Apoptotic effects of a chimeric plant virus carrying a mimotope of the hepatitis C virus hypervariable region 1: role of caspases and endoplasmic reticulum-stress.

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.

Cucumber mosaic virus as the expression system for a potential vaccine against Alzheimer's disease.

A primary therapeutic goal in Alzheimer's disease (AD) is to reduce the quantity of amyloid ß protein (Aß) 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 Aß-derived fragments that stimulate mainly humoral immune responses. Six chimeric constructs, bearing the Aß1-15 or the Aß4-15 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 Aß1-15-CMVs were detected by Aß1-42 antiserum in Western blot analysis. Experimental evidence of Immunoelectron microscopy revealed a complete decoration of Aß1-15-CMV(248) and Aß1-15-CMV(392) following incubation with either anti-Aß1-15 or anti-Aß1-42 polyclonal antibodies. These two chimeric CMVs appear to be endowed with features making them possible candidates for vaccination against Alzheimer's disease.

In vitro stability of Cucumber mosaic virus nanoparticles carrying a Hepatitis C virus-derived epitope under simulated gastrointestinal conditions and in vivo efficacy of an edible vaccine.

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.

Structural and biological properties of Cucumber mosaic virus particles carrying hepatitis C virus-derived epitopes.

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.

Cucumber mosaic virus as a presentation system for a double hepatitis C virus-derived epitope.

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.

Immunogenic properties of a chimeric plant virus expressing a hepatitis C virus (HCV)-derived epitope: new prospects for an HCV vaccine.

A vaccine against Hepatitis C virus (HCV) is urgently needed due to the unsatisfactory clinical response to current therapies. We evaluated the immunological properties of a chimeric Cucumber mosaic virus (CMV), a plant virus engineered to express on its surface a synthetic peptide derived from many HVR1 sequences of the HCV envelope protein E2 (R9 mimotope). Evidence was obtained that the chimeric R9-CMV elicits a specific humoral response in rabbits. Furthermore, in patients with chronic HCV infection, purified preparations of R9-CMV down-modulated the lymphocyte surface density of CD3 and CD8, and induced a significant release of interferon (IFN)-gamma, interleukin (IL)-12 p70 and IL-15 by lymphomonocyte cultures. Finally, an R9 mimotope-specific CD8 T-cell response, as assessed by intracellular IFN-gamma production, was achieved in the majority of the patients studied. Our results open up new prospects for the development of effective vaccines against HCV infection. Moreover, the wide edible host range of CMV makes the production of an edible vaccine conceivable.

Cucumber mosaic virus as carrier of a hepatitis C virus-derived epitope.

Cucumber mosaic virus (CMV) is a three component isodiametric plant virus which is common worldwide and has an extremely wide host range. A pseudorecombinant was made, derived from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA1,2 components of the CMV-D strain. This system developed mild mosaic and vein clearing in Xanthi tobacco three weeks after inoculation. The CP gene was then engineered in three different positions, to encode a Hepatitis C virus (HCV) epitope. The selected peptide was the so-called R9 mimotope, a synthetic surrogate derived from a consensus profile of many hypervariable region 1 (HVR1) sequences of the putative HCV envelope protein E2. Serum samples from 60 patients with chronic hepatitis C displayed a significant immunoreactivity to crude plant extracts infected with the chimeric CMV. These results suggest that further investigation should be made into a possible vaccine function for the CMV-HCV mimotope system.

The complete nucleotide sequence of Plum pox virus isolates from sweet (PPV-SwC) and sour (PPV-SoC) cherry and their taxonomic relationships within the species.

Plum pox virus (PPV) sweet (SwC) and sour (SoC) cherry isolates were the first PPV isolates to be recovered from natural infection in sweet and sour cherry plants, respectively. Their complete nucleotide sequences have been determined finding a deduced genome organisation typical for PPV species. Both genomes are 9795 nucleotides long, excluding the 3' terminal poly(A) tail, and contain an open reading frame of 9432 nt, encoding a polyprotein of 3143 amino acids. The nucleotide and predicted amino acid sequences of PPV-SwC and SoC have been pairwise compared with available sequences of different PPV strains. Although a very high similarity exists between the whole genomes and polyproteins of the two cherry isolates, high levels of divergence have been calculated with sequences of PPV-M, D and EA isolates. In particular, the most considerable divergence has been found in part of 5' non coding region, in regions encoding P1, P3 + 6K1, 6K2 and NIa-VPg proteins as well as in the N-terminal domain of the coat protein. Phylogenetic analysis have been undertaken in order to establish the taxonomic localisation of SwC and SoC isolates within PPV species, showing that they are always clustered together and separated from the rest of PPV strains, being clearly the most distant.

[Radio-guided parathyroidectomy. A prospective study in 54 patients with primary hyperparathyroidism]. / Paratiroidectomia radioguidata. Studio prospettico in 54 pazienti affetti da iperparatirodismo primitivo.

BACKGROUND: The contribution of nuclear-medical mapping using 99mTc-MIBI (MIBI) and the use of an intraoperative probe in primary hyperparathyroidism (I degrees HPT) surgery was evaluated prospectively in a series of patients undergoing parathyroidectomy. METHODS: Fifty-four patients, who were operated between May 1999 and July 2000, under-went a systematic preoperative evaluation using scintigraphy with a dual tracer 99Tc04/MIBI and image subtraction, and high-resolution neck ecotomography. Surgery was performed using a mini-invasive technique through an incision measuring 2-2.5 cm at the base of the neck in 46 patients; the other 8 patients underwent open surgery with bilateral exploration of the neck. MIBI was injected intravenously in the operating theatre following the induction of anesthesia and after 32 minutes on average, radioactivity was measured using a manual gamma probe. Radioactivity was also counted intraoperatively at the tip of the lung contralateral to the pathological gland, a parameter used as the base activity (B), in the presumed seat of the hyperfunctioning parathyroid (P), in correspondence with healthy thyroid tissue (T) and any associated thyroid nodes (N). Radioactivity was also recorded at the level of the empty parathyroid compartment after removal of the corresponding gland, and on the parathyroid removed ex vivo . RESULTS: The ratio between the three main parameters, T/B, P/B and P/T was respectively 1.6 (range=1.5 - 1.8), 2.7 (range=1.6-4.0) and 1.6 (range=1.1-2.8). In 4 cases (7.4%), the small size of the parathyroids, adjacent to thyroid nodes, meant that the parathyroid measurement of MIBI was smaller than the thyroid measurement. The histological finding was consistent with: single parathyroid adenoma in 49 cases, multiple adenomas in 3 cases, parathyroid carcinoma in 2 cases. Rapid intraoperative PTH normalised in all patients. CONCLUSIONS: The significant difference in radioactivity levels recorded in the patients, showed that the technique is useful to the surgeon as a means of intraoperative assay for hyperfunctioning parathyroids, even if it cannot obviously replace experience or the value of preoperative scientigraphic and ecotomographic imaging.

Structural studies of cucumber mosaic virus: Fourier transform infrared spectroscopic studies.

The secondary structure of cucumber mosaic virus (CMV) was investigated in solution using Fourier transform infrared (FT-IR) spectroscopy. The amide I region of intact CMV revealed a doublet at 1671 cm-1 and 1653 cm-1, respectively. In order to isolate the IR bands arising from the protein backbone of CMV, the FT-IR spectra of the RNA component, isolated by phenol-SDS treatment of purified CMV and subsequent precipitation by ethanol, was obtained separately and digitally subtracted from the intact CMV spectra. After digital subtraction, the amide I region contained two bands at 1682 cm-1 and 1644 cm-1. The former band was ascribed to beta-sheet structures, while the later band occurs in the region between alpha-helix and "unordered" structures. Resolution enhancement of the finger print amide I region was accomplished using Fourier self-deconvolution of the digitally subtracted FT-IR spectrum of CMV which further confirmed the presence of anti-parallel beta-sheet structure in the protein coat of CMV. Chou-Fasman predictions on the the coat protein also revealed the presence of beta-sheet structure in agreement with FT-IR studies.

Circular dichroism studies of CMV-D and CMV-S: two strains of cucumber mosaic cucumovirus with a different biological behaviour.

Cucumber mosaic cucumovirus is a plant virus in which a typical satellite RNA system is present, displaying a dualistic biological behaviour. In fact, it has been shown that satRNA is able either to aggravate or attenuate the viral disease symptomatology with a modulating capability going from death of the host plant to a surprising absence of symptoms. D-satRNA and S-satRNA have been considered the prototype necrogenic and non necrogenic satRNAs respectively. On the basis of circular dichroism spectroscopy, it is suggested that the different biological behaviours can be explained by taking into account the different capabilities exerted by S- and D-satRNAs in inducing structuring effects onto CMV-S and CMV-D genomic RNAs.

Production of strain specific antibodies against a synthetic polypeptide corresponding to the N-terminal region of the plum pox potyvirus coat protein.

Comparison of the predicted coat protein amino acid sequence of the 'sweet cherry' strain of plum pox potyvirus (PPV-SwC) with the corresponding regions of several other PPV strains indicated that the main differences are in the N-terminal region. Polyclonal antibodies were produced against a synthetic peptide corresponding to the 1-14 sequence of the N-terminal region of PPV-SwC coat protein. They specifically detected PPV-SwC in different immunochemical tests.

Characterization of the Sweet Cherry Isolate of Plum Pox Potyvirus.

An isolate of plum pox potyvirus from sweet cherry (PPV-SwC) in southern Italy was investigated. The isolate was mechanically or graft transmissible to different Prunus and Nicotiana spp. but not to Chenopodium spp. It was transmitted also by Aphis fabae and Myzus persicae in a nonpersistent manner. Restriction fragment length polymorphism analysis indicated and nucleotide sequencing confirmed that the isolate lacks AluI and RsaI sites in the C-terminal region of the coat protein (CP) gene. Western immunoblot results showed that the PPV-SwC CP has an electrophoretic mobility similar to that of strain PPV-D and faster than that of strain PPV-M. Double-antibody sandwich indirect enzyme-linked immunosorbent assay of the CP showed that PPV-SwC, although reacting with universal monoclonal antibodies to PPV, failed to react with antibodies specific to strains M and D. Results indicate that PPV-SwC is different from conventional strains of PPV but closely related to the sour cherry isolate of PPV from Moldova.

Cucumber mosaic virus from Ixora: infectious RNA transcripts confirm deficiency in satellite support and unusual symptomatology.

A cucumber mosaic virus isolate from an Ixora plant originating in the Philippines and first described 20 years ago (CMV-Ix) by Waterworth and Povish differs from other characterized CMV strains in its anomalous satellite support and in several biological and molecular properties. We describe the preparation of infectious transcripts from cloned complementary DNA and the characterization of progeny virus. The results confirm that CMV-Ix has a more limited host range than most CMV isolates. Virions of CMV-Ix are smaller than those of the control CMV-S, independent of the type of negative stain used. Furthermore, CMV-Ix from transcripts supports the replication of the satellite T-CARNA 5 but not of D-CARNA 5.

Cucumber mosaic virus-associated RNA 5. IX. The overtaking of viral RNA synthesis by CARNA 5 and dsCARNA 5 in tobacco.

Cucumber mosaic virus (CMV) and CMV-associated RNA 5 (CARNA 5)-related RNA synthesis was monitored under conditions of semisynchronous infection using a differential temperature inoculation technique (Dawson and Schlegel, 1973). Leaf strips sampled at specific intervals between 0 and 100 hr after temperature shift were vacuum infiltrated with [32P]phosphoric acid and actinomycin D and incubated during 4-hr periods. Total nucleic acid extracts were analyzed on polyacrylamide gels to compare the relative rates of 32P incorporation into CMV-RNA 3, CARNA 5, and dsCARNA 5. During the first 24 hr there was a rapid increase in the rate of 32P incorporation into all three RNAs. During the next 10-20 hr the rate of 32P incorporation into RNA 3 declined to minimal levels while that of CARNA 5 stayed at a plateau or declined slowly. In contrast, the rate of 32P incorporation into dsCARNA 5 increased steadily well beyond the first 48 hr after temperature shift. The distribution of radioactivity among its (+) and (-) strands was determined by isolating the dsCARNA 5 in each nucleic acid extract obtained from the 4hr-labeled tissues and determining its radioactivity after hybridization in the presence and in the absence of a large excess of unlabeled CARNA 5. It appeared that throughout the 100-hr experiment about 60% of the radioactivity of dsCARNA 5 was in its (+) strands. The rates of 32P incorporation into virus and CARNA 5-related RNAs were also compared in leaf strips from plants inoculated with the genomic CMV-RNAs alone and with a mixture of genomic RNAs and CARNA 5. Although in the infection with the genomic RNAs alone the synthesis of CARNA 5 and dsCARNA 5 was not prevented, the increase in their relative rates of synthesis seemed significantly slower and the relative rate of RNA 3 synthesis much greater than when the inoculum contained detectable CARNA 5.

[Electrophysiologic demonstration of dual atrioventricular nodal pathways and final common pathway in a case of A-V nodal paroxysmal tachycardia. Considerations on the functional complexity of the A-V node (author's transl)]. / Dimostrazione elettrofisiologica di doppia via nodale e di una via finale comune in un caso di tachicardia parossistica da rientro nel nodo A-V.

The AA. studied the A-V nodal conduction using the technique of induced PAB in a patient with A-V reentrant paroxysmal tachycardia. They observed that the conduction through the A-V node failed when coupling intervals A1-A2 were between 280 and 260 msec and, after, recovered, with consistent slackening, when A1-A2 intervals were shortened, until the atrial ERP was reached. This uncommon response indicates the functional complexity of the A-V node and, particularly, suggests the presence of a final common pathway distal to the fast and slow A-V pathways, that are the anatomic-functional basis of the reentry circuit in A-V nodal paroxismal tachycardia.