clearing-associated virus (AVCaV) is proposed. Its genome comprises 7315 nt, excluding the poly(A) tail, covering four open reading frames (ORFs). The putative virus-encoded proteins, i.e., replicase (REP), movement protein (MP), coat protein (CP) and nucleic acid binding protein (NB), had an estimated molecular weight of 192.5, 32.15, 25.5 and 16.1 kDa, respectively and shared the highest identity (ca. 40%) with citrus leaf blotch virus (CLBV) and with orthologs of other known members of the family Betaflexiviridae. The phylogenetic trees constructed with the sequences of the entire replication-associated polyproteins and the putative CP showed incongruent allocations of AVCaV within the genus Citrivirus or as an outgroup species close to the genus Vitivirus, respectively. The peculiar organization of its genome (four ORFs), different from that typical of members of Citrivirus (three ORFs) and Vitivirus (five ORFs) genera, makes likely AVCaV a novel member of an unassigned genus of the family Betaflexiviridae. In RT-PCR assays, AVCaV was found to infect only one out of 39 varieties of apricot tested; thus, suggesting to be limitedly spread.

Alzheimer's disease (AD) onset and progression are associated with the dysregulation of multiple and complex physiological processes and a successful therapeutic approach should therefore address more than one target. Two new chemical entities, the easily accessible heterocyclic scaffolds 1,3-diphenylbenzo [e][1,2,4]triazin-7(1H)-one (benzotriazinone I) and 2-phenyl-6H-[1,2,4]triazino[5,6,1-jk]carbazol-6-one (triazafluoranthenone II), were explored for their multitarget-directed inhibition of beta-amyloid (Ab) fibrillization and acetyl- (AChE) and/or butyryl- (BChE) cholinesterase, three valuable targets for AD therapy. Introduction of appropriate amine substituents at positions 6 and 5 on scaffold I and II, respectively, allowed the preparation of a series of compounds that were tested as Ab1e40 aggregation and cholinesterase inhibitors. Potent inhibitors of Ab self-aggregation were discovered and among them benzotriazinone 7 exhibited an outstanding IC50 equal to 0.37 mM. Compounds bearing a basic amine linked to the heterocyclic scaffold through a linear alkyl chain of varying length also afforded good ChE inhibitors. In particular, benzotriazinone 24 and triazafluoranthenone 38 were endowed with an interesting multiple activity, the former displaying IC50 values of 1.4, 1.5 and 1.9 mM on Ab aggregation and AChE and BChE inhibition, respectively, and the latter showing IC50 values of 1.4 and an outstanding 0.025 mM in the Ab aggregation and BChE inhibition, respectively. Benzotriazinone 24 and triazafluoranthenone 29, selected owing to their suitable aqueous solubility and Ab aggregation inhibition, were submitted to a time course kinetic assay followed with thioflavin T (ThT) spectrofluorimetry, circular dichroism (CD) and transmission electron microscopy (TEM). Experimental data indicated that 24 acted at a low concentration ratio (10 mM 24 vs. 50 mM Ab), stabilizing the unstructured Ab peptide and inhibiting fibrillogenesis, and that 29 also acted as fibrillization inhibitor, but likely enhancing and stabilizing the b-sheet arrangement of Ab to yield protofibrillar species as detected by TEM

Biological screening of (hetero)aromatic compounds allowed the identification of some novel inhibitors of Ab1–40 aggregation, bearing indane and indole rings as common scaffolds. Molecular decoration of lead compounds led to inhibitors exhibiting a potency, measured by the Thioflavin T fluorimetric assay, ranging from high to low micromolar IC50. The 2-(p-isopropylphenyldiazenylmethylene)indolone derivative 6c resulted as the most potent aggregation inhibitor exhibiting an IC50 of 1.4 mM, with complete lack of fibril formation as confirmed by transmission electron microscopy. Structure–activity relationships suggested that binding to the Ab peptide may be largely guided by p-stacking and hydrogen bond interactions.

Two dsRNA molecules with an estimated lenght of 1.5 Kbp were identified and characterized from leaves of a Japanese persimmon (Diospyros kaki) tree, showing veinlets necrosis on both sides of leaf blades.DOP-PCR recognized two genomic fragments of a bipartite cryptic virus, for which the name of Persimmon cryptic virus (PeCV) is proposed. RLM-RACE leaded to the sequencing of a 1,510 bp contig identified as dsRNA-1 and a 1,491 bp complete segment identified as dsRNA-2. The two genomic fragments resulted both monocistronic and harbored conserved domains related to RNA-dependent RNA polymerase (RdRP) and capsid protein (CP) of species associated to Alphacryptovirus genus. Phylogenetic analysis of RdRP sequence showed highest amino acid identity with Black raspberry cryptic virus (BrCV, 63 %), Pepper cryptic virus 2 (PCV-2, 52 %) and -1 (PCV-1, 46 %). Whereas the CP putatively encoded by dsRNA-2 shared highest identity with Mulberry cryptic virus 1 (MCV-1, 49 %), PCV-2 (39 %) and PCV-1 (32 %). N-J phylogenetic analysis confirmed those relationships and delivered PeCV in a cluster with phyto-cryptoviruses belonging to genera Alpha- and Betacryptovirus, quite far distinguished from myco-cryptoviruses, gathered in genus Partitivirus.Virus-specific primers for RT-PCR were successfully designed inside the CP region to detect PeCV in several symptomless trees found in different orchards of Apulia (Southern Italy), thus proving that infection may be fairly common and presumably latent.Policlonal antiserum (kindly provided by Dr. M. Turina, CNR-IVV, Torino, Italy) specific to the CP of family-related Beet cryptic virus 2 (BCV-2) was profitably used for western blot detection of a 45-50 KDa band, coherent with predicted size of dsRNA-2 product. Furthermore, antibodies were useful for ISEM observation and subsequent decoration of PeCV particles, proved to be isometric, around 30 nm in diameter, with rounded shape lacking in fine structural details, not easily permeable by negative stain.

Through the application of next generation sequencing, in synergy with conventional cloning of DOPPCR fragments, two double-stranded RNA (dsRNA) molecules of about 1.5 kbp in size were isolated from leaf tissue of a Japanese persimmon (accession SSPI) from Apulia (southern Italy) showing veinlets necrosis. Highthroughput sequencing allowed whole genome sequence assembly, yielding a 1,577 and a 1,491 bp contigs identified as dsRNA-1 and dsRNA-2 of a previously undescribed virus, provisionally named as Persimmon cryptic virus (PeCV). In silico analysis showed that both dsRNA fragments were monocistronic and comprised the RNAdependent RNA polymerase (RdRp) and the capsid protein (CP) genes, respectively. Phylogenetic reconstruction revealed a close relationship of these dsRNAs with those of cryptoviruses described in woody and herbaceous hosts, recently gathered in genus Deltapartitivirus . Virus-specific primers for RT-PCR, designed in the CP cistron, detected viral RNAs also in symptomless persimmon trees sampled from the same geographical area of SSPI, thus proving that PeCV infection may be fairly common and presumably latent.

Phlomis mottle virus (PhMV), a putative member of the familyFlexiviridae, was isolated in 2008 from Phlomis fruticosa L.S4.74 Journal of Plant Pathology (2010), 92 (4, Supplement), S4.71-S4.105(family Lamiaceae), a native Mediterranean plant widespread inthe Greek Ionian islands and Epirus, on which it causes mottlingand deformation of the leaves. More recently, similar Symptoms were observed in five species of Phlomis

Long term storage of viable pollen is important for bank germplasm constitution to preserve resources that can be used in breeding programs, biotechnologies and genetic engineering. Pollen from 12 olive (Olea europaea L.) cultivars was stored for 1 year in liquid nitrogen at -196°C. The morphology of pollen grains and germination rates on fresh and long term stored pollen were observed. Results on in vitro pollen germinability, both before and after cryopreservation, showed highly significant responses among the 12 cultivars. The relationship between germinability and pollen grain size did not reveal any significant relationships in both treatments. Our findings will contribute to the improvement of olive pollen preservation and lead to find a more efficient method for its long term storage, while retaining its germinability

The partial genome sequence of a putative new closterovirus with particles ca. 200 nm long, was determined. The genome organization of FMMaV is the same as that of mrmbers of the genus Closterovirus. This taxonomic position was confirmed by comparative analyses of the RdRp, HSP70h and CPm amino acid sequenceswhich clustered FMMaV in a clade comprising members of the genus Closterovirus in phylogenetic trees constructed with these sequences.

Eggplant mottled dwarf virus (EMDV) is a member of the genus Nucleorhabdovirus in the family Rhabdoviridae (order Mononegavirales) (Tordo et al., 2005). The EMDV genome is composed of a linear, single stranded and negative-sense molecule of RNA, contained in enveloped bacilliform particles of 220-232 × 66-72 nm (Martelli et al., 2011). The virus is transmitted in nature by the leafhoppers Anacerato-gallia laevis, A. ribauti and Agallia vorobjevi (Della Giustina et al., 2000; Babaie & Izadpanah, 2003), but the mode of transmission is still unclear. Natural host range of EMDV comprises crops (eggplant, tomato, potato, pepper, tobacco), ornamentals (pittosporum, China rose, honeysuckle, pelargonium), and wild plant species (caper, Solanum nigrum and S. sodomaeum) (Roggero et al., 1995; Martelli et al., 2011). The prevalence of infections is generally very low (1-5%), with some exceptions recorded in Algeria and Jordan where moderate infections of about 20% have been reported on eggplant (Martelli & Hamadi, 1986; Al-Musa & Lockhart, 1990). More recently, EMDV was demonstrated to be the causal agent of vein yellowing disease in China rose (Hibiscus rosa-sinensis L.) plants growing in southern Italy and in imported plantlets (De Stradis et al., 2008). Vein yellowing symptoms similar to those observed in Italy were noticed for the last few years in China rose plants growing in public gardens in southern Spain (Fig. 1). During summer 2011, three locations from Málaga province (Rincón de la Victoria, Torre del Mar and Caleta de Vélez) and one from Granada province (La Herradura), were surveyed to study the prevalence, distribution and association of EMDV infections with vein yellowing disease in H. rosa-sinensis by serological and molecular methods.

In October 2013, unusual chlorotic patches were observed on the middle leaves of a few tomato plants cv. Lotty grown in a greenhouse located in the countryside of Fasano (Apulia, Southern Italy). Younger leaves and fruits were symptomless and no aggravation of symptoms was detected as time went by. Electron microscope observations of leaf dips revealed the presence of filamentous virus particles ca. 520 nm in length. Mechanical inoculations with leaf extracts from symptomatic tomatoes elicited a mosaic reaction in Nicotiana benthamiana, but not in tomato plants cv. UC82, which, however, were systemically infected, as shown by RT-PCR. A RT-PCR product of the expected size (ca. 700 bp) was obtained using the degenerate broad-spectrumpotexvirus primers Potex5/Potex1RC (van der Vlugt and Berendsen, 2002). The amplicon was custom sequenced (BMR Genomics, Italy) and the sequence deposited in GenBank under the accession No. KM923762. BLAST alignment showed that the 700 bp amplicon shared 97-99% homology with the RNA-dependent RNA polymerase (RdRp) gene of several isolates of Pepino mosaic virus (PepMV) genotype CH2, 82% homology with the LP and EU genotypes, and 79-80% with the US genotype. A RT-PCR-restriction fragment length polymorphism (RFLP) analysis of the RdRp amplicon with EcoRI and BglII restriction endonucleases confirmed that our isolate, designated PUG1, belongs to the CH2 genotype (Hanssen, 2010). Our observations and assays are consistent with the presence of PepMV in the tomato plants tested, and the fact that PUG1 is a mild isolate of the virus. PepMV has previously been recorded from tomato in three Italian regions, i.e. Sardinia, Sicily and Campania, but this the first report from Apulia.

In this study, stable high-five insect cell line constitutively expressing rotavirus (RV) VP2 was co-transfected with VP6 and VP7-recombinant plasmids. The presence of RV proteins in stably transfected high-five cells was verified by molecular and protein analyses. To yield self-assembled triple-layered RV-like particles (tlRLPs), a stable insect high-five cell line was generated to produce RV VP6 and VP7 besides VP2. Self-assembled tlRLPs were observed by transmission electron microscopy (TEM), and enzyme-linked immunosorbent assay (ELISA) was used to assess their antigenicity in vivo. The results suggest that the stable transfected high-five cells are able to generate tlRLPs with the efficient antigenicity. J. Med. Virol. 87: 102-111, 2015. (c) 2014 Wiley Periodicals, Inc.

Peptide based vaccines may suffer from limited stability and inefficient delivery to professional antigenpresenting cells (APCs), such as dendritic cells (DCs). In order to overcome such limitations, several types of biodegradable nanoparticles (NPs) have been developed as carrier system for antigens. The present study describes for the first time the extensive biological characterization of cationic NPs made of poly (D,L-lactide-co-glycolide) (PLGA) and polyethylenimine (PLGA/PEI) as delivery system for protein/peptide antigens, with potential in therapeutic cancer vaccine development. Flow cytometry as well as confocal laser scanning microscopy (CLSM) showed that PLGA/PEI NPs are more readily taken up than PLGA NPs by both human CD14+ monocytes and mouse Hepa 1-6 hepatoma cell line. No signs of toxicity were observed in either cellular setting. Sequential image acquisition by TEM showed an intracellular apical localization for PLGA NPs and a perinuclear localization for PLGA/PEI NPs. Both NPs showed a clathrin-dependent as well as a caveolin-dependent internalization pathway and, once in the cells, they formed multivesicular endosomes (MVE). Finally, an ex vivo priming experiment showed that PLGA/PEI NPs are comparable to PLGA NPs in delivering a non-self antigen (i.e., ovalbumin - OVA) to immature dendritic cells (imDCs), which matured and induced autologous naïve CD4+ T cells to differentiate to memory (i.e., central memory and effector memory) cells. Such a differentiation was associated with a Th1 phenotype suggesting a downstream activation and amplification of a CD8+ T cell cytotoxic response. The same OVA antigen in a soluble form was unable to induce maturation of DCs, indicating that both NP formulations provided an intrinsic adjuvanting effect combined to efficient antigen delivery. Our study represents the first report on side-by-side comparison of PLGA and PLGA/PEI NPs as strategy for protein antigen delivery. PLGA/PEI NPs are superior for cellular uptake and antigen delivery as compared to PLGA NPs. Such an evidence suggests their great potential value for vaccine development, including therapeutic cancer vaccines.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

Agroinfiltration of Nicotiana benthamiana leaves with a construct expressing the coat protein of Cymbidium ringspot virus (CyRSV) resulted in the production of virus-like particles (VLPs) which showed a preferential localization within mitochondria with an apparently intact bounding membrane. VLPs were either non penetrated or penetrated by the negative stain. The former had the same outward aspect and size of wild type CymRSV and were assumed to encapsidate the CP messanger RNA, which was recovered from virus preparations purified from agroinfiltrated tissues. In addition, VLPs were shown to be able to encapsidate tombusviral satellite RNAs. Immunoblot analysis of dissociated VLPs showed that they were made up of a protein indistinguishable from the native CymRSV CP. A mitochondrial targeting signal was identified in the N-terminal region of CymRSV CP at amino acid (aa) position 10-27. The fusion protein CPMyc, containing the 10-aa Myc epitope fused to the CymRSV CP C-terminus, formed VLPs that were decorated by a Myc-specific antiserum.

Biofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels.

Early diagnosis of plant virus infections before the disease symptoms appearance may represent a significant benefit in limiting disease spread by a prompt application of appropriate containment steps. We propose a label-free procedure applied on a device structure where the electrical signal transduction is evaluated via impedance spectroscopy techniques. The device consists of a droplet suspension embedding two representative purified plant viruses i.e.,Tomato mosaic virus and Turnip yellow mosaic virus,put in contact with a highly hydrophobic plasma textured silicon surface. Results show a high sensitivity of the system towards the virus particles with an interestingly low detection limit,from tens to hundreds of attomolar corresponding to pg/mL of sap,which refers,in the infection time-scale,to a concentration of virus particles in still-symptomless plants. Such a threshold limit,together with an envisaged engineering of an easily manageable device,compared to more sophisticated apparatuses,may contribute in simplifying the in-field plant virus diagnostics.

We have previously described the establishment and characterization of a stably transfected insect cell line for the constitutive and efficient expression of Pr55 HIV Gag proteins, which auto-assemble into enveloped Virus-Like Particles (VLPs) released into the cell culture supernatant. Such HIV-Gag VLPs have been shown to elicit a specific systemic humoral response in vivo, proving the appropriate antigenic presentation of the HIV Gag protein to the immune system. Here we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55Gag- VLPs expressing on their surface trimeric forms of HIV-1 envelope glycoproteins. The persistence of HIV coding genes has been verified in clonal resistant insect cells, the protein expression and conformation has been verified by Western blot analysis. The resulting HIV-VLPs have been visualized by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. This represents, to our knowledge, the first example of stable double transfected insect cell line for the constitutive production of enveloped HIV-Gag VLPs presenting trimeric HIV-gp140 on their surface

Heterologous protein scaffolds engrafted with structurally defined HIV Env epitopes recognized by broadly neutralizing monoclonal antibodies (MAbs) represent a promising strategy to elicit broad neutralizing antibodies. In such regards, a protein scaffold based on the HIV p24 CA protein is a highly attractive approach, providing also Gag epitopes for eliciting HIV non-neutralizing protective antibodies and specific CD4+ and CD8+ T cell responses. In the present study, computational techniques were employed to verify the presence of acceptor sites for conformational HIV Env epitopes and, as proof of concept, the analysis of HIV p24 CA-based scaffolds using a complete V3 loop in a MAb-bound conformation is presented. The V3-p24 epitope-scaffold proteins show the formation of capsomers made of hexamers similarly to the p24 wild type protein. Moreover, the conformational V3 loop presented on p24 scaffold is recognized by a panel of anti-V3 MAbs. The results suggest that HIV p24 CA protein has suitable acceptor sites for engrafting foreign epitopes, without disrupting the formation of capsomer hexamer structures, and that the V3 epitope does retain its antibody-bound conformation. This strongly support the feasibility of developing a scaffolding strategy based on p24 CA proteins displaying conformational minimal structural, antigenic HIV Env epitopes.

Molecular features and genomic organization were determined forCitrus yellow vein clearing virus (CYVCV), the putative viral causalagent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consistingin a positive-sense RNA of 7,529 nucleotides and containing sixpredicted open reading frames. The CYVCV genome organization and size resembled that of flexiviruses, and search for sequence homologies revealed that Indian citrus ringspot virus (ICRSV) (Mandarivirus, Alphaflexiviridae) is the most closely related virus. However, CYVCV had an overall nucleotide sequence identity of ?74% with ICRSV. Although the two viruses were similar with regard to genome organization, viral particles, and herbaceous host range, CYVCV caused different symptoms in citrus and was serologically distinct from ICRSV. Primer pairs were designed and used to detect the virus by conventional and quantitative reverse transcription-polymerase chain reaction on yellow vein clearing symptomatic field trees as well as graft- and mechanically inoculated host plants. Collectively, these data suggest that CYVCV is the causal agent of yellow vein clearing disease and represents a new species in the genus Mandarivirus.

Electron microscope observation of leaf dips from a mulberry (Morus alba) tree from Lebanon showing leaf mottling and vein yellowing disclosed the presence of bacilliform particles ca. 150x30 nm in size, resembling those of members of the genus Badnavirus. BLAST analysis of the sequence of a DOP-PCR clone (810 bp) generated from DNA extracts from partially purified particle preparation disclosed a 67% (nucleotide) and 63% (amino acid) identity with the reverse transcriptase/RNase H gene (ORF3) of the badnavirus Cacao swollen shoot virus (CSSV). In a phylogenetic tree constructed with the amino acid sequence obtained, the putative mulberry badnavirus clustered close to CSSV and Citrus mosaic virus (CiMV) in a branch comprising also Fig badnavirus 1 (FBV-1) and Dioscorea bacilliform virus (DBALV). PCR analyses showed that the virus occurs with high infection rates in Lebanon (7 out of 13 trees examined), Turkey (2 out of 3) and Italy (29 out of 39). However, except for one, all PCR-positive trees were symptomless. Thus, since there is no clear-cut association of the virus in question with a specific disease, we propose for it the provisional name of Mulberry badnavirus 1 (MBV-1).

Discovery of Xylella fastidiosa from olive trees with "Olive quick decline syndrome" in October 2013 on the west coast of the Salento Peninsula prompted an immediate search for insect vectors of the bacterium. The dominant xylem-fluid feeding hemipteran collected in olive orchards during a 3-mo survey was the meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Aphrophoridae). Adult P. spumarius, collected in November 2013 from ground vegetation in X. fastidiosa-infected olive orchards, were 67% (40 out of 60) positive for X. fastidiosa by polymerase chain reaction (PCR) assays. Euscelis lineolatus Brullé were also collected but tested negative for the pathogen. Transmission tests with P. spumarius collected from the Salento area were, therefore, conducted. After a 96-h inoculation access period with 8 to 10 insects per plant and a 30-d incubation period, PCR results showed P. spumarius transmitted X. fastidiosa to two of five periwinkle plants but not to the seven olive plants. Sequences of PCR products from infected periwinkle were identical with those from X. fastidiosa-infected field trees. These data showed P. spumarius as a vector of X. fastidiosa strain infecting olives trees in the Salento Peninsula, Italy. © 2014 Entomological Society of America.

Enation disease of the grapevine is an erratic disorder, whose symptoms recall a teratological condition possibly deriving from hormonal unbalance. Even though graft transmissibility of enations supports a viral aetiology of the disease, its putative agent has not yet been identified.

In Alzheimer's disease (AD), native A beta protein monomers aggregate through the formation of a variety of water-soluble, toxic oligomers, ultimately leading to insoluble fibrillar deposits. The inhibition of oligomers formation and/or their dissociation into non-toxic monomers, are considered an attractive strategy for the prevention and treatment of AD. A number of studies have demonstrated that small molecules, containing single or multiple (hetero)aromatic rings, can inhibit protein aggregation, being potentially effective in AD treatment.Starting from previously reported data on the antiamyloidogenic activity of a series of 3-hydrazonoindolinones, compound PT2 was selected to deeply investigate the inhibitory mechanism in the A beta aggregation cascade. We compared data from DLS, NMR, CD, TEM and ThT fluorescence measures to ascertain the interactions with amyloidogenic species formed in vitro during the aggregation process, and confirmed this feature with cell viability tests on HeLa cultured cells. PT2 was effective in disrupting toxic oligomers and mature amyloid fibrils, stabilizing A beta as non-toxic, beta-sheet arranged, ThT-insensitive protofilaments. It also strongly reduced cellular toxicity caused by A beta and showed good antioxidant properties in two radical scavenging tests. Taken together, these data confirmed that PT2 is a small molecule inhibitor of A beta oligomerization and toxicity, displaying also additional activity as antioxidant.

Citrus ringspot is a graft-transmissible disease and at least two taxonomically distinct viral species are associated with this syndrome: Citrus psorosis virus (CPsV) and Indian citrus ringspot virus (ICRSV). Neither of these two viruses was detected, however, by serological or molecular assays, in symptomatic tissues from citrus trees in southern Iran, where the ringspot syndrome is widespread. By contrast, electron microscopy and molecular assays revealed the presence of a rhabdovirus-like virus, which was graft-transmitted to several citrus species and mechanically to herbaceous hosts. Virus particles were bacilliform and resembled rhabdovirus nucleocapsids deprived of the lipoprotein envelope. Partial sequences of the viral nucleoprotein and RNA polymerase genes showed a distant genetic relatedness with cytorhabdoviruses. This virus appears to be a novel species for which the name Iranian citrus ringspot associated virus (IrCRSaV) is suggested.

The isolation in pure culture of the Xylella fastidiosa strain associated with the quick decline syndrome of olive, recently observed in Apulia (Salento peninsula, southern Italy) was attempted from symptomatic, naturally infected olive and oleander plants, and a periwinkle seedling that had been exposed to, and was infected by Xylella-positive spittlebugs. Prior to isolation, the presence of Xylella was ascertained in all donor hosts by PCR, indirect immunofluorescence and electron microscopy. Isolations from olive failed because of the heavy contamination by bacteria other than Xylella. By contrast, pure bacterial cultures were obtained from oleander and periwinkle extracts plated in periwinkle wilt gelrite (PWG) and buffered cysteine-yeast extract (BCYE) media. In both media, colonies were slow-growing, small-sized (less than 1 mm 25 days from plating), non pigmented, opalescent and exhibited the same morphology, except for the margin that was entire in BCYE and somewhat irregular in PWG. Bacterial cells were rod-shaped with rounded ends, had a thick and rippled cell wall, an average width of 0.35 mu m, and a maximum length of ca. 5 mu m. They gave a positive reaction in immunofluorence assays and were clearly decorated by colloidal gold in immunogold labelling tests. Sequenced PCR products amplified from periwinkle and oleander colonies shared 97-99% sequence identity with known X. fastidiosa strains from database and were 100% identical to one another and to comparable sequences obtained from infected olive trees. These sequences grouped in a distinct cluster of a branch comprising X. fastidiosa isolates belonging to the subspecies pauca.

Istra?ivanje doma?ina B-virusa vinove loze (GVB) provedeno je na 24 zeljaste biljne vrste. U fazi pogodnoj za mehani?ku inokulaciju po 4 biljke svake vrste inokulirane su sa dva razli?ita GVB izolata: PM i MA. Mjesec dana nakoninokulacije uspje?nost prijenosa provjerena je metodom ELISA. Uspje?anprijenos ostvaren je kod vrsta Nicotiana cavicola N. T. Burb. i N. occidentalis H.-M.Wheeler. Dvije biljke (N. glutinosa L. inokulirana izolatom MR i Impatienswalleriana cv. Jambalaya(TM) inokulirana izolatom PM) s upitnim ELISA-rezultatimaprovjerene su metodom RT-PCR i elektronskom mikroskopijom uz dobivene negativne rezultate. Provedenim istra?ivanjem nisu utvr?eni novi zeljasti doma?ini osim onih ve? ranije poznatih.

The use of extracellular vesicles (EVs) from body fluids as "liquid biopsies" is emerging as a promising approach for the diagnosis, prognosis and therapeutic monitoring of cancer patients. MicroRNA-155 (miR155), a non-coding transcript of the B-cell integration cluster (BIC) gene, has been reported to play a critical role in the pathogenesis of several types of hematologic malignancies (HMs) in which high miR155 levels have been found. At yet, however, the EV miR155 level and its putative clinical relevance in sera of HM patients have not been reported.

In the Summer of 2012, Erysimum linifolium L. pot plants produced at an ornamental grower in Liguriaregion (northern Italy), showed unusual virus-like disease of dark mottle and stripes on mauve-purplepetals. A virus was mechanically transmitted from symptomatic flowers to several test plant species belonging to Chenopodiaceae and Brassicaceae families. This virus was identified as an isolated ofturnip mosaic virus (TuMV) by PAS-ELISA analysis, electron microscopy negatively stained crud extracts and immuno-electron microscopy (IEM) tests. In the naturally infected E. linifolium plants,TuMV occurred alone, since any other viruses either by electron microscopy or mechanical inoculations were detected. By applying RT-PCR a fragment of 862 bp was amplified corresponding toall coat protein (CP). The comparison of CP gene showed no correlations between their genetic variation and geographical origins. The diversity in southern Europe appeared very low, most likely due to the rapid growth of TuMV in relation to trade between different Countries. The consequent exchangeof infected propagation material shows that some lineages are adapted to particular crop species, andthat recombination is a significant generator of the genetic diversity in populations of this virus. This is the first report of TuMV in E. linifolium worldwide

Multitarget therapeutic leads for Alzheimer's disease were designed on the models of compounds capable of maintaining or restoring cell protein homeostasis and of inhibiting ?-amyloid (A?) oligomerization. Thirty-seven thioxanthen-9-one, xanthen-9-one, naphto- and anthraquinone derivatives were tested for the direct inhibition of A?(1-40) aggregation and for the inhibition of electric eel acetylcholinesterase (eeAChE) and horse serum butyrylcholinesterase (hsBChE). These compounds are characterized by basic side chains, mainly quinolizidinylalkyl moieties, linked to various bi- and tri-cyclic (hetero)aromatic systems. With very few exceptions, these compounds displayed inhibitory activity on both AChE and BChE and on the spontaneous aggregation of ?-amyloid. In most cases, IC50 values were in the low micromolar and sub-micromolar range, but some compounds even reached nanomolar potency. The time course of amyloid aggregation in the presence of the most active derivative (IC50=0.84 ?m) revealed that these compounds might act as destabilizers of mature fibrils rather than mere inhibitors of fibrillization. Many compounds inhibited one or both cholinesterases and A? aggregation with similar potency, a fundamental requisite for the possible development of therapeutics exhibiting a multitarget mechanism of action. The described compounds thus represent interesting leads for the development of multitarget AD therapeutics.

Potato (Solanum tuberosum subsp. tuberosum) alterations characterized by severe necrosis of vascular rings and adjacent flesh were observed in potato cv. Vivaldi in Emilia Romagna (northen Italy). The presence of Eggplant mottled dwarf virus (EMDV) was demonstrated in diseased plants by bioassays, electron microscopy, serology and RT-PCR. Symptoms similar to those observed in cv. Vivaldi were observed in other potato cultivars and areas of central and southern Italy. This is the first report of natural occurrence of EMDV in potato in Italy.

Since 2004, a potato disease consisting of a severe necrosis of vascular rings and adjacent flesh of tubers was observed in several potato-growing areas of Italy. Electron microscopy (EM) and immuno-electron microscopy (IEM) showed the presence of rhabdovirus-like particles in extracts of potato tuber necrotic tissues and symptomatic leaves of N. glutinosa.

In the spring of 2011, a necrotic disease was observed in yellow melon plants grown in western Sicily (Italy). An infection of Watermelon mosaic virus (WMV) was detected and transmitted to herbaceous hosts, using infected tissues collected from young and mature leaves and from symptomatic fruits. Analyses based on molecular hybridization and observations with the electron microscope excluded the presence of Zucchini yellow mosaic virus, Papaya ringspot virus and of viruses with isometric particles. Although based on preliminary evidences, the results of this study suggest that a necrogenic isolate of WMV for which the name of WMV-Si is proposed, was involved in the severe disease observed on yellow melon plants.

Different Citrus species used as ornamentals and some trifoliate rootstocks were recently found infected by Citrus leaf blotchvirus (CLBV), the type species of the genus Citrivirus, family Betaflexiviridae. This virus is currently detected by RT-PCR following nucleic acid extraction. For a faster virus detection fromcrude plant extracts, the feasibility of raising a CLBV-specific antiserum to be utilized for serological testing was investigated.

Positive-strand RNA virus replication always occurs in association with rearranged host cell membranes. In infected plants, replication of tombusviruses takes place in membranous structures, known as multivesicular bodies (MVBs) which originate from vesiculation of the limiting membrane of peroxisomes or of the mitochondrial outer membrane. We investigated the mechanism of vesicle formation on mitochondria in Carnation Italian ringspot virus (CIRV) infections. The genome of CIRV consists of a 4.8 kb single-stranded RNA molecule encapsidated in icosahedral particles. The genome lacks a 5' cap structure, is not polyadenylated at the 3' end and contains five open reading frames (ORFs). ORF1- and ORF2-encoded p36 and p95 proteins are essential for viral replication. In particular, p95 contains the conserved motifs of RNA-dependent RNA polymerases and p36 the motifs for viral RNA binding and recruitment to replication sites. ORF 3 codes for the p41 coat protein, ORFs 4 and 5 encode p22, required for cell-to-cell movement of the virus in infected plants, and p19, which is a suppressor of virus induced gene silencing, respectively. The requirements for the formation of MVBs were studied in cells infected by cis replicating wild type or defective CIRV genomes, or expressing the p36 and p95 replicase proteins from a non replicatable genome but able to support in trans the replication of defective interfering RNAs. It was ascertained that MVB developed in cells transfected with CIRV defective genomes that, although expressing only p36 and p95, were able to replicate.

Heat therapy, meristem tip culture in vitro and a combination of both techniques were used for obtaining fig (Ficus carica) plants free from some viruses associated with fig mosaic, a disease with a worldwide distribution. Source plants were two field-grown adult fig accessions from a germplasm repository of the University of Bari (Italy) that showed severe and mild symptoms of mosaic, respectively, and two symptomless fig seedlings grown under screen. Adult plants were infected by Fig mosaic virus (FMV), Fig latent virus 1 (FLV-1) and Fig badnavirus 1 (FBV-1). Seedlings were infected by FLV-1 and FBV-1. Progeny of explants subjected to meristem tip culture were still infected by FMV (93.8% elimination). This virus, however, was eradicated (100% sanitation) by shoot tip culture combined with heat therapy, or in vitro heat therapy. High sanitation rates from FLV-1 (81 to 100%) were also registered with all sanitation procedures employed, such as in vitro heat therapy alone (two cycles) or combined with tissue culture. By contrast, the DNA virus FBV-1 resisted all attempts of elimination, a behaviour that confirms indirectly its hypothesized integration in the fig genome.

Mulberry is a deciduous tree belonging to the Moraceae family. Although its economical importance and spreading all over the world, due mainly to the domesticated silkworm (Bombyx mori L.) breeding, to date, only few information are available about viral and virus-like diseases affecting this plant. In 2012 a small fragment of a Badnavirus DNA genome was sequenced after a DOP-PCR assay conducted on a mulberry plant, originated from Lebanon, showing symptoms of leaf mottling and vein yellowing. The virus, whose particles were observed with electron microscopy from a partially purified preparation and in tissue thin sections, was provisionally named Mulberry badnavirus-1 (MBV-1). Since different attempts of completing the full-length genome sequence through a conventional approach failed, a small RNA library was constructed for deep sequencing and run according to Illumina protocol ssRNAs analysis allowed the design of a set of primers used in PCR for the achievement of the full length sequence. The complete genome contains all the sequence features and the characteristic functional domains of the genus Badnavirus (highest nucleotide similarity shared with FBV-1 at 54%). By contrast to the badnaviruses, with genomes encoding for 3-4 ORFs, MBV-1 resembles genome organization of Petunia vein clearing virus (PVCV), which bears a single ORF. The study of the distribution of sRNA on the MBV-1 complete genome showed a tidy prevalence of 21- and 22-nt reads, differently from other viruses in the Caulimoviridae family, featuring a nuclear replication and typically supporting an accumulation of the 24-nt sRNAs involved in methylation processes.

The recent introduction of Xylella fastidiosa in Europe and its involvement in the Olive Quick Decline Syndrome (OQDS) in Apulia (Salento, Lecce district, South Italy) led us to investigate the biology and transmission ability of the meadow spittlebug, Philaenus spumarius, which was recently demonstrated to transmit X. fastidiosa to periwinkle plants. Four xylem-sap-feeding insect species were found within and bordering olive orchards across Salento during a survey carried out from October 2013 to December 2014: P. spumarius was the most abundant species on non-olive vegetation in olive orchards as well as on olive foliage and was the only species that consistently tested positive for the presence of X. fastidiosa using real-time PCR. P. spumarius, whose nymphs develop within spittle on weeds during the spring, are likely to move from weeds beneath olive trees to olive canopy during the dry period (May to October 2014). The first X. fastidiosa infective P. spumarius were collected in May from olive canopy: all the individuals previously collected on weeds tested negative for the bacterium. Experiments demonstrated that P. spumarius transmitted X. fastidiosa from infected to uninfected olive plants. Moreover, P. spumarius acquired X. fastidiosa from several host plant species in the field, with the highest acquisition rate from olive, polygala and acacia. Scanning electron microscopy (SEM) revealed bacterial cells resembling X. fastidiosa in the foreguts of adult P. spumarius. The data presented here are essential to plan an effective IPM strategy and limit further spread of the fastidious bacterium.

In this work the effects of the pressure between 1-150 Bar on Pulsed Laser Ablation in Liquids (PLAL) during the production of Silver Nanoparticles (AgNPs) in water was investigated. The produced NPs are the results of two different well-known stages which are the plasma and the bubble evolution occurring until the generated material is released into the solution. The main aim of this work is to show which roles is played by the variation of water pressure on the laser induced plasma and the cavitation bubble dynamics during the NPs formation. Their implication on the comprehension of the as-produced NPs formation mechanisms is treated. The typical timescales of the different stages occurring in water at different pressures have been studied by Optical Emission Spectroscopy (OES), imaging and shadowgraph experiments. Finally Surface Plasmon Resonance (SPR) spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM) for characterization of the material released in solution, have been used.

Xylella fastidiosa (Xf) was identified in September 2013 in olive trees affected by the Olive quick decline syndrome (OQDS) in the Salento peninsula (southern Italy) and denoted Xf strain CoDiRO. Xf is comprised of a group of genetically diverse bacteria in the class Gammaproteobacteria that causes severe plant diseases in many crops and ornamentals. The bacterium is acquired and transmitted by xylem-sap feeding hemipterans such as sharpshooter leafhoppers (Cicadellidae, Cicadellinae), froghoppers and spittlebugs (Aphrophoridae and Cercopidae) and, possibly, cicadas (Cicadidae and Tibicinidae.)

Xylella fastidiosa (Xf) was identified in September 2013 in olive trees affected by the Olive quick decline syndrome (OQDS) in the Salento peninsula (southern Italy) and denoted Xf strain CoDiRO. Xf is comprised of a group of genetically diverse bacteria in the class Gammaproteobacteria that causes severe plant diseases in many crops and ornamentals. The bacterium is acquired and transmitted by xylem-sap feeding hemipterans such as sharpshooter leafhoppers (Cicadellidae, Cicadellinae), froghoppers and spittlebugs (Aphrophoridae and Cercopidae) and, possibly, cicadas (Cicadidae and Tibicinidae.)

A series of isatin-3-arylhydrazones were synthesized and evaluated in vitro as inhibitors of Ab1-40 aggregation using a thioflavin T fluorescence method. An exploration of the effects on Ab1-40 aggregation of a number of diverse substituents at phenylhydrazone group and 5,6- positions of the indolinone nucleus led us to single out some new anti-aggregating compounds with IC50 values in the low micromolar range. The most active compounds carry methoxy- or hydroxy- substituents in the indolinone 5,6-positions and lipophilic groups such as iPr and Cl at 40- and 30-position, respectively, of the phenylhydrazone moiety. Two derivatives are noteworthy, namely 18 (IC50 = 0.4 mM) and 42 (IC50 = 1.1 mM). The in vitro effects of the highly active, water soluble, compound 42 on the temporal evolution of Ab1-40 fibrils formation were further investigated by circular dichroism spectroscopy, transmission electron microscopy and dynamic light scattering studies, which clearly showed that this compound delayed and lowered the amyloid fibril formation

Tepovirus is a new monotypic genus of plant viruses typified by potato virus T (PVT), a virus with helically constructed filamentous particles that are 640 nm long, previously classified as unassigned species in the family Betaflexiviridae. Virions have a single-stranded positive-sense polyadenylated RNA genome that is 6.5 kb in size, and a single type of coat protein with a size of 24 kDa. The viral genome contains three slightly overlapping ORFs encoding, respectively, the replication-related proteins (ORF1), a putative movement protein of the 30 K type (ORF2) and the coat protein (ORF3). Its structure and organization (number and order of genes) resembles that of trichoviruses and of citrus leaf blotch virus (CLBV, genus Citrivirus) but has a smaller size. Besides potato, the primary host, PVT can experimentally infect herbaceous hosts by mechanical inoculation. No vector is known, and transmission is through propagating material (tubers), seeds and pollen. PVT has a number of biological, physical and molecular properties that differentiate it from betaflexiviruses with a 30K-type movement protein. It is phylogenetically distant from all these viruses, but least so from grapevine virus A (GVA), the type member of the genus Vitivirus, with which it groups in trees constructed using the sequences of all of the genes.

A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providingintriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as themost likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of theother nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptationoccurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNAsegments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid andspecific detection methods developed here on citrus sanitation and certification are also discussed.

Chloroplast genetic engineering has long been recognised as a powerful technology to produce recombinant proteins. To date, however, little attention has been given to the causes of pleiotropic effects reported, in some cases, as consequence of the expres- sion of foreign proteins in transgenic plastids. In this study, we investigated the phenotypic alterations observed in transplastomic tobacco plants accumulating the Pr55gag polyprotein of human immunodeficiency virus (HIV-1). The expression of Pr55gag at high levels in the tobacco plastome leads to a lethal phenotype of seedlings grown in soil, severe impairment of plastid development and photosynthetic activity, with chloro- plasts largely resembling undeveloped proplastids. These alterations are associated to the binding of Pr55gag to thylakoids. During particle assembly in HIV-1 infected human cells, the binding of Pr55gag to a specific lipid [phosphatidylinositol-(4-5) bisphosphate] in the plasma membrane is mediated by myristoylation at the amino-terminus and the so-called highly basic region (HBR). Surprisingly, the non-myristoylated Pr55gag expressed in tobacco plastids was likely able, through the HBR motif, to bind to non phosphorous glycerogalactolipids or other classes of lipids present in plastidial membranes. Although secondary conse- quences of disturbed chloroplast biogenesis on expres- sion of nuclear-encoded plastid proteins cannot be ruled out, results of proteomic analyses suggest that their altered accumulation could be due to retrograde control in which chloroplasts relay their status to the nucleus for fine-tuning of gene expression.

Mulberry badnavirus 1 (MBV1) has been characterized as the aetiological agent of a disease observed on a mulberry tree in Lebanon (accession L34). A small RNA next-generation sequencing library was prepared and analysed from L34 extract, and these data together with genome walking experiments have been used to obtain the full-length virus sequence. Uniquely among badnaviruses, the MBV1 sequence encodes a single ORF containing all the conserved pararetrovirus motifs. Two genome sizes (6 kb and 7 kb) were found to be encapsidated in infected plants, the shortest of which shares 98.95% sequence identity with the full L34 genome. In the less-than-full-length deleted genome, the translational frame for the replication domains was conserved, but the particle morphology, observed under electron microscopy, was somehow altered. Southern blot hybridization confirmed the coexistence of the two genomic forms in the original L34 accession, as well as the absence of cointegration in the plant genome. Both long and deleted genomes were cloned and proved to be infectious in mulberry. Differently from other similar nuclear-replicating viruses or viroids, the characterization of the MBV1-derived small RNAs showed a reduced amount of the 24-mer class size.

Fig (Ficus carica L.) is one of the most widespread fruit tree in the Mediterraneanbasin. Despite the significance of the crop in some areas,its sanitation status has deteriorated as several new viruses affecting this perennial woody host haverecently been discovered and characterized.

A virus with filamentous articles ca. 700 nm long, denoted Fig latent virus 1 (FLV-1) is widespread in Apulian(southern Italy) fig orchards, in trees showing or not mosaic symptoms and in symptomless seedlings. The virus wastransmitted by sap inoculation to a very restricted range of herbaceous hosts without inducing apparent symptoms andwas transmitted through fig seeds to a very high percentage (80 to 100 %). It was successfully purified from root tissues of infected figs. A virus-specific antiserum raised in rabbits, proved useful for its detection in fig leaf dips byimmunosorbent electron microscopy (ISEM), Western Blot, dot immuno-binding (DIBA), ELISA.The viral genomestructure resembles that of members of the genus Trichovirus in the family Flexiviridae.

The discovery of Xylella fastidiosa (Xf) in olive trees affected by the quick decline syndrome (OQDS) in the Salento peninsula of Apulia (southern Italy) had prompted a series of studies which, among other aspects, investigated the presence of the bacterium in the xylem vessels of infected olives. These observations have now been extended to hosts other than olive that showed leaf scorching and desiccations of the canopy. In these hosts (Acacia saligna, Nerium oleander, Polygala myrtifolia, Prunus amygdalus) the presence of X. fastidiosa had previously been ascertained by PCR and ELISA.