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- Occurrence of genetic bottlenecks during citrus tristeza virus acquisition by Toxoptera citricida under field conditionsPublication . Nolasco, Gustavo; Fonseca, Filomena; Silva, GoncaloIn this study, we address the involvement of T. citricida in strain segregation and genetic bottleneck events by comparing the nucleotide diversity of CTV coat protein (CP) gene variants present in field-grown trees with that of variants retrieved from single apterous aphids. Plant material and aphids were collected in orange orchards in the northern part of Portugal. Shoots from two trees that were found to be positive using ELISA and twenty-four apterous aphids from these same trees were selected for individual molecular assays. CTV was detected in 60% of the aphids by amplification of a 417-bp fragment of the CP gene. Analysis of molecular variance (AMOVA) of this fragment revealed that most of the variation of the virus was found among individual aphids (FSC: 0.766) within each location. Nucleotide diversity comparison between the pool of sequences obtained from a given shoot and sequences obtained from individual aphids present on that shoot showed a reduction of more than one order of magnitude in most cases. Computer simulations of random virus acquisition by single aphids showed that in 54% of the cases only a single CP gene phylogenetic group was acquired. However, a small number of aphids (e.g. 6) was enough to acquire the full complement of phylogenetic groups present.
- Presence of Citrus tristeza virus in Angola and São Tomé e Princípe: characterization of isolates based on coat protein gene analysisPublication . Silva, Goncalo; Fonseca, Filomena; Santos, C.; Nolasco, GustavoA first report is given of the presence of Citrus tristeza virus (CTV) in Angola and Sao Tome e Principe. Two out of twenty citrus samples from Sao Tome e Principe and all of the seven samples from Angola were shown by ELISA to contain CTV. The capsid protein gene variants obtained by IC/RT-PCR were characterized by SSCP analysis to test for the presence of different haplotypes in each isolate and nucleotide sequence analysis of each variant leading to a different SSCP pattern, followed by in silico comparison with previously characterized CTV groups. The results confirmed that each isolate could contain different variants, which clustered with the different groups. Variants obtained from each country fitted into the same three clusters.
- Development of an asymmetric PCR-ELISA typing method for citrus tristeza virus based on the coat protein genePublication . Nolasco, Gustavo; Santos, C.; Silva, Goncalo; Fonseca, FilomenaThe coat protein gene of isolates of citrus tristeza virus (CTV) from 20 citrus-producing regions around the world was amplified by RT-PCR, TA cloned, and characterized by SSCP. Haplotypes that produced different patterns within each geographic region were sequenced and a database of 153 accessions of CTV was assembled. Phylogenetic analysis revealed the existence of seven well-defined clusters (Coefficient of differentiation 0.78). An asymmetric PCR-ELISA typing (APET) assay was developed in the frame of this clustering pattern using a set of eight hybridisation probes. The membership of any unknown haplotype is determined by comparing its pattern of reaction against the whole set of probes and not, as previously done in hybridisation assays, in an all-or-nothing basis. Interpretation of the results is objective and done through a visual basic application that compares the rates of hydrolysis of the ELISA substrate of an assayed isolate to a matrix of rates of hydrolysis obtained from standard haplotypes. This assay was validated and showed a better ability to resolve haplotypes than other assays to which it was compared experimentally. It may be automated to the same extent as any ELISA. (c) 2008 Elsevier B.V. All rights reserved.
- Epidemiological situation of Citrus tristeza virus in mainland PortugalPublication . Silva, Goncalo; Nolasco, GustavoThis 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 reached the Algarve. Although most of the isolates harbour haplotypes from group M, haplotypes from the remaining phylogenetic groups were also identified and characterized.
- Comparing p20's RNA silencing suppressing activity among five phylogenetic groups of Citrus Tristeza virusPublication . Marques, N T.; Costa, A. A.; Lopes, D.; Silva, Goncalo; Nolasco, GustavoThe 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 revealed a much reduced activity when compared with the others. At present we still don't know whether this property is a characteristic of this group or an atypical feature due to a unique point mutation. The differences in the symptom type and intensity originated by isolates belonging to the phylogenetic groups assayed could not be related to differences to the p20 suppressor's activity.
- The evolutionary rate of citrus tristeza virus ranks among the rates of the slowest RNA virusesPublication . Silva, Goncalo; Marques, N T.; Nolasco, GustavoCitrus 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) nt per site year(-1) (95% highest posterior density, 1.73x10(-6)-3.16x10(-4) nt per site year-1). For an unbiased comparison with other plant and animal viruses, the evolutionary rate of synonymous substitutions was considered. In a series of 88 synonymous evolutionary rates, ranging from 5.2x10(-6) to 6.2x10(-2) nt per site year(-1), CTV ranks in the 10th percentile, embedded among the slowest animal RNA viruses. At the time of citrus dissemination to Europe and the New World, the major clades that led to the current phylogenetic groups were already defined, which may explain the absence nowadays of geographical speciation.