Mineral nutrition of plum trees as an important factor of protection against plum pox virus disease

The experimental results obtained from a fruit garden at Krajné pointed out a different content of macrobiogenic elements in the leaves of plum trees infected with plum pox virus (PPV) as compared to healthy ones. Mineral nutrition of diseased trees was characteristic by lower relational values of topical levels of macrobiogenic elements especially those of N/P, N/K, (Ca + Mg)/K, and (N/P)/(N/K).     

High resistance and control of biological risks in transgenic plants expressing modified plum pox virus coat protein

Transgenic plums transformed with the plum pox virus coat protein (PPV CP) gene displayed a resistance to the sharka disease (Ravelonandro et al., 1997). However, the expression of PPV CP in transgenic plants may lead to complementation of deficient characteristic of an incoming potyvirus. Indeed, an aphid-intransmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) could be transmitted when encapsidated by the engineered PPV CP (Lecoq et al., 1993). To control such a risk, new PPV CP constructs were designed and introduced into Nicotiana benthamiana genome. In the first construct, the DAG amino acid triplet involved in the potyvirus aphid-transmission was deleted. The second construct encoded a truncated PPV CP lacking its first 140 amino acids. In the last construct, the nucleotides encoding the charged amino-acids R220, Q221 and D264 localized in the core of the PPV CP were removed. A bacterial expression system was developed to show that these deletions prevent the assembly of the PPV CP subunits. For each construct, several transgenic lines were produced and first challenged with several strains of PPV. Two phenotypes of resistance were observed: recovery and immunity. Their biochemical characterization showed that the resistance was RNA-mediated and therefore can be classified as homology-dependent (Jacquet et al., 1998a). Resistant lines producing high level of wild type or modified PPV CP were then inoculated with ZYMV-NAT to perform an aphid-transmission assay. Results of these experiments demonstrated that the use of modified forms of PPV CP genes in transgenic plants provide a good way to control the biological risks associated with heteroencapsidation (Jacquet et al., 1998b).     

New results concerning the plum pox virus epidemiology and resistance of plum cultivars, hybrids and rootstocks

Our recent results on the plum pox virus (PPV) epidemiology show that PPV spreads very rapidly in plum tree plantations in the contaminated areas. A clearing of the PPV-infected trees reduces significantly the spread of the virus but does not eliminate the disease. Some plum tree cultivars, hybrids and rootstocks (Scoldus, Alina, Cristi, BN 1/8Fl, BN 2Gr. etc) showing field resistance could not be infected with PPV by natural way. However, they could be infected with PPV by artificial inoculation except for the plum tree cv. Local of Dragasani and the BN 4Kr myrobalan, which proved to be immune to PPV. PPV was not transmitted through seeds in plum tree and myrobalan in the nursery. The Hyalopterus pruni aphids were found PPV-positive by an enzyme-linked immunosorbent assay (ELISA).     

A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees

A procedure for sensitive detection of plum pox virus RNA in infected bark of trees is described. The method is based on the extraction of bark material with buffer containing proteinase K followed by partial purification of RNA using QUIAGEN anion exchange resin. The RNA is then reverse transcribed, the single stranded cDNA is amplified by the polymerase chain reaction using biotinylated deoxynucleotides as label. The amplified cDNA can subsequently be detected by spotting the reaction mixture onto a nitrocellulose membrane. After fixation and washing the incorporated label is detected enzymatically using streptavidin-alkaline phosphatase. It was shown that this non-radioactive detection system is more sensitive than ELISA and a DNA/RNA hybridization test using 32P-labelled probes. It is also possible to detect plum pox virus infection with this assay in trees in the non-vegetative period.     

Some results of 50 years of research on the resistance to plum pox virus

Over 300 references on the resistance of stone fruit species to plum pox virus (PPV) have been utilized for the summarization of relevant information in this review. Methods of testing PPV resistance, procedures of evaluation and characterization of PPV resistance as well as breeding of PPV-resistant cultivars are briefly discussed. Altogether 370 cultivars, hybrids and clones of plum, peach, apricot, nectarine and wild Prunus species are tabulated together with the authors who have reported on their immunity, resistance and tolerance.     

Immunodetection of the plum pox virus helper component in infected plants and expression of its gene in transgenic plants

Summary Tobacco plants (Nicotiana tabacum cv. Xanthi) have been transformed viaAgrobacterium tumefaciens vectors, with cDNAs corresponding to the plum pox virus (PPV) cistron 2 encoding helper component (HC-Pro) and with the first two and half cistrons of the PPV genome. Presence of the HC-Pro in PPV-infected plants and transgenic plants transformed with the gene coding for this protein was investigated using specific polyclonal antibodies produced against the PPV HC-Pro. The results suggest that two proteases are involved in the processing of the PPV N-terminal polyprotein to yield a protein of 48 k (HC-Pro). HC-Pro autolytically cleaves at its carboxyl-terminus and a proteolytic activity, probably associated with the protein (P 1) encoded by the cistron 1, is required for the cleavage in planta between the proteins derived from cistrons 1 and 2.     

Evaluation of the genetic diversity of Plum pox virus in a single plum tree

Recent research has shown that Coronavirus (CoV) replication depends on active immunophilin pathways. Here we demonstrate that the drug FK506 (Tacrolimus) inhibited strongly the growth of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E at low, non-cytotoxic concentrations in cell culture. As shown by plaque titration, qPCR, Luciferase- and green fluorescent protein (GFP) reporter gene expression, replication was diminished by several orders of magnitude. Knockdown of the cellular FK506-binding proteins FKBP1A and FKBP1B in CaCo2 cells prevented replication of HCoV-NL63, suggesting the requirement of these members of the immunophilin family for virus growth.     

Production of a monoclonal antibody specific to the El Amar strain of plum pox virus

Plum pox virus (PPV) isolates are grouped into three clusters differentiated by biological, serological, molecular and epidemiological characteristics: Marcus (M), Dideron (D) and Cherry (C). The El Amar (EA) isolate that does not fit any of the above groups is also known. Monoclonal antibodies (MAbs) that specifically recognize M, D, and C strains of PPV are already available. To complete the set of PPV strain-specific serological reagents, MAbs against the EA isolate were raised by immunizing BALB/c mice and fusing their spleen cells with NS0/1 myeloma cells. After a preliminary characterization by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), 1 of 13 selected MAbs proved to be EA strain-specific. This MAb (EA24) reacted equally well with a homologous antigen and several PPV isolates from Egyptian apricot trees, supporting the hypothesis of an additional specific PPV group. MAb EA24 did not react either with about a hundred PPV isolates belonging to the D and M groups or with PPV-SwC and PPV-SoC isolates belonging to the C group. The strain specificity of MAb EA24 was confirmed by Western blot analysis and immunoelectron microscopy. We conclude that there is now available a set of MAbs which are highly specific to the four currently known groups of PPV strains.     

The complete genome sequence of an El Amar isolate of plum pox virus (PPV) and its phylogenetic relationship to other PPV strains

Summary. The genomic sequence of an El Amar isolate of plum pox virus (PPV) from Egypt was determined by sequencing overlapping cDNA fragments. This is the first complete sequence of a member of the El Amar (EA) strain of PPV. The genome consists of 9791 nt, excluding a poly(A) tail at the 3′ terminus. The complete nt sequence of PPV EA is 79–80%, 80%, 77%, and 77% homologous with isolates of strains D/M, Rec (BOR3), C, and W, respectively. The polyprotein identity ranged from 87–91%. Phylogenetic analysis using the complete genome sequence of PPV EA confirmed its strain status. No significant recombination signals were identified using PhylPro and SimPlot scans of the PPV EA sequence, however an interesting recombination signal was identified in the P1/HC-Pro region of PPV W3174.     

A generalized stochastic model for simulation of epidemics in a heterogeneous population (model VI)

A new, more general, stochastic model of epidemics is described. This model, one in a continuing series by Elveback and co-workers, includes provision for more complex, pseudo-sociologically stratified populations. Other major new features include the use of several types of conditional probabilities and of time-varying patterns of infectiousness to enable a more detailed simulation of individual and time-related differences in reactions to the infectious agent. A specific computer implementation of one and two-agent versions of this model is illustrated. Plans for additional computer-based simulation studies are discussed, for this and related models.     

Chicken pox infection (varicella zoster virus) and acute monoarthritis: evidence against a direct viral mechanism.

A 9 year old boy developed acute monoarthritis of the left knee concurrent with the appearance of a varicella zoster virus (VZV) rash. Repeated VZV DNA hybridisation of the cells within the synovial fluid and synovial membrane failed to show any evidence of intracellular virus. Virus was isolated from synovial fluid 24 hours after the start of clinical infection but not later. These findings suggest that the mechanism of the arthritis is not due to viral replication inside the swollen joint.     

Consequences of dendritic cell (DC)-immunodeficiency virus interactions: chemically inactivated virus as a model for studying antigen presentation and virus transmission by primate DCs

We have established a model approach to study DC-virus communication, in which classically immature and mature DCs can be compared. The striking observation that macropinocytically poor mature DCs capture and then internalize whole virus particles has substantial implications for both antigen processing and presentation as well as cell to cell transmission of virus from DCs to nearby Tcells (as well as other cell types). Studies are ongoing using this system to determine what molecules and mechanisms are involved in virus binding and internalization by immature versus mature DCs. Discerning the consequences of the differential fates of virus in immature versus mature monocyte-derived DCs should provide important information on how virus captured by DCs is processed for immune activation versus virus dissemination.

While there are many features shared by monocyte-derived DCs and DCs directly isolated from blood or tissues, there are some important distinctions between these DC subsets and their activation stage. These traits have considerable influence on immune activation by DCs as well as how the individual DC subset handles an immunodeficiency virus. Thus, this approach is ultimately being applied to specific DC subsets, especially those found at the body surfaces where the first DC-virus interactions most frequently occur invivo. Utilizing this system, we hope to better comprehend the initial events of DC-virus communication to (i) facilitate the development of strategies to block these events and prevent the onset of infection and (ii) identify how to augment the generation of broad anti-viral immunity.     

Detection of chemical mutagens using Muta(R) Mouse: a transgenic mouse model

A transgenic mouse strain with a high copy number of rescuablelac Z sequences was evaluated for its effectiveness in detectinglacZ^– mutations in selected tissues. Procarbazine, cyclophosphamide,ethylnitrosourea, 7, 12-dimethylbenz [a] anthracene (DMBA),acrylamide and chlorambucil were tested following either singleor repeated dosing regimens. Bone marrow, liver, skin and testistissues were selected to assess as target sites for mutation.Bone marrow, liver and testis tissues were examined for mutationfollowing exposures to ethylnitrosourea and chlorambucil. Increasedmutant frequencies were found for both chemicals in all threetissues. Bone marrow tissue was examined for mutation followingprocarbazine, cyclophosphamide and acrylamide exposures, andskin was examined for mutation following dermal applicationof DMBA. Mutation induction was observed in all cases. The resultsbtained from this investigation demonstrate the applicabilityof this transgenic mouse as an effective model to detect andanalyze gene mutation in selected organs including germinaltissues. Studies of organotrophic chemical mutagens and carcinogensare possible with this model as are studies of the susceptibilityof germinal tissues to mutagen exposures. ²To whom correspondence should be addressed     

Chemical genetics approach to hepatitis C virus replication: cyclophilin as a target for anti-hepatitis C virus strategy

Hepatitis C virus (HCV) is a major causative agent of liver diseases such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Because the current standard therapy, interferon (IFN) or pegylated-IFN alone or in combination with ribavirin, is ineffective on approximately half of the HCV-infected patients, alternative therapeutics are greatly needed. The chemical genetics method is a useful strategy to elucidate molecular mechanisms of the viral life cycle and screen for anti-viral agents. This review focuses on the use of chemical genetics approach to virology, which could be called 'chemical virology', and introduces an example of such analysis. From a cell culture-based screening, an immunosuppressant cyclosporin A (CsA) was identified as an anti-HCV compound. Analysis using CsA as a bioprobe showed that cyclophilin (CyP) B, a cellular target of CsA, regulates the function of HCV RNA polymerase NS5B, which is essential for efficient viral genome replication. By targeting CyP, HCV genome replication was drastically suppressed. Thus, chemical genetics analysis identified CyPB as a cellular cofactor of HCV genome replication and a target for novel anti-HCV agents.     

The use of N-(aminobenzoyloxy) succinimide as a two-level heterobifunctional agent for the preparation of hapten-protein conjugates. Daunomycin as a model hapten with an amino group

Three geometric ortho-, meta-, and para-isomers of N-(aminobenzoyloxy)succinimide (ABS) were synthesized, and their usefulness as a two-level heterobifunctional cross-linking agent in the preparation of hapten-protein conjugates was evaluated. The conjugation was based on the principle that ABS reacts immediately with an amino group of a hapten, and an aminobenzoyl group incorporated into the hapten is then activated by diazotization to a functional diazobenzoyl group acting on tyrosine or histidine residues of the protein. Using the anti-tumor antibiotic daunomycin (DM) as a model hapten, the three isomers of ABS were compared for their ability to conjugate DM with bovine serum albumin (BSA); DM incorporation onto a BSA molecular was found to occur to the highest degree with m-ABS, followed by p-ABS. while o-ABS completely failed to conjugate under the same coupling conditions. Using m-ABS it was possible to introduce more than 10 molecules of DM per BSA molecule. One of the DM-BSA samples was used as the immunogen for the production of anti-DM serum in a rabbit. The antibody specificity was shown to be direct to DM but not to other anti-cancer drugs (bleomycin, mitomycin C, actinomycin D and 5-fluorouracil) by the double antibody enzyme immunoassay (DEIA) using DM-beta-galactosidase conjugate as a label. An enzyme-linked immunosorbent assay (ELISA) for anti-DM IgG was developed using a DM-human serum albumin (DM-HSA) conjugate similarly prepared with m-ABS and horseradish peroxidase-conjugated goat anti-rabbit IgG as the solid-phase antigen and the labelled second antibody, respectively. This ELISA permitted us to measure accurately as little as 50 ng of anti-DM IgG per ml using a standard anti-DM IgG which had been purified from the anti-DM serum using an affinity column of Sepharose 4B with DM-HSA as the ligand. Using this ELISA as well as a sandwich enzyme immunoassay (SEIA) for total IgG, serum levels of anti-DM IgG and total IgG levels were easily monitored in a rabbit following immunization with DM-BSA. These results indicate that the use of DBS provides a novel method for preparing hapten-protein conjugates which will be useful in biochemistry and immunochemistry.     

The vomeronasal organ of wild canids: the fox (Vulpes vulpes) as a model

The vomeronasal system (VNS) has been extensively studied within specific animal families, such as Rodentia. However, the study of the VNS in other families, such as Canidae, has long been neglected. Among canids, the vomeronasal organ (VNO) has only been studied in detail in the dog, and no studies have examined the morphofunctional or immunohistochemical characteristics of the VNS in wild canids, which is surprising, given the well-known importance of chemical senses for the dog and fox and the likelihood that the VNS plays roles in the socio-reproductive physiology and behaviours of these species. In addition, characterising the fox VNS could contribute to a better understanding of the domestication process that occurred in the dog, as the fox would represent the first wild canid to be studied in depth. Therefore, the aim of this study was to analyze the morphological and immunohistochemical characteristics of the fox VNO. Tissue dissection and microdissection techniques were employed, followed by general and specific histological staining techniques, including with immunohistochemical and lectin-histochemical labelling strategies, using antibodies against olfactory marker protein (OMP), growth-associated protein 43 (GAP-43), calbindin (CB), calretinin (CR), α-tubulin, Gαo, and Gαi2 proteins, to highlight the specific features of the VNO in the fox. This study found significant differences in the VNS between the fox and the dog, particularly concerning the expression of Gαi2 and Gαo proteins, which were associated with the expression of the type 1 vomeronasal receptors (V1R) and type 2 vomeronasal receptors (V2R), respectively, in the vomeronasal epithelium. Both are immunopositive in foxes, as opposed to the dog, which only expresses Gαi2. This finding suggests that the fox possesses a well-developed VNO and supports the hypothesis that a profound transformation in the VNS is associated with domestication in the canid family. Furthermore, the unique features identified in the fox VNO confirm the necessity of studying the VNS system in different species to better comprehend specific phylogenetic aspects of the VNS.     

The preparation of purified bluetongue virus group antigen for use as a diagnostic reagent

Summary Soluble group antigen isolated from bluetongue virus infected BHK cells has been shown to be a single protein with a molecular weight of 38,000 and to comigrate on polyacrylamide gels with protein 7 isolated from virus particles. Purification by exclusion chromatography and isoelectric focussing yields a protein that appears to be antigenicaly identical to the native group antigen found in virus harvests; it is non infectious for sheep and is thought to be largely free from host cell and other virus specified proteins. It is suggested that this antigen could be used in diagnostic procedures with safety.With 4 Figures