*Institut "Cavanilles" de Biodiversitat i Biología Evolutiva and Departament de Genètica, Universitat de València, Spain;
and
Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Spain
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Abstract |
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Introduction |
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Foot-and-mouth disease virus (FMDV) is an economically important animal pathogen that belongs to the genus Aphthovirus of the Picornaviridae family (Rueckert 1996
). Its genome consists of a positive single-stranded RNA molecule of 8,500 nt that is translated to give a polyprotein, which is proteolytically processed to yield the mature structural and nonstructural viral proteins (Belsham 1993
; Rueckert 1996
). The coding region can be divided into three distinct regions: P1 (encoding capsid proteins VP4, VP2, VP3, and VP1, in that order), P2 (nonstructural proteins), and P3 (nonstructural proteins including the viral replicase).
In the present work, we studied the evolutionary dynamics of FMDV under different experimental regimes: cytolytic passages involving different viral population sizes, and persistent infections in which both host cells and FMDV survive and coevolve for many generations (de la Torre et al. 1985, 1988
). Since in all cases the starting genome was the same FMDV clone, this experimental design allowed an evaluation of the evolutionary processes acting on this virus under different population dynamics by comparing the patterns of divergence between sequences from different FMDV isolates. This kind of study is of particular interest to unveil (1) the importance of convergent evolution in organisms with high mutation rates and huge population sizes, (2) the adaptive significance of convergent evolution, and (3) the effect of convergent evolution on phylogenetic reconstructions.
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Materials and Methods |
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cDNA Synthesis, PCR Amplifications, and Nucleotide Sequencing
Viral RNA extraction, reverse transcription for the synthesis of cDNA, PCR amplification, and the subsequent direct sequencing of the amplified product to obtain consensus sequences of the viral isolates or clones were performed as previously described (Escarmís et al. 1996
; Escarmís, Dávila, and Domingo 1999
). The accession numbers of the complete viral genome sequences available are as follows: C-S8c1, AJ133357; Rp99, AJ133358; Rp146, AJ133359; MARLS, AF274010.
Phylogenetic Analysis
Nucleotide sequences were aligned using CLUSTAL X, a Windows version of the CLUSTAL W program (Thompson, Higgins, and Gibson 1994
). Alignments are available from the authors on request.
For phylogenetic inference, we used three different methods: (1) the neighbor-joining (NJ) distance-based method (Saitou and Nei 1987
), (2) maximum-parsimony (MP) analysis (Fitch 1971
), and (3) maximum-likelihood (ML) procedures (Felsenstein 1981
). The MEGA program (Kumar, Tamura, and Nei 1993
) was used to calculate distances and to construct NJ trees with bootstrap support values based on 1,000 replications. MP and ML trees were constructed by using the computer programs DNAPARS and DNAML, respectively, from the PHYLIP package, version 3.5 for Windows (Felsenstein 1993
).
Alternative topologies were viewed with the TreeView program, version 1.5.2 (Page 1992), and compared using the test of MP proposed by Templeton (1983)
and developed by Felsenstein (1985)
. This test was also performed with the program DNAPARS from the PHYLIP package.
For distance estimation and ML analysis, different models of evolution were compared using conventional likelihood ratio tests (Huelsenbeck and Crandall 1997
). The models tested for a given phylogenetic tree of FMDV sequences were those described by Jukes and Cantor (1969)
, Kimura (1980)
, Felsenstein (1981, 1984
[described in Felsenstein 1993
]), Hasegawa, Kishino, and Yano (1985)
, Tamura and Nei (1993)
, and Yang (1994)
. In addition, models assuming equal substitution rates among sites (Poisson distribution) or heterogeneous substitution rates (the
-distributed rates model) were also compared using likelihood ratio tests. The nucleotide changes experienced by the capsid-coding region were better explained by Tamura and Nei's (1993)
model with a gamma distribution of substitution rates across sites (
= 0.02). the model that best accounted for the evolution of the L protein region was Kimura's (1980)
two-parameter model with a Poisson distribution. Log likelihood values were obtained using the BASEML program of the PAML, version 3.0 package (Yang 1997
).
Detecting Positive Selection
Different codon-based models for the evolution of protein-coding sequences that allow for variable selection intensity among sites (Nielsen and Yang 1998
; Yang et al. 2000
) were also tested with the use of conventional likelihood ratio tests. In these models, the nonsynonymous/synonymous substitution rate ratio (
= dN/dS), which reflects the selection intensity at the amino acid level, was allowed to vary among amino acid sites.
The 13 different models tested were those described in Nielsen and Yang (1998)
and Yang et al. (2000)
. Several models assumed discrete distributions to model heterogeneous
among sites. These models were the "neutral" model (M1 according to Yang et al. 2000
), with two categories of
ratio sites (
= 0 and
= 1); the "selection" model (M2), including a third class of codons with the underlying
ratio estimated from the data; the "discrete" model (M3), with an unconstrained discrete distribution of K
ratios; and the "freqs" model (M4), with five classes of codons with prespecified values of
.
Other models assumed continuous distributions for heterogeneous ratios among sites, although they used a discrete distribution of 10 categories with the same probabilities as an approximation in the likelihood calculation. These models were the "gamma" model (M5), assuming a simple gamma distribution; the "double gamma" model, which used a mixture of two gamma distributions; and the "beta" model (M7), which did not allow for positive selected sites (
> 1). The following models (M8M11) accounted for positively selected sites: the "beta &
" model (M8); the "beta & gamma" model (M9), the "beta & gamma + 1" model (M10), with a gamma distribution shifted to the right by one unit, accounting for positively selected sites (
> 1); the "beta & normal > 1" model (M11), with the distributions left-truncated by 1; the "0 & 2 normal > 0" model (M12), which assumed a proportion of sites with
= 0 and a proportion from a mixture of two normal distributions truncated at
= 0; and the "3 normal > 0" model (M13), which used a mixture of three normal distributions truncated at
= 0. A more detailed description of the models is given in the original references.
These models, implemented in the CODEML program of the PAML, version 3.0, package (Yang 1997
), are very useful for testing adaptive molecular evolution and also for identifying amino acid sites under positive selection by an empirical Bayes approach (Nielsen and Yang 1998
).
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Results |
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Sequences of the capsid region were obtained by consensus sequencing (i.e., direct sequencing of PCR fragments amplified by RT-PCR from a heterogeneous population of viruses) for each of the 38 FMDV isolates indicated in bold in the scheme presented in figure 1 . A total of 31 of the 38 sequences analyzed were different and yielded 2,142 nucleotide positions in the alignment because there were no indels. In the alignment, 78 sites were variable (3.6%). Of the inferred amino acid positions, 35 were variable (out of 714). There was a very slight predominance of variable sites exhibiting nonsynonymous changes (40 nonsynonymous vs. 38 synonymous).
With respect to the L protein region, sequences also obtained by consensus sequencing were available for the same isolates except for HR and HRp100. Of the 36 L-protein sequences, 22 were different and yielded a 603-nt alignment with no gaps. This set of aligned sequences contained 34 variable sites (5.6%). Of the inferred amino acid positions, 33 were variable (out of 201). In this case, there was a clear predominance of variable nucleotide sites exhibiting nonsynonymous changes (29) over synonymous changes (5).
Phylogenetic analyses of the capsid sequences with three methods (MP, NJ, and ML) gave similar topologies, but relationships differed from those of the actual experimental evolutionary history of the FMDV isolates. The MP method gave eight equally parsimonious trees (but only four alternative topologies) requiring 86 changes. These trees differed in the alternative positions of three pairs of ambiguous changes due to convergent mutations (we use the term "convergence" as including both parallel and reverse changes according to Doolittle [1994
]) that could be interpreted either as two parallel substitutions or as a forward substitution and its subsequent reversion or back mutation. Table 1
describes convergent changes, as well as adjacent (taking place in neighbor positions of the same codon) and multiple-hit changes, which occurred during the evolution of FMDV capsid region sequences according to the MP phylogenetic reconstructions. Of these changes, 14 correspond to nonsynonymous changes, and only 1 corresponds to a synonymous substitution.
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The topologies obtained with the three methods of phylogenetic inference (MP, ML, and NJ) corresponded to a similar phylogenetic reconstruction. In fact, the parsimony-based method of Templeton (1983)
(modified by Felsenstein 1985
), used to test if alternative topologies were significantly better or worse, indicated that the five topologies were not significantly different (results not shown). However, the same test suggested that the tree corresponding to the experimental history of the isolates required significantly more nucleotide changes to explain the evolution of the capsid sequences than the trees obtained with the different methods of phylogenetic reconstruction.
The application of the parsimony method to the L-coding region gave a unique MP tree of length 36 (fig. 3
). Convergent, adjacent, and multiple-hit changes, accumulated during the evolution of FMDV L-protein sequences according to the MP phylogenetic reconstructions, are also given in table 1
, and all correspond to nonsynonymous changes. As in the case of the capsid-coding region, the ML tree (not shown) had the same topology as the MP tree, and the NJ method gave a slightly different tree (results not shown). The Templeton (1983)
test indicated that the topologies of the ML and MP trees were better than that of the distance-based tree, but the difference was not significant. Again, the trees obtained with the different methods of phylogenetic reconstruction were significantly better for explaining the evolution of the sequences of the L-protein region than was the tree corresponding to the experimental history of the isolates.
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With respect to L protein, convergent changes occurred only twice in the persistence lineage and corresponded to nonsynonymous substitutions; the other nonsynonymous changes were single events per codon, and there was no concentration of nonsynonymous changes in adjacent codons.
Tests of Positive Selection and Identification of Positively Selected Sites
To determine the possible action of positive selection on subregions of the FMDV capsid and L-protein regions, different codon-based models for the evolution of protein-coding sequences that allowed for variable selection intensity among sites (Nielsen and Yang 1998
; Yang et al. 2000
) were compared with the use of likelihood ratio tests.
The log likelihood values obtained from the phylogenetic analysis of the two FMDV protein-coding regions under different models (Nielsen and Yang 1998
; Yang et al. 2000
) are listed in tables 2 (capsid region) and 3 (L protein). When two models are nested, they can be compared using the likelihood ratio test; twice the log likelihood difference follows a
2 distribution, with the number of degrees of freedom being the difference in the numbers of free parameters between the two models. However, nonnested models can also be compared using the Akaike Information Criterion (AIC; Akaike 1974
): AIC = -2 (estimated log likelihood of the model) + 2 (number of free parameters of the model), which is also given in table 2
. A model that minimizes the AIC is considered the most appropriate model.
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Models that allowed for positively selected sites (M2, M3, M5, M6, and M8M13), all suggested the presence of positively selected sites. For example, the selection model (M2) suggested 1.3% of sites under strong positive selection, with
2 = 10. Model M3 (discrete) suggested an important fraction of sites (4.2%) under strong diversifying selection, with
2 = 10.45. Both models had significantly higher likelihood values than models M0 and M1, providing evidence for adaptive evolution. The likelihood ratio test statistics for the comparison of M2 (selection) to M0 (one-ratio) and M1 (neutral) were 48.0 (P = 3.8 x 10-11, df = 2) and 25.8 (P = 2.5 x 10-6, df = 2), respectively. The likelihood ratio test statistics for the comparison of M3 (discrete) to M0 (one-ratio) and M1 (neutral) were 48.6 (P = 6.9 x 10-10, df = 4) and 26.5 (P = 2.6 x 10-5, df = 4), respectively. Similarly, M8 (beta &
) suggested
1.4% of sites under very strong positive selection, with
2 = 17.2. The likelihood ratio test statistic for the comparison of M7 (beta) and M8 (beta &
) was 24.7 (P = 4.3 x 10-6, df = 2). No other continuous models involving a beta distribution (M9M11) fitted the data significantly better than M7; however, these models (with the exception of M10), together with models M5 (gamma) and M6 (double gamma), did not allow for positively selected sites.
Because positive-selection models (M2, M3, and M8) provided a better explanation of the evolution of the capsid sequences than neutral models (M1 and M7), an identification of individual positively selected amino acid positions was performed by the empirical Bayesian approach (Nielsen and Yang 1998
). According to this approach, the category (with the associated dN/dS ratio) that maximizes the posterior probability is the most likely category for the amino acid site. The posterior probabilities provide a measure of the accuracy of that inference.
Posterior probability distributions for sites along the FMDV capsid region were plotted for the three best fitting models, M2 (selection), M3 (discrete), and M8 (beta & ), as shown in figure 4
. At the 99% level, model M2 identified three positively selected codons: 277, 310, and 568. At the same level, model M3 identified codons 310, 312, and 568. At the 95% level, both models also identified codons 666 and 670. Amino acid 277 was placed within the VP2 protein, sites 310 and 312 were located in the N-terminal region of the VP3 protein, amino acid 568 corresponded to the central region of the VP1 protein, and sites 666 and 670 were located within antigenic site A of the VP1 protein, the first one involved in the generation of the Mab escape mutant MARLS. Codons with a posterior probability smaller than 95% but larger than 90% according to model M3 were 668 (P = 0.949), also located within antigenic site A of the VP1 protein and involved in the generation of the Mab escape mutant HR, and 716 (P = 0.936), the single codon located between antigenic sites D1 and C. Model M8 (beta &
) identified fewer sites because the parameter estimates under this model suggested a smaller proportion of very strongly selected sites (
= 17.2). Other models including components specifically designed to allow for positively selected sites identified all codons exhibiting nonsynonymous substitutions as being under positive selection (
= 1.5 for models M5, M9, and M11;
= 3 for model M4; and
= 4.04.2 for models M12 and M13, respectively) with a posterior probability of >0.99, except M6, which identified the same eight codons as model M3 as being under positive selection (
= 3.8) with a posterior probability of >99%.
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Discussion |
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In other cases, discrepancies can only be attributed to convergent evolution. Thus, for both gene regions, series L sequences are closely related and series S sequences are identical according to the reconstructed trees, but, according to the experimental procedure, S-1 and L-1 share a common experimental history, and the same goes for S-2 and L-2 and for S-3 and L-3. These discrepancies can be explained either by convergent nucleotide changes or by the presence of several variants in the parental VR100 population, with one variant becoming more frequent under one of the infection conditions (series S sequence) and the other becoming more frequent under the new infection conditions (the ancestor of sequences L-1, L-2, and L-3). Two of the most relevant differences between series L and series S sequences are the two nonsynonymous substitutions located in the antigenic A region of VP1 involving two adjacent amino acid replacements (codons 666 and 667) from Leu-Ala to Val-Pro in series L sequences. A sequence analysis of this region from isolates at intermediate passages was performed by Sevilla and Domingo (1996)
. In their sequencing gels, bands diagnostic of the two changes were not visible at passages 6080 and increased from passage 82 to passage 100. An analysis of individual biological clones derived from L-1 at different intermediate passages showed the presence of the intermediate form Leu-Pro and confirmed the progressive increase of the frequency of the variant Val-Pro (0%; 11%, 68%, and 100% at passages 80, 85, 90, and 100, respectively). The results are compatible with both alternative hypotheses, but if several variants were present in the parental VR100 isolate, the derived variants had to be present at very low frequencies and increase in a parallel fashion in the three series L.
Finally, in other examples, the wrong interpretation of convergent nucleotide changes as homologies in the phylogenetic reconstructions is the only explanation. Thus, isolate HR100 was derived from the antibody-resistant clone HR but appears in the MP trees clustered with the persistence lineage. This HR100 sequence shares only one single nucleotide substitution with HR, causing the replacement of His with Arg in the antigenic A region of the VP1 capsid protein selected by the antibody SD6. However, HR100 shares four nonsynonymous substitutions with the sequences of the isolates from the persistence lineage, which explains why they are clustered together in the phylogenetic reconstructions. In this case, ancestral polymorphism and differential sorting of variants cannot be proposed as an alternative hypothesis to the convergent nucleotide changes to explain the incongruence between the experimental tree and the phylogenetic reconstructions, since HR is not a heterogeneous isolate, but a clone.
Eleven other convergences (parallel or reverse changes) are also required to explain the evolution of the persistence lineage, and these were correctly interpreted as homoplasic changes in the phylogenetic reconstructions.
Adaptive Significance of Convergence
Evidence demonstrating that convergent changes play an important evolutionary role has been obtained from both natural populations (Weisblum 1995
; Crandall et al. 1999
) and experimental systems (Bull et al. 1997
; Cunningham et al. 1997
; Wichman et al. 1999
; Crill, Wichman, and Bull 2000
). Also, phylogenetic reconstructions based on molecular data always reveal homoplasy, indicating that a certain level of convergence is common, if not universal (Sanderson and Donoghue 1996
; Wells 1996
). Although a certain amount of convergence is expected even under neutral random substitutions, the rate of convergence is higher when strong mass selection is operating on very large populations, e.g., drug resistance in bacteria (Weisblum 1995
) or viruses (Crandall et al. 1999
) or viral adaptation to different hosts (Bull et al. 1997
; Cunningham et al. 1997
; Wichman et al. 1999
; Crill, Wichman, and Bull 2000
).
In the case of the FMDV capsid region, convergent changes (all but one nonsynonymous) occurred in short sequence stretches coding for antigenic sites of the capsid proteins and are involved in the generation of MAb escape mutants during the evolution of the persistence lineage or after the subsequent recovery of the cytolytic capability. Under the restrictive conditions imposed either by the presence of the SD6 monoclonal antibody, by the persistent infections, or by the competition processes established between different variants of the viral population, amino acid replacement in the capsid-coding region can therefore be positively selected and thus increase fitness. These processes imply viral adaptation to changing conditions that results in the generation of C-S8c1 derivates with increased virulence (de la Torre et al. 1988
; Sevilla and Domingo 1996
; Baranowski et al. 1998
).
Adaptive Evolution in FMDV
Rapid divergence driven by positive selection has rarely been demonstrated at the molecular level, since it can be confounded with variation at RNA and protein sites that tolerate genetic drift. However, the convergent evolution of the FMDV lineages may imply adaptive change. In this study, adaptive molecular evolution in FMDV has been demonstrated by the application of ML methods that allow for variable intensity of selection pressure among codons. Codons of the FMDV genome undergoing positive selection correspond to those where convergent and adjacent nonsynonymous substitutions accumulate (table 1
and fig. 4
), which supports the adaptive value of such convergences.
The application of the likelihood ratio tests to determine the evolutionary dynamics of the capsid-coding region strongly suggests that positive selection is operating on the antigenic A region located in the VP1 capsid protein. Several other codons under positive selection (666, 668, and 670) are located within the region coding for this antigenic site at the G-H loop of the VP1 capsid protein. Previous studies have shown that large population passages may produce FMDV variants with unique amino acid substitutions in this antigenic A region of the VP1 protein and drastic alterations in antigenic specificity even in the absence of immune selection (Díez et al. 1990
; Sevilla, Verdaguer, and Domingo 1996
; Holguín et al. 1997
). In fact, evidence of selection acting on this antigenic A region can be deduced from the study by Holguín et al. (1997)
, where the nonsynonymous/synonymous mutant frequencies ratios in the absence of immune selection are
1 and 1 for antigenic sites C and D, respectively, but 16.9 for site A.
Moreover, the same Thr-to-Lys-or-Ala replacements in amino acid position 670 (148 in VP1) have been found in an independent number of passage series of FMDV C-S8c1 or derivatives (Díez et al. 1990
; Borrego et al. 1993
; Holguín et al. 1997
), including its fixation in the process of fitness gain of a weakened FMDV clone (Escarmís, Dávila, and Domingo 1999
). Something similar happens with position 666 (144 in VP1): Leu is replaced by Ser in MAR mutants derived both from C-S8c1p100 (Martinez et al. 1997
) and from C-S8c1p213 (virus MARLS), or by a Val in MAR mutants derived from C-S8c1p100 (Martinez et al. 1997
), and in the L series isolates.
These results are not unexpected, because capsid proteins establish contacts with the host cell surface as the first step in the intracellular replication of FMDV, especially the highly conserved Arg-Gly-Asp triplet of VP1 (positions 663665), which mediates cell adhesion by being the major determinant in the interaction of protein ligands with cell surface receptors of the integrin superfamily (Mateu et al. 1996
) not only in FMDV, but also in other viral pathogens. In FMDV, it has further been demonstrated that the contiguous residues 666668, on which selection is acting according to the present study, are also involved in cell recognition (Mateu et al. 1996
).
In addition, it has recently been shown that FMDV harbors the potential for adaptation to multiple cellular receptors, including heparan sulfate and several integrin molecules (Jackson et al. 1996, 2000a, 2000b
). In the case of FMDV C-S8c1, changes in receptor specificity have been associated with various capsid surface alterations (Baranowski et al. 1998, 2000
), such as that at position 666 (144 in VP1), where positive selection has been demonstrated.
In this study, we also observed that positive selection was operating on codons located in other capsid regions, such as sites 277 (located in VP2), 310, 312 (located at the 5' end of the VP3 region), and 568 (located in VP1). The antigenic importance of these regions has not been demonstrated. However, site 568 is located in a region, called antigenic site 3, that has been demonstrated to be antigenic in FMDV type O. In addition, Escarmís et al. (1998)
selected variants of FMDV C3Arg/85 (a type C clone isolated in Argentina) in a single infection of partially resistant BHK-21 cells (obtained after persistent infection with C-S8c1 and subsequent elimination of virus from the cells with ribavirin) and found that the selected variants (C3-Rb) showed increased virulence for BHK-21 cells, were able to overcome the resistance of modified BHK-21 cells to infection, and had acquired the ability to bind heparin and to infect other cells (Chinese hamster ovary cells). The comparison of the genomic sequences of the parental (C3Arg/85) and the modified (C3-Rb) clones revealed only two amino acid differences, the fixed Asp-9 to Ala in VP3 (codon 312 in the numbering used in the present study), and either His-108 to Arg or Gly-110 to Arg in VP1 (codons 613 and 615). The same phenotypic and genotypic modifications occurred in a number of independent infections with C3Arg/85 or with clones derived from it. Of the two amino acid differences observed, the constant Asp to Ala in VP3 involved the same nonsynonymous substitution occurring in the persistence lineage analyzed in the present study (table 1
, codon 312). This convergence proves that adaptation to the same cells promotes the selection of virus variants showing the same alterations in cell tropism.
Finally, evidence of positive selection acting on the FMDV genome was obtained from the statistical analysis of the ratio between nonsynonymous and synonymous substitution rates in coding regions. However, an excess of nonsynonymous substitutions is an indicator of positive selection acting at the amino acid level, but genomic RNA is involved in many RNA-RNA and RNA-protein interactions that affect viral replication. This is obvious for noncoding, regulatory regions, but there is increasing evidence that capsid-coding regions in picornaviruses may also have an effect on viral replication (McKnight and Lemon 1998
). Therefore, the RNA itself (apart from its protein-coding capacity) may contribute to the viral phenotype, and fitness may also be affected by synonymous replacements not detected according to this kind of analysis.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: FMDV, foot-and-mouth disease virus; MAb, monoclonal antibody; m.o.i., multiplicity of infection; nt, nucleotide(s); PCR, polymerase chain reaction; PFU, plaque-forming unit.
2 Keywords: foot-and-mouth disease virus
experimental phylogeny
convergent evolution
parallel evolution
positive selection
3 Address for correspondence and reprints: Eladio Barrio, Institut "Cavanilles" de Biodiversitat i Biologia Evolutiva, Universitat de València, Edifici d'Instituts del Campus de Paterna. P.O. Box 2085, E-46071 València, Spain. E-mail: eladio.barrio{at}uv.es
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