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Journal of Virology, September 2001, p. 8054-8062, Vol. 75, No. 17
Plant Pathology Department, University of
California, Davis, California 956161;
Instituto Valenciano de Investigaciones Agrarias, Valencia,
Spain2; and Central California Tristeza
Eradication Agency, Tulare, California 932243
Received 9 March 2001/Accepted 18 May 2001
We examined the population structure and genetic variation of four
genomic regions within and between 30 Citrus tristeza
virus (CTV) isolates from Spain and California. Our analyses
showed that most isolates contained a population of sequence
variants, with one being predominant. Four isolates showed two major
sequence variants in some genomic regions. The two major variants of
three of these isolates showed very low nucleotide identity to each other but were very similar to those of other isolates, suggesting the
possibility of mixed infections with two divergent isolates. Incongruencies of phylogenetic relationships in the different genomic
regions and statistical analyses suggested that the genomes of some CTV
sequence variants originated by recombination events between diverged
sequence variants. No correlation was observed between geographic
origin and nucleotide distance, and thus from a genetic view, the
Spanish and Californian isolates analyzed here could be considered
members of the same population.
Citrus tristeza
virus (CTV) is distributed worldwide and is the causal agent of
one of the most economically important diseases of citrus. CTV, a
member of the genus Closterovirus within the family
Closteroviridae, is phloem limited and is transmitted by aphids in a semipersistent manner. CTV virions are filamentous flexuous
particles about 2,000 nm long, with two coat proteins (CP and CPm)
covering 95 and 5% of the particle length, respectively (8). The CTV genome is a single-stranded, positive-sense
RNA of 19,226 to 19,296 nucleotides (nt) (18, 27, 48, 51) organized in 12 open reading frames encoding at least 19 proteins. These include two papain-like proteases, replication-associated proteins (RNA polymerase, helicase, and methyltransferase), a homologue
of the HSP70 protein, two coat proteins (CP and CPm), RNA-binding
protein p23 (23), a p20 protein that accumulates in the
amorphous inclusion bodies (14), and other proteins of so
far unknown function (p61, p13, and p18) (Fig.
1). CTV-infected plants contain, in
addition to the genomic RNA, 3'-coterminal subgenomic RNAs
(15) and defective RNAs (D RNAs), the latter resulting
from extensive internal deletions of the genomic RNA (2,
26, 28, 50).
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.8054-8062.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Variation of Citrus Tristeza Virus
Isolates from California and Spain: Evidence for Mixed Infections
and Recombination
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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FIG. 1.
Unrooted maximum-likelihood phylogenetic trees of
genomic regions A, F, C, and P (see also Table 2) of 34 CTV
isolates (Table 1), constructed using the PHYLIP program DNAML.
Bootstrap values of between 600 and 800 for 1,000 replicates are
indicated by #, and values greater than 800 are indicated by *.
Branch lengths are proportional to the genetic distances. Boxes include
sequences with a nucleotide identity of greater than 99%. Above is a
layout of the CTV genome, with the regions analyzed in black boxes.
When an isolate contained more than one major sequence, these are
indicated by a letter (corresponding to the genomic region) and
a number (corresponding to the sequence variant), e.g., 65A1 and 65A2
occurred in genomic region A of isolate 65.
CTV isolates differing in the type and intensity of symptoms induced in different citrus species and cultivars and in their aphid transmissibility have been reported worldwide (38). In the last two decades, efforts have been taken to develop molecular techniques for rapid differentiation of CTV isolates and identification of molecular markers related to CTV-induced symptoms. Variation in serological reactivity, peptide maps of the coat protein, double-stranded RNA (dsRNA) patterns, hybridization with cDNA probes, restriction fragment length polymorphism, and single-strand conformation polymorphism (SSCP) have been described in attempts to differentiate CTV isolates (29).
Nucleotide sequence analysis is the most accurate procedure for CTV differentiation and estimation of molecular or genetic variation. To date, the complete genome nucleotide sequences of the five CTV isolates T36 and T30 from Florida (1, 18, 34), VT from Israel (27), SY568 from California (51), and T385 from Spain (48) have been reported. Also the partial nucleotide sequences of several CTV isolates have been reported (1, 17, 22, 25, 35, 36). Recently, it has been shown that individual CTV isolates are composed of a population of sequence variants (3, 19, 22). These reports showed the genetic differences between CTV isolates but did not estimate the genetic diversity of natural populations of CTV. Only Moya and García-Arenal (31) estimated the genetic diversity of CTV in Spain based on the number and position of D RNAs associated with Spanish CTV isolates (14). However, for CTV, nucleotide identity seems not to be correlated with the similarity of D RNA patterns (2).
In this study, we assessed the structure and genetic diversity of four genomic regions from two natural CTV populations located in two important citrus-growing areas, Spain and California. By SSCP analysis we estimated the population structure of sequence variants within individual isolates. Nucleotide analysis was used to estimate the genetic distance between sequence variants. These analyses open new insights about mixed infections, recombination, and spatial population structure in the effort to understand CTV complexity.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Virus isolates.
Nineteen Californian and 11 Spanish CTV
isolates were obtained from field trees and maintained in sweet orange
(Citrus sinensis) plants in insect-proof greenhouses.
Symptom evaluation was performed for these isolates in sweet orange
plants and in graft-inoculated Mexican lime (Citrus
aurantiifolia) and grapefruit (Citrus paradisi) plants.
Geographic origin and symptoms induced by these CTV isolates are
summarized in Table 1. More detailed
information about the biological characteristics of CTV Spanish
isolates can be found in Ballester-Olmos et al. (4).
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Purification of CTV dsRNA. CTV-infected sweet orange bark was pulverized with nitrogen liquid, and total nucleic acids were extracted with phenol-detergent buffer. The dsRNAs were then purified by column chromatography on nonionic cellulose and precipitated as previously described (30).
cDNA synthesis and PCR amplification.
Four pairs of primers
(A, F, C, and P) were designed for performing reverse transcription
(RT)-PCR of different regions of the CTV genomic RNA (Table
2). Approximately 200 ng of dsRNA was
denatured at 95°C for 5 min and reverse transcribed by incubation at
42°C for 45 min in a reaction mixture (20 µl) containing 1× avian
myeloblastosis virus (AMV) buffer, 200 µM each of the four deoxynucleoside triphosphates (dNTPs), 40 ng of reverse primer, 2 U of
RNasin (Promega Corp.), and 0.3 U of AMV reverse transcriptase. An
aliquot (1/10) of cDNA product was PCR amplified in a 20-µl reaction
mixture containing 1× PCR buffer, 1.5 mM MgCl2,
200 µM each dNTPs, 20 ng of each primer, and Taq DNA
polymerase (Promega). The following PCR conditions were used: 94°C
for 2 min; 30 cycles each of 94°C for 30 s, 50°C for 30 s, and 72°C 40 s; and 72°C for 5 min. The resulting RT-PCR
products were separated by electrophoresis in a 2% agarose gel and
detected by ethidium bromide staining.
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Cloning and SSCP analysis.
The cDNAs obtained by RT-PCR were
cloned into PGEM-T (Promega) using T4 DNA ligase (Promega) according to
the manufacturer's instructions, followed by transformation into
Eschericia coli DH5
(44). Ten clones
obtained from each cDNA product were selected and PCR amplified using
the same conditions as the PCR described above. SSCP analysis was
performed on the resulting PCR products as previously described
(19, 40). Denatured samples were electrophoresed in
nondenaturing 8% polyacrylamide gels at 200 V for 3 h
(genomic regions A and F), 2 h (region P), or 1 h
(region C).
Nucleotide sequences and statistical analysis. Nucleotide sequences of cDNAs were determined in both directions by means of an ABI Prism DNA sequencer 377 (Perkin-Elmer, Foster City, Calif.). Multiple alignments of the nucleotide sequences were realized with the program CLUSTAL W (47). Numbers of synonymous and nonsynonymous nucleotide substitutions were determined using the program DIVERGE in the Genetics Computer Group (GCG) package (7), which is based on the method described by Pamilo and Bianchi (33) and Li (21) (PBL method). Nucleotide identity of pairs of sequences over the entire sequence length was plotted using GCG's program PLOTSIMILARITY (7). Nucleotide distances were estimated by using the DNADIST program of PHYLIP package version 3.573 (9) using the Jukes and Cantor method for correction of superimposed substitutions. Phylogenetic relationships were inferred using the PHYLIP programs DNAML, based on the maximum-likelihood method, and NEIGHBOR, which implements the neighbor-joining method from a nucleotide distance matrix. SEQBOOT (1,000 repetitions) and CONSENSE were used for bootstrap analysis. Genetic diversity (average number and standard errors of the number of nucleotide substitutions for each pair of sequence variants) and the degree of population subdivision were calculated by the method of Lynch and Crease (24). Recombination events between diverged nucleotide sequences were explored with the programs GENECONV (45) and PHYLPRO (49).
Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper have been deposited in the GenBank database under accession numbers AF356226 to AF356329. Other CTV nucleotide sequences used in our analyses were obtained from the indicated GenBank entries: isolate T36 (U16034), isolate VT (U56902), isolate T385 (Y18420), isolate SY568 (AF001623), isolate T30 (AF260651), Japanese isolates (AB011185 to AB011197), Portuguese isolates (AF184113 to 184118), and Californian isolates 65, 107, 122, 173, and 190 (AF203035 to AF203037, AF203040, AF203043, AF203047, AF203048, AF203051, AF203053, AF203055, AF203057, AF203060, AF203062, AF203063, AF203066, AF203070, AF203073, AF203075, AF203077, AF203079, and AF203081).
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RESULTS AND DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Genetic variation within CTV isolates: evidence of mixed infections. RNA viruses have a great potential for genetic variation due to their error-prone RNA replication, large populations, and short replication times. As a consequence, each isolate of RNA virus is expected to consist of a population of genetically related variants, termed quasispecies (16). To estimate the within-isolate population structure, we performed RT-PCR of four genomic regions (Table 2) of 11 Spanish and 19 Californian CTV isolates and analyzed the SSCP patterns of 10 clones obtained from each genomic region and isolate. Previously we assessed the accuracy of these SSCP analyses (19). The average number of nucleotide differences per site between pairs of CTV sequence variant clones was only 0.001346 ± 0.002126, supporting SSCP analysis as a precise tool for population studies. To minimize primer-directed selection of sequence variants within a isolate, the primers used here (Table 2) were designed from nucleotide sequences conserved for the five CTV isolates whose complete genome sequences are known (1, 18, 27, 34, 48, 51). Previously, we also found that the intensities of the DNA bands in SSCP profiles reflected the relative proportion of CTV RNA variants within an isolate, suggesting that these primers do not bind preferentially to some CTV sequence variants (42). Furthermore, to minimize the possibility that nucleotide incorporation errors in the initial phases of the RT-PCR give rise to a detectable subpopulation of false mutants (20), we used as a template a large amount of dsRNAs (~200 ng). Nonetheless, because we analyzed only the major sequence variants, RT-PCR-induced errors, if present, did not affect our analysis. Finally, to minimize possible variations due to irregular distribution of sequence variants in infected tissues, the dsRNAs were extracted from different branches of a large plant and pooled.
Our results and the results reported previously (19) showed that 26 of 30 field CTV isolates, in the four genomic regions analyzed, had a within-isolate population structure consisting of one major variant (frequency greater than 0.7) and other sequence variants of lower frequencies (minor variants), a typical quasispecies structure (16). In contrast, genomic region A of isolate 65 (19), genomic region C of isolates T398 and 386, and genomic region P of isolates T398 and T405 had two major variants with frequencies of 0.4 or greater. SSCP frequencies and nucleotide distances for all variants from Californian isolates 65, 107, 122, 173, and 190 have been described in detail by Kong et al. (19). We determined the nucleotide sequences of all CTV major variants and estimated the nucleotide distances between them. The nucleotide distances between sequences are represented in Fig. 1 as branch lengths of phylogenetic trees (see next section). Interestingly, for 65 A, T398 and 386 C, and T398 P, the nucleotide distances between the two major variants within the same isolate were high (Fig. 1). For example, the nucleotide distance of genomic region P between the two major variants of isolate T398 (T398P1 and T398P2) was 0.0795. Furthermore, within these isolate regions, each major variant was significantly more similar to major variants of other isolates than to the other major variant from the same isolate (Fig. 1). For example, the major variant T398P2 and that of isolate T300 showed a nucleotide distance of 0.0019, whereas T398P1 and T346 had a nucleotide distance of 0.0039. These data suggest that 65 A, T398 and 386 C, and T398 P could have originated from mixed infections of two CTV isolates with diverged sequence variants. Also, the fact that some genomic regions in the same isolate have two diverged major variants whereas other genomic regions have only one suggests the possibility of recombination events between sequence variants of the original coinfecting CTV isolates. Mixed infections are possible, as CTV hosts are long-lived perennial plants (some living 100 years or more), allowing the possibility of repeated inoculations of CTV by viruliferous aphids. Although we have evidence for mixed infections in a small proportion of CTV isolates here analyzed (3 of 30), it is probable that mixed CTV infections occur more frequently in nature. With the approaches used here, mixed infections could only be detected between two isolates with diverged sequence variants, and both sequences should be in relatively high proportion when they were sampled. The coexistence of two quasispecies in the same host is probably not common, as differences in fitness would likely cause the displacement of one quasispecies.Genetic variation in different genomic regions: evidence
for natural recombination events.
The major variant sequences from
the CTV isolates analyzed here and the sequences of CTV isolates T36,
VT, T385, and SY568 (18, 27, 34, 48, 51) were used to
estimate the nucleotide diversity (average number of nucleotide
substitutions per site in each pair of sequence variants) for each
genomic region. These analyses showed appreciable differences
between the different genomic regions. Genomic region A showed
the greatest diversity, twice that for F and P and three times that for
the C genomic region (Table 3).
To estimate the degree of selective constraints on each genomic
region, nonsynonymous and synonymous substitutions were computed
separately. The number of synonymous substitutions per synonymous site
(dS) was similar over the four genomic regions analyzed (Table 3). However, the number of nonsynonymous substitutions per nonsynonymous site (dN) was smaller than dS
and varied considerably between genomic regions (Table 3). This
suggests a negative selective pressure for most amino acid changes
(functional constraints) and that the degree of the functional
constraints varies for the different genomic regions. Genomic
regions F and P had a ratio dN/dS in the range of most DNA
and plant RNA virus protein-coding sequences (12, 32). F
corresponds to part of the methyltransferase domain, involved in virus
replication, and P corresponds to gene p20, coding for a protein of
unknown function that accumulates in amorphous inclusion bodies
(13). Genomic region A, which so far is not known to be
part of any known functional domain, showed the greatest
dN/dS ratio. Finally, C, corresponding to a portion of the
coat protein gene, showed the greatest functional constraints. The CTV
coat protein, in addition to constraints related to virion structure
and stability, might play a critical role in interactions with its
aphid vector and/or plant host. The dN/dS ratio of CTV
genomic region C was very low in comparison with that of other
plant virus coat protein genes (12).
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Genetic variation with respect to geographic distribution: absence of correlation between genetic and geographic proximity. According to CTV genomic RNA 5' untranslated region nucleotide identity, Lopez et al. (22) classified clones from 11 CTV isolates in three groups: I, represented by isolate T36; II, represented by VT; and III, represented by T385. According to our analyses, genomic regions A and F (both located in the 5' half of the genome; Fig. 1) of all the Spanish and Californian isolates analyzed here can be included in these three groups. The major variants of Spanish isolates T346 and T373 are included in group I, three to five Californian isolates are in group II, and about 80% of the Spanish and California isolates belonged to group III (Fig. 1). For genomic regions C and P, the CTV isolates could not be readily assigned to these three discrete groups. Curiously, group III isolates showed a nucleotide identity of greater than 99% in the four genomic regions (Fig. 1). Group III isolates, with a very high nucleotide identity, has been also found in Florida, Taiwan, and Colombia (1).
For a better comparison of CTV populations, we estimated the nucleotide diversity (average nucleotide distance between two pairs of CTV major sequences chosen randomly) of Spanish and Californian CTV isolates (including T385 and SY568) for each genomic region (Table 4). We found that in each genomic region, the nucleotide diversity of CTV isolates from the same geographic population was remarkably higher than that assessed between the two geographic populations (Table 4). For example, the nucleotide diversity of CTV genomic region P within California and within Spain was about 0.050 for both, whereas the nucleotide diversity considering only nucleotide distances between Spanish and Californian isolates was less than 0.004 (Table 4). Application of the D statistic (24) showed that the Spanish and Californian CTV populations were not significantly genetically different (D = 0.0000), and hence, from a genetic view, the Spanish and Californian CTV isolates can be considered part of the same population. We wanted to know if this was also true for other CTV geographic populations. Unfortunately, not many CTV nucleotide sequences from the same geographic region are available. When genomic region C of six Portuguese isolates (GenBank accession nos. AF184113-AF84118) was included in the analysis, we found that the Portuguese CTV isolates were part of the same genetic population as the Spanish and Californian CTV isolates (Table 4). Analysis of the first 462 nt of the coat protein gene (C2) of 6 Portuguese and 13 Japanese CTV isolates (17) also showed higher diversity within a geographic population than between the two geographic populations (Table 4). Low genetic variation among geographically distant isolates has also been observed for Curcubit yellow stunting disorder virus and Beet pseudo-yellows virus in the genus Crinivirus, the other genus of the family Closteroviridae (41, 43), and for Tobacco mild green mosaic virus and Pepper mild mottle virus in the genus Tobamovirus (11, 37).
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ACKNOWLEDGMENTS |
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Part of this work was supported by grants from the USDA special grants Citrus Tristeza Virus Research Program, the California Tristeza Research Coalition, and the University of California to B.W.F. Part of this work was supported by projects SC93-111 and SC97-098 (INIA). L.R. was supported in part by a postdoctoral fellowship from Ministerio de Educación y Ciencia, Spain.
We acknowledge the technical assistance of T. Olupona, M. Boil, E. Estela, and M. Martínez. We thank F. Garcia-Arenal for excellent critical review of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Plant Pathology, One Shields Ave., University of California-Davis, Davis, CA 95616. Phone: (530) 752-0302. Fax: (530) 752-5674. E-mail: bwfalk{at}ucdavis.edu.
Present address: Citrus Research and Education Center, Department
of Plant Pathology, University of Florida, Lake Alfred, FL 33850.
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Abstract
Introduction
Materials and Methods
Results and Discussion
References
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Journal of Virology, September 2001, p. 8054-8062, Vol. 75, No. 17
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.17.8054-8062.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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