Mostrando recursos 1 - 20 de 44

  1. Typing of Egyptian Citrus tristeza virus (CTV) isolates based on the capsid protein gene

    Amin, H. A.; Fonseca, Filomena; Santos, C.; Nolasco, Gustavo
    The capsid protein gene of three Egyptian CTV isolates from two locations was amplified by immunocapture RT-PCR and analysed by single stranded conformation polymorphism and sequencing. The CTV isolates studied did not differ significantly in sequence composition and each isolate consisted of very similar haplotypes. Comparison with reference sequences from isolates elsewhere in the world showed that these haplotypes clustered very close to the severe strain T3 from Florida causing quick decline and stem pitting. Analysis of the deduced amino acid sequence showed the epitope characteristic of reactivity with the MCA13 antibody. Sequence comparison with the sequence of an Egyptian...

  2. East Adriatic: a reservoir region of severe Citrus tristeza virus strains

    Cerni, S.; Skoric, D.; Ruscic, J.; Krajacic, M.; Papic, T.; Djelouah, K.; Nolasco, Gustavo
    Citrus tristeza virus (CTV) represents one of the major threats to citrus production worldwide. In the East Adriatic region, CTV symptoms are mostly absent due to traditional citrus grafting on trifoliate orange (Poncirus trifoliata), a CTV-tolerant rootstock. Therefore, the virus has been continuously spreading by the propagation of infected material. The genetic variability of CTV was studied on nineteen citrus samples, collected from orchards in the coastal region of Croatia, Montenegro and Albania, that previously tested positive by ELISA and immunocapture RT-PCR. Single-strand conformation polymorphism of the amplified coat protein gene demonstrated the presence of different CTV variants in each...

  3. Citrus variegation virus: Molecular variability of a portion of the RNA 3 containing the coat protein gene and design of primers for RT-PCR detection

    Bennani, B.; Mendes, C.; Zemzami, M.; Azeddoug, H.; Nolasco, Gustavo
    The coat protein gene and part of the intergenic region of the RNA 3 of several isolates of Citrus variegation virus (CVV) producing either infectious variegation or crinkly leaf symptoms were amplified by RT-PCR, cloned and sequenced. Some isolates were composed of a mixture of sequence variants. The coat protein gene appeared to be highly conserved (lowest similarity among all CVV sequences 93%), especially at the N-terminal, indicating at the molecular level that both types of symptoms are indeed produced by the same virus species. No relationship could be found with the geographic origin. Sequences obtained from isolates producing infectious...

  4. Genomic variability of Citrus tristeza virus (CTV) isolates introduced into Morocco

    Lbida, B.; Fonseca, Filomena; Santos, C.; Zemzami, M.; Bennani, A.; Nolasco, Gustavo
    Genomic variability of the coat protein gene of Citrus tristeza virus isolates obtained from old Meyer lemon introductions in Morocco and more recent budwood introductions from Spain were studied. The coat protein gene of the virus was amplified directly from infected tissue by immunocapture RT-PCR and analysed by single stranded conformation polymorphism (SSCP) and sequencing. Each isolate consisted of several related genomic variants, typical of a quasi-species. Although SSCP analysis has only limited typing ability it could be used in an initial screening to discriminate between isolates of different origin and to analyse the genomic structure of each isolate. Sequence...

  5. Five phylogenetic groups identified in the coat protein gene of grapevine leafroll-associated virus 3 obtained from Portuguese grapevine varieties

    Gouveia, P.; Santos, M. T.; Eiras Dias, J. E.; Nolasco, Gustavo
    The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure....

  6. Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISA

    Bailey, A. M.; Mitchell, D. J.; Manjunath, K. L.; Nolasco, Gustavo; Niblett, C. L.
    The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Phythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual Cultures or front mixed samples of the DNAs of two different species. This approach to species...

  7. Bacterial expressed coat protein: development of a single antiserum for routine detection of Citrus tristeza virus

    Sequeira, Z.; Nolasco, Gustavo
    Citrus tristeza virus (CTV) causes one of the most important citrus diseases world-wide and has recently been detected in Portugal. Early diagnosis based on immunoenzymatic techniques requires significant amounts of reagents. This research describes the isolation of the coat protein gene of CTV, its expression in Escherichia coli as a fusion protein containing an N-terminal (His)6 region and its use to raise rabbit polyclonal antibodies. These antibodies could be used at dilutions higher than 1/80,000 at the detection stage in indirect ELISA tests and were also efficient for trapping the virus in ELISA and Immunocapture RT-PCR. These characteristics allowed the...

  8. Asymmetric PCR ELISA: increased sensitivity and reduced costs for the detection of plant viruses

    Nolasco, Gustavo; Sequeira, Z.; Soares, C.; Mansinho, A.; Bailey, A. M.; Niblett, C. l.
    PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan(TM), a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the...

  9. Genetic diversity of the coat protein of Olive Mild Mosaic Virus (OMMV) and Tobacco Necrosis Virus D (TNV-D) isolates and its structural implications

    Varanda, C. M. R.; Machado, M.; Martel, P.; Nolasco, Gustavo; Clara, M. I. E.; Felix, M. R.
    The genetic variability among 13 isolates of Olive mild mosaic virus (OMMV) and of 11 isolates of Tobacco necrosis virus D (TNV-D) recovered from Olea europaea L. samples from various sites in Portugal, was assessed through the analysis of the coat protein (CP) gene sequences. This gene was amplified through reverse transcriptase polymerase chain reaction (RTPCR), cloned, and 5 clone sequences of each virus isolate, were analysed and compared, including sequences from OMMV and TNV-D isolates originally recovered from different hosts and countries and available in the GenBank, totalling 131 sequences. The encoded CP sequences consisted of 269 amino acids...

  10. The evolutionary rate of citrus tristeza virus ranks among the rates of the slowest RNA viruses

    Silva, G.; Marques, N. T.; Nolasco, Gustavo
    Citrus tristeza virus (CTV) has been studied intensively at the molecular level. However, knowledge regarding the dynamics of its evolution is practically non-existent. In the past, diverse authors have referred to CTV as a highly variable virus, implying rapid evolution. Others have, in recent times, referred to CTV as an exceptionally slowly evolving virus. In this work, we used the capsid protein (CP) gene to estimate the rate of evolution. This was obtained from a large set of heterochronous OP gene sequences using a Bayesian coalescent approach. The best-fitting evolutionary and population models pointed to an evolutionary rate of 1.58x10(-4)...

  11. Citrus tristeza virus p23 may suppress systemic silencing but is not related to the kind of viral syndrome

    Costa, A.; Marques, N.; Nolasco, Gustavo
    Citrus tristeza virus (CTV) the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins namely p20, p23 and p25 protein, previously reported for the monophyletic isolate T36. In this study the p23 suppressing activity from isolates of each of seven phylogenetic groups recognized for CTV was characterized in Nicotiana benthamiana 16C plants expressing GFP. Data revealed that the p23 protein from each phylogenetic group was able to transiently suppress the local but not the short-range silencing. p23 from Group 5 was the most efficient local suppressor followed by Groups 1, 3a, 3b and...

  12. Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3

    Gouveia, P.; Dandlen, S. A.; Costa, A.; Marques, N. T.; Nolasco, Gustavo
    GLRaV-3, a member of the Closteroviridae family and type member of the genus Ampelovirus, is involved in the grapevine leafroll disease. Until now no RNA silencing suppressor has been found among viruses of this genus, contrary to what happens with a large number of other viral genera. In the sister genus Closterovirus, RNA silencing suppressors are present in the 3' end of the genome and have molecular weights close to 20 KDa. To test for RNA suppressing activity screening of p21, p19.6 and p19.7 proteins, coded for in an analogous genomic location of the GLRaV-3 was undertaken. Only p19.7 revealed...

  13. Cellular location of Prune dwarf virus in almond sections by in situ reverse transcription-polymerase chain reaction

    Silva, C.; Tereso, S.; Nolasco, Gustavo; Oliveira, M. M.
    In situ reverse transcription-polymerase chain reaction (RT-PCR) was used in young leaves (from trees and in vitro shoots) and flower buds of almond (Prunus dulcis), a stone fruit, for cellular location of Prune dwarf virus (PDV, a member of the genus Ilarvirus). Sections obtained from samples fixed in formaldehyde and embedded in paraffin were refixed in formaldehyde to increase tissue preservation in the RT-PCR steps. The coat protein gene of PDV was used as the target to produce a cDNA copy that was amplified by PCR and visualized using a direct detection method with digoxigenin-labeled nucleotides. Protein digestion, PCR, and...

  14. Genetic diversity of the coat protein of olive latent virus 1 isolates

    Varanda, C. M. R.; Nolasco, Gustavo; Clara, M. I.; Felix, M. R.
    The CP gene variability among 21 olive latent virus 1 (OLV-1) isolates obtained from different hosts and locations and at different times was assessed. Amplicons obtained by RT-PCR were cloned, and at least 10 sequences from each isolate were analyzed and compared. OLV-1 sequences available in GenBank were included. The encoded CPs consisted of 270 amino acids, except those of isolates G1S and C7 (269 aa) and G6 (271 aa). Comparison of CP genomic sequences of the isolates under study showed very low values of nucleotide diversity, 0.02, and maximum nucleotide distances between (0.087) or within isolates (0.001). Although very...

  15. Comparing p20's RNA silencing suppressing activity among five phylogenetic groups of Citrus Tristeza virus

    Marques, N. T.; Costa, A. A.; Lopes, D.; Silva, G.; Nolasco, Gustavo
    The p20 protein encoded by the Citrus Tristeza Virus (CTV) was previously identified as a RNA silencing suppressor. In this study, we analyzed the p20's suppressing activity from five phylogenetic groups of CTV, using the co-infiltration assay of Green fluorescence protein (GFP) gene and the suppressor gene in 16C line Nicotiana benthamiana plants. Green fluorescence, GFP mRNA relative levels and GFP specific siRNAS were compared showing in most cases, only slight differences. Contrary to previous studies, the p20 suppressor was not able to impede neither short range nor systemic spreading of RNA silencing. The suppressor from the phylogenetic group 4...

  16. Towards an integrated diagnosis of filamentous viruses affecting grapevine

    Nolasco, Gustavo
    Certification is of overwhelming importance in the modern viticulture. Its implementation is dependent of the availability of reliable methods for virus detection. The increasing number of viral species that are found affecting grapevine is a serious limitation due to difficulties of obtaining suitable reagents for immunodiagnosis. Genomic methods targeting several viruses at once might be an economical alternative to ELISA. In this paper we discuss the advantages and problems posed by genomic targeted methods.

  17. Detection of a new variant of Citrus tristeza virus in Greek citrus crops

    Chatzivassiliou, E. K.; Nolasco, Gustavo
    Citrus tristeza virus (CTV), the most destructive virus of citrus, is a quarantine pathogen in Greece. Since 2000, several accidental imports of infected propagation material have been detected in the country, and while eradication measures were applied, a few disease foci still remain. CTV isolates were collected from Chania (Crete) and the "lemonwood" of Poros (Peloponnese), and their genetic variability was studied using single-strand conformation polymorphism (SSCP). One previously characterized isolate from Argolida grafted on a Mexican lime (GR3) and two Italian isolates from Calamondin were also included in the study. ELISA and RT-PCR tests confirmed CTV presence, and SSCP...

  18. Molecular characterization and potential sources of Citrus tristeza virus in Oman

    Al Sadi, A. M.; Al Hilali, S. A.; Al Yahyai, R. A.; Al Said, F. A.; Deadman, M. I.; Al Mahmooli, I. H.; Nolasco, Gustavo
    A study was conducted to characterize occurrence, molecular variability and potential sources of Citrus tristeza virus (CTV) in Oman. A survey during 2009 and 2010 showed that CTV occurs in most (91%) of the surveyed districts. Moderate to high levels of infection with CTV in lime were detected in northern Oman (1563%) compared to the south (012%). This could be related to the continuous introduction of infected citrus germplasm in the north from abroad, where CTV was detected in 45% of citrus seedlings imported from Syria, Lebanon, India, Pakistan and Egypt. CTV was detected for the first time in sweet...

  19. Occurrence of stem-pitting strains of Citrus tristeza virus in Croatia

    Cerni, S.; Skoric, D.; Krajacic, M.; Gatin, Z.; Santos, C.; Martins, V.; Nolasco, Gustavo
    Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30'N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field- infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof...

  20. Epidemiological situation of Citrus tristeza virus in mainland Portugal

    Silva, G.; Nolasco, Gustavo
    This study was conducted to update the occurrence and molecular variability of Citrus tristeza virus (CTV) isolates recently obtained from surveys in different orchards in mainland Portugal. The asymmetric PCR-ELISA typing method based on the coat protein (CP) gene was used to characterize CTV isolates. Most isolates of the virus found in the Algarve region, where major citrus producing zones exist, belonged to the mild phylogenetic group (GpM). The prevalence of haplotypes from this group suggests that the aphid vector Toxoptera citricidus, which is present in the northern region of the country associated to diverse severe strains, has not yet...

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