Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

Identification of As accumulation plant species growing on highly contaminated soils.

Soils from the alluvial flats of the Turia River, Valencia, Spain, which were highly contaminated by decades of industrial activity, were surveyed for native plant species that could be candidates useful in phytoremediation. Concentrations of heavy metals and arsenic (As) in soils reached 25,000 mg Kg(-1) Pb, 12,000 mg Kg(-1) Zn, 70 mg Kg(-1) Cd, and 13500 mg Kg(-1) As. The predominant vegetation was collected and species identified. Soil samples and the corresponding plant shoots were analyzed to determine the amount of As accumulated by the various plant species. Several were able to tolerate more than 1000 mg Kg(-1) As in the soil. Bassia scoparia (Chenopodiaceae) survive in soil with 8375 mg Kg(-1) As. Arsenic accumulation in shoots of the various plant species investigated ranged from 0.1 to 107 mg Kg(-1) dw. Bassia scoparia (Chenopodiaceae), Inula viscosa (Asteraceae), Solanum nigrum (Solanaceae), and Hirschfeldia incana (Brassicaceae) had the highest values for As accumulation.

Fuga de triamcinolona intravítrea a cámara anterior / Leak of intravitreal triamcinolone to the anterior chamber

Caso clínico: Un hombre de 78 años de edad, fáquico, es sometido a terapia fotodinámica seguida de una inyección de triamcinolona intravítrea. Durante la inyección se observó el paso de una sustancia blanca a cámara anterior. En el examen en la lámpara de hendidura se observó una opacidad blanca en el ángulo inferior de la cámara anterior pero sin signos inflamatorios ni alteración corneal. Discusión: La triamcinolona intraocular se ha usado para el edema macular y diversas enfermedades inflamatorias retinianas. Describimos el paso de esta sustancia a cámara anterior como una complicación muy poco frecuente, probablemente por un defecto de la zónula

Case report: A 78-year-old man with phakic eyes underwent photodynamic therapy followed by intravitreal injection of triamcinolone. During the injection a white solution was observed in the anterior chamber. Slit lamp examination revealed white opacities in the inferior anterior chamber angle and neither inflammatory activity nor corneal changes were noticed. Discussion: Intravitreal triamcinolone has been used for treating macular edema and various retinal vascular and inflammatory diseases. We describe the passage of triamcinolone to the anterior chamber during the injection in a phakic eye as a very unusual adverse effect, observed probably as a result of a zonular defect

[Leak of intravitreal triamcinolone to the anterior chamber]. / Fuga de triamcinolona intravítrea a cámara anterior.

CASE REPORT: A 78-year-old man with phakic eyes underwent photodynamic therapy followed by intravitreal injection of triamcinolone. During the injection a white solution was observed in the anterior chamber. Slit lamp examination revealed white opacities in the inferior anterior chamber angle and neither inflammatory activity nor corneal changes were noticed. DISCUSSION: Intravitreal triamcinolone has been used for treating macular edema and various retinal vascular and inflammatory diseases. We describe the passage of triamcinolone to the anterior chamber during the injection in a phakic eye as a very unusual adverse effect, observed probably as a result of a zonular defect.

Resultados de un programa de cirugía mayor ambulatoria en oftalmología / Results an outpatient major surgery program in ophthalmology

Objetivo: Evaluar los resultados de un programa de cirugía mayor ambulatoria en pacientes intervenidos quirúrgicamente de diversas patologías oculares en nuestro hospital. Métodos: Este estudio retrospectivo incluye 13.878 pacientes intervenidos de forma programada por el Servicio de Oftalmología desde septiembre de 1998 a diciembre de 2004. En 11.187 pacientes se realizaron diferentes procedimientos quirúrgicos de forma ambulatoria, siendo la cirugía de catarata (facoemulsificación) la operación mayoritaria (8.155 casos). Se han analizado diversos índices (sustitución, suspensión, ingresos, reingresos), así como el rendimiento quirúrgico y las complicaciones sistémicas y oculares que surgieron dentro de las primeras 72 horas tras la cirugía. La medición de las variables se realizó mediante frecuencias relativas. El análisis utilizado para la evolución de las complicaciones en el período de estudio fue la Chi cuadrado de tendencias. Resultados: 13.878 pacientes fueron intervenidos en el período indicado, de los que 11.187 se operaron de forma ambulatoria (índice de sustitución global del 80,6%). El rendimiento quirúrgico medio fue 74,36%. El índice de ingresos tras la cirugía fue 4,46% (499 pacientes), siendo ingresos inmediatos en el 92,18% de los casos (460). 21 pacientes sufrieron complicaciones graves (cardiovasculares, neurológicas, metabólicas, infecciosas), representando un riesgo proporcional de 1:532. En 45 pacientes aparecieron complicaciones de menor gravedad (hipertensión arterial, nauseas, vómitos, síncope vasovagal) que requirieron su ingreso hospitalario. Las complicaciones oftalmológicas ocurrieron en 79 casos (0,56%). Conclusiones: La cirugía mayor ambulatoria (CMA) es un excelente modelo organizativo de asistencia quirúrgica multidisciplinar que permite tratar pacientes bien seleccionados de una manera efectiva, segura y eficiente. A pesar del cumplimiento de los requisitos óptimos, existe un porcentaje pequeño de complicaciones postoperatorias de gravedad variable, aunque afortunadamente la mortalidad es prácticamente nula

Purpose: To evaluate the outcomes in our hospital of an ambulatory major surgery program in patients with a variety of different ocular pathologies. Method: This retrospective study includes 13,878 patients who underwent programmed surgery by the Department of Ophthalmology between September 1998 and December 2004. Different ophthalmological surgical procedures were performed as outpatient surgery in 11,187 patients, with cataract surgery (phacoemulsification) being the most frequent operation performed (8,155 cases). We have analysed several indicators (substitution, suspension, admission and readmission rates), as well as surgical yield and systemic and ocular complications which appeared within 72 hours after surgery. The variables were measured as relative frequencies. The evolution of complications during the study period was analysed by the Chi-square trend test. Results: 13,878 patients had ophthalmic surgery during the study period; 11,187 had outpatient surgery with a global substitution ratio of 80.6%. The median surgical yield was 74.36%. The admission rate after surgery was 4.46% (499 patients), with 92.18% (460) of these requiring immediate admission. Twenty-one patients suffered from severe complications (cardiovascular, neurological, metabolic, infectious), representing a proportional risk of 1:532. Forty-five patients had less severe complications (arterial hypertension, nausea, vomiting, vasovagal syncope) that required admission to hospital. Ophthalmologic complications occurred in 79 cases (0.56%). Conclusions: Ambulatory major surgery (AMS) is an excellent organization model of multidisciplinary surgical assistance that makes it possible to treat well selected patients in an effective, safe and efficient manner. There is a low incidence of postoperative complications of variable severity despite following the optimum requisites, although fortunately mortality is practically absent

[Results of an outpatient major surgery program in ophthalmology]. / Resultados de un programa de cirugía mayor ambulatoria en oftalmología.

PURPOSE: To evaluate the outcomes in our hospital of an ambulatory major surgery program in patients with a variety of different ocular pathologies. METHOD: This retrospective study includes 13,878 patients who underwent programmed surgery by the Department of Ophthalmology between September 1998 and December 2004. Different ophthalmological surgical procedures were performed as outpatient surgery in 11,187 patients, with cataract surgery (phacoemulsification) being the most frequent operation performed (8,155 cases). We have analysed several indicators (substitution, suspension, admission and readmission rates), as well as surgical yield and systemic and ocular complications which appeared within 72 hours after surgery. The variables were measured as relative frequencies. The evolution of complications during the study period was analysed by the Chi-square trend test. RESULTS: 13,878 patients had ophthalmic surgery during the study period; 11,187 had outpatient surgery with a global substitution ratio of 80.6%. The median surgical yield was 74.36%. The admission rate after surgery was 4.46% (499 patients), with 92.18% (460) of these requiring immediate admission. Twenty-one patients suffered from severe complications (cardiovascular, neurological, metabolic, infectious), representing a proportional risk of 1:532. Forty-five patients had less severe complications (arterial hypertension, nausea, vomiting, vasovagal syncope) that required admission to hospital. Ophthalmologic complications occurred in 79 cases (0.56%). CONCLUSIONS: Ambulatory major surgery (AMS) is an excellent organization model of multidisciplinary surgical assistance that makes it possible to treat well selected patients in an effective, safe and efficient manner. There is a low incidence of postoperative complications of variable severity despite following the optimum requisites, although fortunately mortality is practically absent.

Construction of a stable and highly infectious intron-containing cDNA clone of plum pox potyvirus and its use to infect plants by particle bombardment.

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.

Biotechnological aspects of plum pox virus.

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.

Bioluminescence and secondary structure properties of aequorin mutants produced for site-specific conjugation and immobilization.

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.

Preservation of 5'-end integrity of a potyvirus genomic RNA is not dependent on template specificity.

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.

Context of the coat protein DAG motif affects potyvirus transmissibility by aphids.

Previous work with tobacco vein mottling virus (TVMV) has established that a highly conserved three amino acid motif, asp-ala-gly (DAG), located near the N terminus of the coat protein (CP), is important for aphid transmission. However, several other potyviruses which have motifs other than DAG are aphid-transmissible. Creation of these motifs in TVMV through site-directed mutagenesis failed to render TVMV aphid-transmissible from infected plants, and the creation of a putative complementary motif in the helper component did not restore transmissibility. In an isolate of tobacco etch virus (TEV) that contains two consecutive DAG motifs separated by a single ala, transmissibility was abolished or reduced by mutations affecting the first motif, whereas mutations in the second motif had little or no effect. In a TEV mutant made non-transmissible due to an altered first motif, substitution of val for ala in the position immediately before the second DAG restored transmissibility, whereas changing val to ala in the location prior to the first DAG resulted in reduced TEV transmissibility. In contrast, a val to ala change in the position preceding the single DAG motif of TVMV did not affect transmission. Creation of another DAG motif at the beginning of the TVMV CP core, in a position where certain other potyviruses have a second DAG motif, did not restore transmissibility. Our results suggest that the mere presence of a DAG motif does not guarantee transmissibility and that the context in which the DAG or equivalent motif is found plays a role in the process.

Mitotic stability of infection-induced resistance to plum pox potyvirus associated with transgene silencing and DNA methylation.

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.

Susceptibility to recombination rearrangements of a chimeric plum pox potyvirus genome after insertion of a foreign gene.

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.

Charge changes near the N terminus of the coat protein of two potyviruses affect virus movement.

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.

Serological characterization of Hungarian plum pox virus isolates.

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.

Two separate regions in the genome of the tobacco etch virus contain determinants of the wilting response of Tabasco pepper.

Infection of Tabasco pepper by the tobacco etch virus (TEV) typically causes wilting associated with root necrosis. However, a strain of TEV, designated TEV nonwilting (TEV NW), is able to infect Tabasco pepper plants but does not cause wilting. In order to locate the genetic determinants responsible for the wilting response, a full-length cDNA clone of TEV NW from which infectious transcripts can be derived was made. A number of chimeric constructs were prepared by substituting cDNA fragments between TEV HAT (which causes wilting) and TEV NW clones. This approach was used to identify two wilting determinants in TEV HAT: one encompasses the 3' one-third of the P3 encoding region; the other spans the 3' end of the CI, the 6-kDa protein, and the 5' end of the VPg-NIa coding regions. Substitutions of both these TEV NW fragments into TEV HAT resulted in infection but not wilting of Tabasco pepper, while the replacement of either of the fragments alone did not alter the wilting response. This indicates that both TEV NW regions contain determinants necessary but not sufficient to alter the wilting response and that both must be present in order to avoid the wilting response. There was no difference between the in vitro transcription-translation products derived from constructs containing these regions from TEV HAT and TEV NW.

A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus.

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.

Loss of potyvirus transmissibility and helper-component activity correlate with non-retention of virions in aphid stylets.

The hypothesis that loss of aphid transmissibility of potyvirus mutants is due to non-retention of virions in the mouthparts was tested by feeding aphids through membranes on purified virions of aphid transmissible (AT or HAT) and non-aphid-transmissible (NAT) tobacco vein mottling virus (TVMV) or tobacco etch virus (TEV), in the presence of functional [potato virus Y (PVY) HC or TVMV HC] or non-functional (PVC HC) helper component (HC). TVMV virions were detected, by electron microscopic examination of immunogold-labelled thin sections, in the food canal or cibarium of 57% of 28 aphids fed on the transmissible combination of TVMV-AT and functional HC, while no virions were found in these structures in 25 aphids fed on the non-transmissible combinations: TVMV-NAT and PVY HC, or TVMV-AT and PVC HC. Autoradiography of intact stylets allowed the examination of much larger numbers of aphids, fed on 125I-labelled TEV; 48% of 523 aphids fed on the TEV-HAT and PVY HC combination retained label in the stylets: this correlated well with the percentage transmission in bioassays. In contrast, in non-transmissible combinations, label was found in the stylets of 0.77% of 389 aphids fed on TEV-NAT and PVY HC, and 1.35% of 223 aphids fed on TEV-HAT and PVC HC. No differences were found in the overall amount of label in the bodies of aphids fed on the transmissible and non-transmissible combinations. There was a strong tendency for virions to be retained in the distal third of the stylets; 56% of aphids positive for TVMV, and 82% of those positive for TEV, had label in this area. These data support the concept that virions retained within the stylets are those that are primarily involved in potyvirus transmission.

Transmission by aphids of a naturally non-transmissible plum pox virus isolate with the aid of potato virus Y helper component.

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.

Mutational analysis of the coat protein N-terminal amino acids involved in potyvirus transmission by aphids.

The nature of the amino acids in the N-terminal 'DAGX' motif of the coat protein of tobacco vein mottling virus (TVMV) that have a direct effect on aphid transmissibility of the virion were further defined by site-directed mutagenesis. In the first position of the DAGX motif, Asp or Asn are required for aphid transmissibility. In the second position, the nonpolar residue Ala, but not the nonpolar Gly or Val or the polar Thr and Ser, is compatible with transmissibility. In the third position, the small, neutral, nonpolar Gly appears to be critical; even substitution of Ala, with a minimal side-chain, drastically reduces transmissibility. Although the amino acid following the DAG sequence is not highly conserved among potyviruses, the presence of an acidic Glu or Asp residue at this position in the TVMV coat protein drastically reduces or abolishes aphid transmissibility. An attempt was made to test the hypothesis that trypsin cleavage of the N terminus is involved in the aphid inoculation process by destroying a trypsin cleavage site downstream from the DAGX motif. While the predicted decrease in transmission occurred from infected plants, there was no effect on the transmission of purified virus. Of the 23 mutations in the DAGX region of TVMV reported here and previously, only two, substitutions of Lys and Arg for Asp, had a detectable adverse effect other than on aphid transmissibility. These, and perhaps other, residues near the N terminus function in some phase of the TVMV life cycle, in addition to aphid transmission.