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An infectious plum pox potyvirus cDNA clone was constructed placing a copy of the full-length sequence of the virus genome between an enhanced cauliflower mosaic virus 35S promoter and a nopaline synthase termination signal. Stabilization of the clone and faster growth of bacteria, in addition to higher plasmid yield, followed a modification consisting of the insertion of an intron which interrupted the viral open reading frame at the P3 region. This intron-containing clone was infectious when inoculated into plants after undergoing in vivo transcription and splicing. Particle bombardment delivery of the cDNA greatly increased the efficiency of plant infection.

Full-length in vitro transcripts of plum pox potyvirus (PPV) genomic RNA with mutations altering the number of 5'-terminal adenosine residues were able to infect Nicotiana clevelandii plants, whereas a mutant with a substitution of adenosine in position 2 by guanosine failed to infect. The genomic 5' end was template-independently repaired during in vivo RNA synthesis producing wild-type viral progeny. Putative models of replication initiation are discussed., (Copyright 2000 Academic Press.)

Plum pox potyvirus (PPV), the causal agent of a devastating disease that affects stone fruit trees, is becoming a target of intense studies intended both to fight against viral infection and to develop practical applications based on the current knowledge of potyvirus molecular biology. This review focuses on biotechnological aspects related to PPV, such as novel diagnostic techniques that facilitate detection and typing of virus isolates, strategies to implement pathogen-derived resistance through plant transformation, the potential use of genetic elements derived from the virus, and the recent development of PPV-based expression vectors.

Aequorin is one of several photoproteins that emits visible light upon binding to calcium ions. It has been widely used as a Ca(2+)-indicator and as an alternative highly sensitive bioluminescent label in binding assays. The apoprotein of aequorin binds an imidazopyrazine compound (coelenterazine) and molecular oxygen to form a stable photoprotein complex. Upon addition of calcium, the photoprotein undergoes a conformational change leading to the oxidation of the chromophore with the release of CO(2) and blue light. To gain more information of structure-function relationships within the photoprotein that will aid in the design of mutants suitable for site-specific conjugation and immobilization, polymerase chain reaction (PCR)-based site-directed mutagenesis was employed to produce five different aequorin mutants. The five mutants included a cysteine-free mutant and four other mutants with single cysteine residues at selected positions within the protein. The aequorin mutants exhibited different bioluminescence emission characteristics with two mutants showing a decrease in relative light production in comparison to the cysteine-free mutant. Additionally, circular dichroism (CD) spectra revealed that the single amino acid substitutions made for two of the aequorin mutants did alter their secondary structures.

Plum pox potyvirus (PPV) infection of transgenic Nicotiana benthamiana plants that expressed the PPV NIb RNA replicase carrying a Gly to Val mutation at the GDD motif (NIbV lines) induced a phenotype of virus resistance and transgene silencing, which was not transmissible to the progeny after self-fertilization (H. S. Guo and J. A. García, Mol. Plant-Microbe Interact. 10:160-170, 1997). Here, we demonstrate that the induced resistance of NIbV plants is mitotically stable after plant propagation by grafting and by in vitro regeneration. Virus replication or residual virus RNA seem not to be required to maintain transgene silencing and virus resistance. Analysis by PCR (polymerase chain reaction) amplification after treatment with methylation-sensitive restriction nucleases indicates that DNA methylation is associated with establishment and maintenance of transgene silencing and virus resistance. Restoration of transgene activity and susceptibility to PPV in sexual progeny correlated with resetting of transgene DNA methylation. On the basis of these and other published results, we present a general model for post-transcriptional gene silencing in which RNA signals, generated either by a silenced nuclear gene or by virus replication, both activate a specific cytoplasmic RNA degradation pathway and induce changes (in particular, DNA methylation) in homologous nuclear genes that switch them from an active to a silenced status.

Infectious RNA transcripts were generated from a chimeric cDNA clone of the plum pox potyvirus (PPV) genome containing the bacterial beta-glucuronidase (GUS) gene inserted between the sequences coding for the P1 and HC proteins. An artificial cleavage site specific for the NIa viral proteinase was engineered between the GUS and HC sequences to produce free GUS and HC proteins. The resulting virus PPVGus/ was stably maintained during the first round of infection, although plants remained symptomless and virus accumulation was delayed with respect to wild-type infection. PPVGus/ deleted variants, missing between 645 and 1779 nt, were detected in a subsequent plant passage. PPVGus/ deletions were confined inside the GUS gene, never affecting the P1 and HC coding regions, in contrast with previous reports of deletions in other potyvirus-based vector, in which deletions frequently reached the HC gene. These results suggest that the N-terminus of the PPV HC protein may be essential for virus viability. Analysis of the deletion endpoints showed short stretches of similarity in donor and acceptor RNAs, as well as oligo A tracts conserved in most junction sites, suggesting that deletions in PPVGus/ might take place by similarity-assisted recombination events.

Mutants of tobacco vein mottling virus (TVMV) with substitutions of Lys or Arg for Asp in the DAG motif at position 5 in the coat protein (CP) failed to infect tobacco plants systemically, but replicated and produced virions in protoplasts. Occasional systemic infections occurred when Nicotiana benthamiana or transgenic tobacco plants expressing wild-type TVMV CP were inoculated with these mutants, but viral progeny contained reversions to negatively or non-charged amino acids at position 5 or substitutions of Glu for Lys at position 8. The compensatory nature of these mutations was demonstrated by recreating one of the most common alterations. Tobacco etch virus (TEV) mutants with substitutions of Lys for Asp in the two DAG motifs near the CP N terminus also failed to infect tobacco plants systemically, and in situ histochemical analysis showed limited movement. A certain net charge evidently must be maintained near the CP N terminus for systemic movement to occur.

Three Hungarian (no. 2, 4 and 9), and a Moldavian (K) plum pox virus isolates were compared with a characterized Spanish isolate (5.15) by RT-PCR, ELISA, dot-blot and Western blot analysis. Monoclonal antibodies prepared against the external, intermediate and internal sequences of the coat protein of the Spanish isolate were able to differentiate the four isolates. Hungarian isolate No. 2 proved to be serologically identical to the Spanish isolate, while No. 4 showed appreciable differences and No. 9 could be recognized only by the monoclonal antibodies representing the intermedial and internal parts of the coat protein. K isolate showed a more distant relationship to other isolates. Our experiment provided the first demonstration of the presence of D type isolates in Hungary.

Specific binding between the coat protein (CP) and the helper component (HC) of the tobacco vein mottling potyvirus (TVMV) was characterized using a protein blotting-overlay protocol. In this in vitro assay, HC interacted with either virions or CP monomers originating from the aphid-transmissible TVMV-AT but not from the non-aphid-transmissible TVMV-NAT. There was a strong correlation between the aphid transmissibility of a series of TVMV variants having mutations in the DAG motif of the CP and their ability to bind HC. Expression of TVMV CP derivatives in bacteria allowed a precise determination of the minimum domain mediating HC binding. This domain is composed of seven amino acids, including the DAG motif (DTVDAGK), located in the N-terminus of the TVMV CP at amino acid positions 2 to 8.

Two Spanish plum pox virus (PPV) isolates, 5.15 and 3.3, were used in transmission experiments involving the aphid vector Myzus persicae, with woody and herbaceous host plants. These isolates differ in the size of their coat protein (CP) and sequence analysis revealed that isolate 3.3 has a 15 amino acid deletion near the N terminus of the CP, affecting the same positions as in a previously reported non-aphid-transmissible PPV isolate from Germany. Aphid transmission experiments showed that isolate 5.15 was transmitted from infected plants whereas isolate 3.3 was not. In contrast, both isolates were readily aphid-transmitted when acquired through artificial membranes from purified virus preparations supplemented with purified helper component (HC) obtained from potato virus Y-infected plants. This indicates that non-transmissibility of isolate 3.3 may be due to a defect in the HC rather than in the CP.

Hypoacusia is a common audiologic sequela of hypoxia associated with fetal suffering. Nonetheless, the structural features of the cochlear lesions associated with the disorder have not been studied much. We analyzed histopathologically the cochleas of 21 newborns diagnosed as fetal suffering. Each cochlea was dissected from the temporal bone, decalcified in EDTA, included in methacrylate, sliced into serial 10 microns-thick sections, and stained with hematoxylin and eosin. The cochleas of eight newborns were eliminated because of intense autolysis. Of the 13 newborns whose cochlea were considered suitable for histopathologic study, seven had hemorrhage of the vestibular ramp and one had a ruptured Reissner's membrane. The other cochlea was were within the limits of normality. The histologic technique is discussed and the critical factor judged to be the post mortem period prior to sample fixation.

The detection for the first time of a plant virus in a single aphid by the high sensitivity polymerase chain reaction (PCR) technology is reported. The application of PCR for the detection of viruses in their vectors will aid the understanding of the complex virus-vector relationship and therefore allow the development of new approaches for control of spread of plant virus diseases.

We study 65 patients coming from neonatology service with a diagnostic of prematurity and we test of audiometric level with brain stem response audiometry during three years, giving importance to the clinics characteristics that have affected during the story in the hospital. We diagnose hearing loss in a 41% of the patients, and in one case (5.88%) is retrocochlear (17.6%) as with no valuable. After three years only one patient presents persistent hearing loss; while in the first exploration 17 are considered pathologics. The prematurity by itself and without others clinic factors doesn't involve an important risk in the generation of hypoacusy.

A population of 80 patients affected with foetal distress is analyzed classifying it according to a qualitative variable (Apgar test) to which we assigned three category levels: normal/low/very low; and a quantitative variable, the pH from the umbilical artery being more or less than 7.15. We also studied all the clinical variables and complications derived from the base illness and their relationship. Four subpopulations were established which were studied by means of brain stem potential audiometry over 3 years, establishing that in those patients whose pH is less than 7.15 and their Apgar test is low/very low there exists a very high incidence of hypoacusis, characterized as sensorineural in 13.3%, conductive in 13.3%, not classifiable in 60%, and retrocochlear in 13.3%. There was a tendency to improve in 26.6%. Four of the 24 patients that are in this group wear an auditory prosthesis as an indication of the important damage suffered. There is no relationship in our study between the hypoacusis and the tests of the clinical variables which confirms the foetal distress.