High genetic variability and evidence for plant-to-plant transfer of Banana mild mosaic virus

Pierre-Yves Teycheney1, Nathalie Laboureau2, Marie-Line Iskra-Caruana2 and Thierry Candresse3

1 CIRAD, UPR 75, Station de Neufchâteau, Sainte-Marie, F-97130 Capesterre Belle-Eau, Guadeloupe, French West Indies
2 CIRAD/UMR BGPI, TA 41/K, Campus International de Baillarguet, F-34398 Montpellier Cedex, France
3 UMR GD2P, INRA et Université Bordeaux 2, IBVM, Campus INRA de la Grande Ferrade, BP 81, F-33883 Villenave d'Ornon Cedex, France

Correspondence
Pierre-Yves Teycheney
teycheney{at}cirad.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A total of 154 partial nucleotide sequences within the Banana mild mosaic virus (BanMMV) ORF1, which encodes the viral RNA-dependent RNA polymerase (RdRp), was obtained from 68 distinct infected banana accessions originating from various locations worldwide. The 310 nt sequences displayed a high level of variability with a mean pairwise nucleotide sequence divergence level of 20·4 %. This situation resulted essentially from a high rate of synonymous mutations. A similar analysis was performed for a limited selection of 10 banana accessions (30 sequences) on the region comprising approximately the last 310 nt of the BanMMV genome. This region corresponds to the 3' end of ORF5, which encodes the coat protein (234 nt), and to the 3' non-coding region. This analysis confirmed the high level of diversity observed in the RdRp dataset, characterized by a high level of synonymous mutations. Analysis of intra-host diversity indicated the existence of two distinct situations, with some plants containing only closely related sequence variants, whereas others contained widely divergent isolates. Analyses indicated that BanMMV genetic diversity is not structured by the geographical origin of the infected Musa accessions or by their genotype. This situation may be, in part, explained by the exchange of banana germplasm between different parts of the world and also by plant-to-plant transfer of virus isolates, the evidence for which is, for the first time, provided by this study.

The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AY729491–AY729643 (RdRp sequences) and AY730729–AY730758 (CP/3' NCR sequences).

Tables showing the names, genomic groups, origins and sequences generated from banana accessions are available as supplementary material in JGV Online.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Within the past 20 years, numerous studies on the diversity and evolution of viral genomes have highlighted the high evolutionary potential of RNA viruses. This situation results, in particular, from the high error rate of viral RNA-dependent RNA polymerases (RdRp) due to their lack of proofreading activity (Domingo et al., 1996; Drake & Holland, 1999; Malpica et al., 2002). It has also been shown that competition between variants or virus isolates, selection pressures exerted by host(s) defence mechanisms or by vector transmission and genetic drift based, in particular, on transmission bottlenecks (Grenfell et al., 2004; Chare & Holmes, 2004) play a critical role in shaping virus evolution and the ensuing virus populations. Although substantially less well known than their animal and human counterparts, plant viruses, especially RNA plant viruses, and their evolutionary potential have also been studied in this respect (García-Arenal et al., 2001; Chare & Holmes, 2004). The results obtained show that populations of RNA viruses infecting plants generally display levels of genetic variability that fall within the range of those observed for RNA or DNA viruses infecting animals (García-Arenal et al., 2001; Chare & Holmes, 2004). However, plants have no immune system, and most viruses infecting plants establish what can be considered to be chronic infections lasting the lifetime of their hosts. Sexual transmission to the progeny is limited as only approximately 20 % of plant viruses are seed-transmitted, but the absence of sexual reproduction in vegetatively propagated plants enables viruses to establish long-term infections and to be transmitted with high efficiency to daughter plants. Such permanent infections offer ideal conditions for long-term co-evolution of viruses with their host, but limited information is currently available on the variability of RNA plant viruses infecting vegetatively propagated plants.

Banana mild mosaic virus (BanMMV) is a recently characterized flexuous virus infecting bananas (Gambley & Thomas, 2001) and an unassigned member of the family Flexiviridae (Adams et al., 2004). Banana (Musa spp.) is a widely cultivated monocotyledonous crop belonging to the family Musacae. Diploid, triploid or more rarely tetraploid cultivated banana varieties (cultivars) are parthenocarpic and often sterile, and are therefore mostly propagated vegetatively. They originate from two diploid Musa species, Musa acuminata Colla (AA genome) and Musa balbisiana Colla (BB genome) (Simmonds & Shepherd, 1955; Stover & Simmonds, 1987) and are classified in five main genomic groups designated AA, AAA, AAB, ABB and AAAA according to their genetic make-up. The 7352 nt, positive-sense, polyadenylated genomic RNA of BanMMV bears five open reading frames encoding a replication-associated protein with RdRp activity, three small proteins necessary for cell-to-cell movement within its host (TGBp1 to TGBp3) and a coat protein (CP). Vertical transmission by vegetative propagation is currently the only known means of propagation of this virus, since no biological vector has been identified nor has experimental mechanical inoculation on susceptible hosts been successful so far (Thomas et al., 1999).

Here, the first analysis of the genetic variability of BanMMV genomic RNA is reported. The aim of the present study was to assess the molecular diversity of a virus not known to be transmitted from plant to plant in a vegetatively propagated crop. This was achieved through extensive cloning and sequencing of two distinct regions of the BanMMV genomic RNA.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Plant materials.
Most leaf samples used in this study were harvested from the CIRAD Musa ex situ collection of live plants maintained in Guadeloupe (French West Indies). This collection has been compiled over the past 45 years from suckers of various wild accessions and cultivars collected worldwide, and vegetatively propagated ever since. Additional leaf samples shipped from various locations and/or collected from accessions maintained as a greenhouse collection in Montpellier (France) were also used.

Determination of BanMMV sequences.
Immunocapture of BanMMV particles was performed on banana leaf homogenates with purified anti-BanMMV IgGs (kindly provided by B. E. Lockhart, University of Minnesota, St Paul, USA) using the technique of Wetzel et al. (1992) with minor modifications (P.-Y. Teycheney and others, unpublished results).

For amplification of a portion of the ORF1 encoding the viral RdRp, RT-nested PCR with inosine-containing degenerated primers and Biotaq DNA polymerase (Eurobio) was performed on immunocaptured virus particles using the protocol, primers and conditions developed by Foissac et al. (2005). The two nested internal primers used target conserved motifs II and V near the active site of the polymerase (Fig. 1) so that the amplified region (310 nt excluding the primers) contains conserved motifs III and IV of the RdRp (Koonin, 1991; Koonin et al., 1991). As such, this region is expected to correspond to one of the most conserved regions of the BanMMV proteins.



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Fig. 1. Genetic organization of Banana mild mosaic virus genomic RNA and regions analysed in this study. Positions of the ORFs along BanMMV genomic RNA and of conserved domains (I to VII) of the RdRp are indicated (a). Arrows show the position of the primers used for RT-PCR and nested PCR experiments. Inosine-containing primers F1, R3 and R4 were used for amplifying by RT-PCR part of BanMMV ORF1 encoding the viral RdRp (b) and primers F2 and R2 were used in the subsequent nested PCR step (c). Primers CP1 and CP2 were used for amplifying by RT-PCR part of BanMMV ORF5 encoding the viral CP and the adjacent 3' NCR (c). The size of the amplified PCR products whose sequences were further analysed is also given.

 
A second genomic region was analysed for isolates present in a subset of banana accessions. This region (Fig. 1) corresponds to the last 234 nt of the 717 nt coat protein (CP) gene and to the complete 3' non-coding region (NCR; 77 nt in the reference isolate AF314662). This region was chosen because it is known that for several members of the family Flexiviridae, the C-terminal half of the CP gene is significantly more conserved than the N-terminal part (German-Retana et al., 1997; Yoshikawa et al., 2001) and because it is likely to encompass the negative-strand promoter involved in BanMMV replication (Gambley & Thomas, 2001). Amplification of this second genomic region was performed by RT-PCR following immunocapture. Primer BanCP1 (5'-GGATCCCGGGTTTTTTTTTTTTTTTTT-3'), which is simply an oligo(dT) with additional cloning sites at its 5' end, was used for the reverse transcription step. The same primer was also used for PCR amplification, together with primer BanCP2 (5'-TATGCNTTYGAYTTCTTRGAYG-5'), which is complementary to BanMMV genomic RNA positions 7042–7063.

Before the reverse transcription step, encapsidated RNAs were released from the immunocaptured particles by incubation at 95 °C for 2 min in 15 µl DEPC-treated water. First strand cDNAs were prepared using AMV reverse transcriptase (USB) according to the manufacturer's instructions. PCR primers were used at 10 pmol and PCR parameters were as follows: pre-incubation at 95 °C for 3 min; 35 cycles at 95 °C for 30 s, 56 °C for 1 min, 72 °C for 30 s; and a final elongation step at 72 °C for 10 min.

All PCR products were cloned into plasmid pGEM-T (Promega) and one to seven distinct cDNA clones per PCR product were used for sequencing. Sequencing was performed by Genome Express. All mutations were individually confirmed using original electrophoregrams.

To provide an evaluation of the mutations that might be introduced by the nested PCR method used to analyse the RdRp region, amplifications were performed on in vitro transcribed RNA targets and the PCR products obtained were cloned and sequenced as described above.

Nucleotide sequence analysis.
Multiple sequence alignments and initial phylogenetic reconstructions (neighbour-joining) were performed using the program CLUSTAL_X with randomized bootstrapping evaluation of branching validity (Thompson et al., 1997). Mean diversities, genetic distances (p-distances calculated on amino acid or nucleotide identity), Nei–Gojobori synonymous/non-synonymous substitution rates (Nei & Gojobori, 1986) and Nei's Gst coefficient of differentiation (Nei, 1987) were calculated using MEGA2 (Kumar et al., 2001). Potential recombination events in the datasets were evaluated using GENECONV v.1.81 software, available at http://www.math.wustl.edu/~sawyer/geneconv/ (Sawyer, 1989), following manual editing of the CLUSTAL_X alignments, when appropriate.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Generation of sequence datasets for two regions of the BanMMV genomic RNA
Full indexing of the 461 Musa accessions of CIRAD's collection led to the identification of 39 infected accessions which were used in this study. In addition, 29 other BanMMV-infected accessions coming either from another Musa germplasm collection kept under greenhouse conditions by CIRAD in Montpellier (France) or freshly collected in the field were also used (Supplementary Tables S1 and S2 available in JGV Online).

All sequences were obtained from cDNAs amplified from immunocaptured virus particles, which ensures that only encapsidated sequences were analysed. A total of 154 sequences were obtained from the 68 banana accessions for the BanMMV RdRp region and 30 sequences were from 10 accessions for the BanMMV CP/3' NCR (Supplementary Tables S1 and S2 available in JGV Online). The absence of recombination events was verified in both datasets using the program GENECONV (Sawyer, 1989) (data not shown).

Within the RdRp dataset, all sequences are fully collinear, without indels, when compared to the reference BanMMV sequence (Gambley & Thomas, 2001). However, the coding capacity of three of the 154 sequences is affected by point mutations introducing in-frame stop codons. In the CP/3' NCR dataset, a similar situation was observed concerning the CP coding region: no indels are observed but mutations interrupting the reading frame are observed in two cDNA clones. The genomes containing such stop codon-introducing mutations are likely to produce non-functional proteins and, particularly in the case of the RdRp, to represent non-viable sequences unless they can be complemented by other sequence variants.

The 3' NCR presented a different situation (Fig. 2) with the simultaneous presence of highly conserved regions and three zones of indel polymorphism. Although such polymorphisms are frequent in the non-coding regions of RNA viruses, the situation reported here appears to indicate a high level of variability. In particular, the indel polymorphism region immediately following the stop codon varied in size from 1 to 20 nt, whereas that adjacent to the poly(A) tail varied in size from 2 to 4 nt.



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Fig. 2. Alignment of the nucleotide sequences of the 3' NCR of BanMMV isolates. The stop codon is indicated in bold and dashes indicate gaps inserted in the alignment. Asterisks indicate fully conserved nucleotide positions. The poly(A) tail has been removed for clarity. Nucleotides involved in the formation of a conserved hairpin are in bold and underlined.

 
Molecular variability of two regions of the BanMMV genomic RNA
Analysis of the RdRp and CP/3' NCR revealed an elevated level of sequence variation, as indicated for instance by the fact that only 23·8 % nucleotide positions and 14·6 % amino acid positions within the RdRp dataset are fully conserved. In the CP/3' NCR, using a dataset of only 30 sequences derived from 10 distinct banana accessions, those values were higher, reaching 51 % fully conserved nucleotide positions and 64 % amino acid positions.

In order to evaluate the potential effects of the RT-nested PCR procedure used for analysis of the RdRp region on the sequence variability observed, control amplifications were performed on in vitro transcribed RNA targets. The RT-PCR products were cloned and 10 cDNA clones were sequenced. Sequence analysis revealed an overall mutation rate introduced by the RT-nested PCR procedure of 0·47 % (result not shown). Given the mean divergence between isolates (see below), this error rate can be estimated to contribute to a little less than 5 % of the total observed diversity. Pairwise divergence levels (nucleotide identity) were calculated for all sequences from the RdRp dataset. The genomic sequence from another member of the family Flexiviridae, Apple stem pitting virus (ASPV, genus Foveavirus; D21829), was added to the dataset in order to provide a comparative estimation of the divergence level against another virus species belonging to the same family. Pairwise values obtained between BanMMV sequences were 0–28·4 %, whereas those calculated between BanMMV sequences and ASPV were 34·7–40·8 %. The distribution of the pairwise percentages of divergence observed is shown in Fig. 3. There is a clear separation between the peaks corresponding to intra-BanMMV values (solid bars) and those corresponding to BanMMV/ASPV values (open bars). This pattern confirms that all the sequences obtained correspond to isolates of BanMMV. The highest level of divergence observed between BanMMV isolates (28·4 %) can be compared with the species discrimination criteria for the family Flexiviridae, which states that isolates represent distinct species when they share less than 72 % identical nucleotides between their complete CP or RdRp genes (Adams et al., 2004).



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Fig. 3. Distribution of the percentages of pairwise divergence (p-distance, nucleotide identity) in the BanMMV RdRp dataset. Each bar represents the total number of pairwise nucleotide comparisons sharing the same percentage of divergence. Solid bars refer to comparisons between BanMMV sequences, whereas open bars refer to comparisons between a BanMMV sequence and the homologous sequence from Apple stem pitting virus (GenBank accession no. NC_003462), used as a control.

 
The frequency of the pairwise divergence values observed between the BanMMV RdRp sequences showed a bimodal distribution (Fig. 3) with two clear but overlapping peaks. The first peak represents comparisons between sequences sharing less than 4–5 % divergence, whereas the other, broader peak represents the bulk of the pairwise values. This latter peak extends from 12–13 to 28–29 % and is centred around an approximately 20–23 % divergence value. The low divergence peak corresponds mostly to within-plant variability. However, in some cases, sequences obtained from the same plant were found to be more divergent or, conversely, sequences from different plants were found to be closely related (see below). Generally speaking, the middle and upper values observed for the two peaks are relatively high when compared with similar data from populations of other plant viruses (Gibbs et al., 1999). They lead to a mean divergence value of 20·4 % between sequences of the RdRp dataset (see Table 1). A similar mean divergence value (19·3 %) was observed when analysing the CP coding region (234 nt) of the 30 sequences from the CP/3' NCR dataset. In contrast, the corresponding value for the 3' NCR (13·3 %; Table 1) was statistically different (P<0·0001 using a Mann–Whitney non-parametric test), providing a strong indication that sequences of the coding and non-coding regions of the BanMMV genome are under different selection pressures.


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Table 1. Mean nucleotide and amino acid diversities for the BanMMV RdRp and CP/3' NCR datasets

 
Encoded proteins and synonymous and non-synonymous mutations
When considering amino acid sequence identities, the mean divergence between sequences within the RdRp and CP datasets were 11·2 and 7·0 %, respectively (Table 1). The reduced size of the CP dataset only partly explains the lower mean pairwise distance observed for the CP sequences, since the 9·6 % value obtained for a reduced RdRp dataset (35 sequences), including only the sequences from the 10 plants used to generate the CP/3' NCR dataset, is still higher and statistically different (P<0·0001 in a Mann–Whitney non-parametric test) than the corresponding value obtained from the CP/3' NCR dataset. It seems therefore that the amino acid sequence of the CP gene targeted in this study could be under stronger evolutionary constraints than the RdRp region analysed, despite the fact that the RdRp region analysed contains highly conserved amino acids motifs.

Overall, the ratio of non-synonymous to synonymous substitution rates (dN/dS), which gives a measure of the evolutionary constraints to variation (Kimura, 1980, 1983), is in the same range for both BanMMV RdRp and CP complete datasets (Table 1). This ratio is well below 0·1 in both cases, i.e. in the range of the lowest values reported for plant viruses (García-Arenal et al., 2001; Chare & Holmes, 2004). The dN/dS value calculated for the BanMMV CP dataset is, in particular, in the range of those calculated for CP sequences of other members of the family Flexiviridae (Chare & Holmes, 2004). This result indicates that despite accumulating synonymous mutations to a high level, the genome of BanMMV seems to be under a level of evolutionary constraint similar to that of other plant viruses.

Very different situations were observed, however, between the various BanMMV sequences. This is illustrated in Fig. 4, which presents a plot of the pairwise percentage of divergence (identity) in the CP amino acid sequences against the pairwise percentage of divergence in the corresponding nucleotide sequences. While there is a general positive correlation between these two measures of divergence, variability in the association is strong and the correlation observed is poor (r2 of only 39 % for the best-fit linear regression), suggesting a complex relationship between synonymous and non-synonymous substitutions. For example, a similar divergence value of 18 % at the nucleotide level was observed for sequences showing 1·5–15·0 % amino acid divergence. Reciprocally, an amino acid sequence divergence value of 1·5 % was observed for pairs of isolates displaying 2–19 % nt divergence. Similar results were obtained when performing the same analysis on the RdRp dataset (data not shown).



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Fig. 4. Plot of the pairwise divergence of the encoded amino acid sequences as a function of the divergence in the corresponding nucleotide sequences for the BanMMV CP/3' NCR dataset. Pairwise divergence values were calculated as strict identity between the amino acid or nucleotide sequences.

 
Patterns of intra-plant and inter-plant BanMMV sequence variability
Comparisons of sequences derived from the same host plant revealed contrasting situations, as reflected by the bimodal distribution of the frequency of pairwise percentages of divergence (results not shown). About 56 and 33 % of all intra-plant comparisons displayed divergence values below 2 % for the RdRp and CP/3' NCR datasets, respectively. Meanwhile, divergence levels in excess of 15 % were observed for about 33 % of intra-plant comparisons from both datasets (results not shown). These observations reflect the fact that only closely related sequence variants (within a range of 2–3 % divergence values) were recovered from some plants, such as plant 44, for which the six variants sequenced displayed a mean divergence value of 1·4 %, whereas other plants were found to contain up to three widely divergent isolates. Of the 34 plants from which three or more RdRp sequences were generated, 15 (44 %) contained only closely related variants, whereas six (18 %) contained three highly divergent isolates (results not shown). For these six plants (plants 1, 2, 7, 21, 30 and 82), intra-plant mean pairwise distances of between 0·687 and 0·804 (dS) and between 0·047 and 0·079 (dN) were observed (data not shown). Such intra-host diversity values compare to those registered for viruses causing chronic infections in animals or humans, such as Hepatitis C virus (Löve et al., 2004).

A significant increase in the dN/dS ratio was observed for the RdRp sequences from plants from which only closely related sequences were recovered, such as plant 44, which displayed a mean dN/dS value of 0·35 between the six sequences obtained (data not shown). This may reflect a relaxation of selection constraints, possibly due to trans-complementation effects between closely related variants present in the same host.

Inter-plant comparisons, which represent over 98 % of all pairwise comparisons, provided generally high divergence between sequences (Fig. 3). Nevertheless, a small proportion of comparisons (0·6 %) yielded low (below 2 %) divergence values. In two cases (sequences 1·1 and 40·6, sequences 8·4 and 22·3), identical sequences were even recovered from different plants. These results indicate that, in some instances, different plants share BanMMV sequences that are as closely related as those observed within individual plants.

Factors structuring the genetic diversity of BanMMV
Mean pairwise distance values obtained using subsets of the BanMMV RdRp dataset were further analysed in order to pinpoint possible factors that might influence the structure of the genetic diversity of BanMMV. Three structuring parameters were evaluated: individual host plant, host plant genomic group (AA, AAA, AAAA, AAB, ABB and TT) and geographical origin of host plant. For each parameter, relevant sequences of the RdRp dataset were pooled in subpopulations and the mean intra- and inter-subpopulation diversities were calculated, as well as the entire population diversity and Nei's Gst coefficient of differentiation between subpopulations (Nei, 1987). Sequences of the CP/3' NCR dataset were also used to estimate host plant influence on the genetic structure of the population. However, the CP/3' NCR dataset could not be used to estimate the effects of host plant genomic group or country of origin due to the low number of plants analysed. The results are shown in Table 2. With each parameter analysed, mean inter-subpopulation diversity was found to be lower or within the range of intra-subpopulation diversity, indicating an overall low level of subpopulation differentiation. The fraction of the overall genetic diversity due to subpopulation differentiation was further evaluated using Nei's Gst parameter (Nei, 1987). Relatively low values of 0·13 and 0·259 were calculated using the host plant genomic group and the host plant country of origin, respectively, as structuring parameters. This result indicates that the subpopulations defined by these two parameters show a low level of differentiation. However, in a few cases, phylogenetically supported clusters of plants originating from the same geographical area were observed (results not shown), resulting in reduced intra-subpopulation diversity values. Such a situation was encountered for isolates from Colombia (plants 87 and 88), Mayotte (plants 100 and 101) and Vietnam (plants 85, 86 and 89). In the majority of cases, however, sequences retrieved from plants with the same geographical origin did not cluster together, strongly suggesting that the diversity of BanMMV is poorly structured by the geographical origin of the host plants.


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Table 2. Analysis of the factors structuring the BanMMV diversity

Subpopulations A, B and C are defined by host plant, host plant genomic group and host plant country of origin, respectively.

 
A different situation was observed when considering individual host plants as the structuring parameter. Gst values of 0·438 (CP/3' NCR dataset) and 0·562 (RdRp dataset) were obtained, indicating considerable differences between the sets of sequences generated from different host plants.

Although not previously documented, the overall limited effect of host or geographical parameters on BanMMV populations could potentially result from plant-to-plant spread of BanMMV isolates within CIRAD's field collection. In order to obtain insights into a possible plant-to-plant transmission of BanMMV, the percentage of plants sharing RdRp sequence variants harbouring less than an arbitrary cut-off of 2 % nucleotide identity divergence were plotted against the distance separating these plants. This cut-off value was retained because it roughly corresponds to the variability observed within plants containing only closely related isolates and may, therefore, represent a conservative measure of the variability envelope of individual virus isolates. Fig. 5 shows the level of infection by identical or closely related variants as a function of the physical distance between plants sharing those variants (expressed as the number of intervening banana plants). A steep gradient was observed, indicating that closely located plants were more likely to share closely related variants than more distant ones.



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Fig. 5. Percentage of plants infected by close or identical BanMMV isolates as a function of the distance separating plants. The size of bars is proportional to the percentage of plants sharing BanMMV RdRp nucleotide sequences diverging by less than 2 %. Between-plant distance classes are expressed as the number of intervening plants.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the molecular variability of BanMMV reported here targeted two separate regions of the viral genome (RdRp region and CP/3' NCR) and a large number of isolates originating from all over the world. Overall, this analysis revealed a high level of nucleotide diversity, in the range of those observed for the most variable plant viruses such as several other members of the family Flexiviridae or a few potyviruses (Chare & Holmes, 2004). This high variability seems, however, in marked contrast to the situation encountered for another member of the family Flexiviridae infecting Musa spp., Banana virus X (Teycheney et al., 2005). The control experiments performed during this study indicate that although the RT-nested PCR procedure used to generate the RdRp dataset has a higher mutation rate than standard PCR procedures (such as the one used to amplify the CP/3' NCR), this higher rate is unlikely to affect the major conclusions of this study.

The results reported here show that the variability of BanMMV results mostly from the accumulation of synonymous mutations. This translates into a very high variability of the third nucleotide of the codons, with only 3 and 6 % of the third bases conserved in the RdRp and CP datasets, respectively. The third base variability contributes to 76 and 81 % of the total variability of the analysed regions of CP and RdRp genes, respectively. In contrast to other plant viruses in which conservation of the encoded proteins cannot fully account for the low overall molecular variability of the viral genome (García-Arenal et al., 2001, 2003), the results reported here can be taken as an indication that little selection pressure is applied on BanMMV coding genomic sequences above that necessary for the conservation of the encoded proteins. This hypothesis is corroborated by observation of similar levels of amino acid conservation between pairs of isolates showing wide variations in their nucleotide sequences (Fig. 3). Similarly, high variabilities and low dN/dS values have been reported for other members of the family Flexiviridae (Chare & Holmes, 2004; Shi et al., 2004), suggesting that this property might be widely shared by members of this family.

Despite the fact that the CP region exhibits a slightly reduced variability (particularly at the amino acid sequence level), the overall trends are similar in the two regions analysed. The relatively close dN/dS values observed (CP, 0·054; RdRp, 0·085) indicate that both regions are similarly constrained. These regions correspond to parts of their respective ORFs likely to be under stronger selection pressure than other regions of the ORFs. This is particularly true for the RdRp region, which is close to the viral polymerase catalytic domain and encompasses previously identified conserved motifs (Koonin, 1991). The results reported here are therefore likely to somewhat underestimate the whole genome variability of BanMMV.

The third functional region analysed in this study, the viral 3' NCR, is usually a highly conserved region of the genome of positive-sense RNA viruses since it frequently contains RNA motifs corresponding to the negative-strand RNA promoter necessary for virus replication. The present results indicate that although this region is more conserved than the RdRp and CP coding regions analysed, it still shows a high variability, including two blocks of extensive indel polymorphism. Gambley & Thomas (2001) postulated that contrary to the situation observed in most positive-sense RNA viruses, the BanMMV negative-strand promoter may be, in part, located further upstream than the 3' NCR, in the 3' end of the CP gene. However, the ACUAAA motif they identified as being possibly involved is not conserved between the isolates of the CP dataset (results not shown) and the region immediately around this putative motif is not significantly more conserved than the whole coding region analysed. It is, therefore, likely that the BanMMV negative-strand promoter is located in the 3' NCR and that the need to maintain a structure and function essential to virus replication accounts for the lower variability observed in that region, compared to the other regions analysed in this study. In this respect, it is worth noting that a 9 bp hairpin (in bold and underlined in Fig. 2) is strongly conserved through co-variation at base-paired positions in almost all the 3' NCR sequences obtained.

When considering intra-plant diversity, two very contrasting situations were encountered: about half (43 %) of the plants analysed yielded only closely related sequence variants, with mean pairwise divergence values below 2–3 %, whereas other plants (57 %) contained two or more highly divergent isolates, resulting in much higher intra-plant diversity values (up to 21·8 % in the case of plant 30). Similar proportions of plants containing only closely related sequences (40 %) or divergent sequences (60 %) were observed when analysing the CP dataset, confirming that co-existence of widely diverging BanMMV isolates in a given Musa genotype is frequent.

The first situation probably reflects the heterogeneous nature of virus populations due to high error rates of viral RNA polymerases. Such a genetic structure comprising a major genotype or ‘master sequence’ and a set of minor variants is well documented for bacterial, animal and plant viruses (Domingo & Holland, 1997; García-Arenal et al., 2001). The second situation, in which highly divergent sequences are simultaneously detected in a single plant, is also frequently observed and might result from two non-exclusive factors: genetic divergence during long-term association of virus isolates with vegetatively propagated banana genotypes or plant-to-plant transfer of virus isolates. Genetic divergence could reflect either a lowering of selection pressures through complementation between co-infecting variants or, alternatively, an increase in positive selection created by the need to escape host defence mechanisms during persistent infections (Simmonds, 2004; Tuplin et al., 2004). Sequence-specific post-transcriptional gene silencing (Voinnet, 2005; Waterhouse et al., 2001), with its ability to counter-select all sequences close to the inducing molecule and, conversely, its inactivity against widely divergent variants, could potentially represent such a driving force in virus evolution. In this respect, it is noteworthy that analysis of the phylogenetic trees built from BanMMV RdRp and CP datasets (not shown) shows that the phylodynamic situation of BanMMV compares to that of other viruses causing persistent infections (Grenfell et al., 2004).

The other possibility to explain the co-existence of widely divergent isolates in a single plant is plant-to-plant transfer of isolates, which has never been reported for BanMMV (Gambley & Thomas, 2001). However, two observations support the existence of horizontal spread of BanMMV isolates in the field. Firstly, identical RdRp sequences were retrieved from two independent pairs of plants. Secondly, the results presented in Fig. 5 are best explained if one postulates the existence of a steep gradient of dispersion of the virus between adjacent plants. These observations do not, however, provide any insight into the mechanism(s) underlying this plant-to-plant transfer, which could represent either an unsuspected consequence of cultural practices or an as yet undescribed vector-borne transmission. Further work is clearly needed to clarify this point.


   ACKNOWLEDGEMENTS
 
The authors thank B. E. Lockhart (University of Minnesota, St Paul, USA) for generously providing the anti-BanMMV serum. The many helpful comments and suggestions of F. Garcia-Arenal, J.-Y. Rasplus, M. Tepfer, S. Blanc and O. Le Gall on the initial drafts of this manuscript are gratefully acknowledged, as well as the help of Kathryn Mayo in improving the English of the manuscript.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Adams, M. J., Antoniw, J. F., Bar-Joseph, M. & 7 other authors (2004). The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation. Arch Virol 149, 1045–1060.[Medline]

Chare, E. R. & Holmes, E. C. (2004). Selection pressures in the capsid genes of plant RNA viruses reflect mode of transmission. J Gen Virol 85, 3149–3157.[Abstract/Free Full Text]

Domingo, E. & Holland, J. J. (1997). RNA virus mutations and fitness for survival. Annu Rev Microbiol 51, 151–178.[CrossRef][Medline]

Domingo, E., Escarmis, C., Sevilla, N., Moya, A., Elena, S. F., Quer, J., Novella, I. S. & Holland, J. J. (1996). Basic concepts in RNA virus evolution. FASEB J 10, 859–864.[Abstract/Free Full Text]

Drake, J. W. & Holland, J. J. (1999). Mutation rates among RNA viruses. Proc Natl Acad Sci U S A 96, 13910–13913.[Abstract/Free Full Text]

Foissac, X., Svanella-Dumas, L., Gentit, P., Dulucq, M.-J., Marais, A. & Candresse, T. (2005). Polyvalent degenerate oligonucleotides reverse transcription-polymerase chain reaction: a polyvalent detection and characterization tool for trichoviruses, capilloviruses, and foveaviruses. Phytopathology 95, 617–625.

Gambley, C. F. & Thomas, J. E. (2001). Molecular characterisation of Banana mild mosaic virus, a new filamentous virus in Musa spp. Arch Virol 146, 1369–1379.[CrossRef][Medline]

García-Arenal, F., Fraile, A. & Malpica, J. M. (2001). Variability and genetic structure of plant virus populations. Annu Rev Phytopathol 39, 157–186.[CrossRef][Medline]

García-Arenal, F., Fraile, A. & Malpica, J. M. (2003). Variation and evolution of plant virus populations. Int Microbiol 6, 225–232.[CrossRef][Medline]

German-Retana, S., Bergey, B., Delbos, R. P., Candresse, T. & Dunez, J. (1997). Complete nucleotide sequence of the genome of a severe cherry isolate of apple chlorotic leaf spot trichovirus (ACLSV). Arch Virol 142, 833–841.[CrossRef][Medline]

Gibbs, A. J., Keese, P. L., Gibbs, M. J. & García-Arenal, F. (1999). Plant virus evolution: past, present and future. In Origins and Evolution of Viruses, pp. 263–285. Edited by E. Domingo, R. Webster & J. J. Holland. San Diego: Academic Press.

Grenfell, B. T., Pybus, O. G., Gog, J. R., Wood, J. L. N., Daly, J. M., Mumford, J. A. & Holmes, E. C. (2004). Unifying the epidemiological and evolutionary dynamics of pathogens. Science 303, 327–332.[Abstract/Free Full Text]

Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[Medline]

Kimura, M. (1983). The Neutral Theory of Molecular Evolution. Cambridge, UK: Cambridge University Press.

Koonin, E. V. (1991). The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J Gen Virol 72, 2197–2206.[Abstract]

Koonin, E. V., Choi, G. H., Nuss, D. L., Shapira, R. & Carrington, J. C. (1991). Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses. Proc Natl Acad Sci U S A 88, 10647–10651.[Abstract/Free Full Text]

Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245.[Abstract/Free Full Text]

Löve, A., Molnegren, V., Månsson, A.-S., Smáradóttir, A., Thorsteinsson, S. B. & Widell, A. (2004). Evolution of hepatitis C virus variants following blood transfusion from one infected donor to several recipients: a long-term follow-up. J Gen Virol 85, 441–450.[Abstract/Free Full Text]

Malpica, J. M., Fraile, A., Moreno, I., Obies, C. I., Drake, J. W. & Garcia-Arenal, F. (2002). The rate and character of spontaneous mutations in an RNA virus. Genetics 162, 1505–1511.[Abstract/Free Full Text]

Nei, M. (1987). Molecular Evolutionary Genetics, pp. 190–191. New York, NY: Columbia University Press.

Nei, M. & Gojobori, T. (1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3, 418–423.[Abstract]

Sawyer, S. (1989). Statistical tests for detecting gene conversion. Mol Biol Evol 6, 526–538.[Abstract]

Shi, B. J., Habili, N., Gafny, R. & Symons, R. H. (2004). Extensive variation of sequence within isolates of grapevine virus B+. Virus Genes 29, 279–285.[CrossRef][Medline]

Simmonds, P. (2004). Genetic diversity and evolution of hepatitis C virus – 15 years on. J Gen Virol 85, 3173–3188.[Abstract/Free Full Text]

Simmonds, N. W. & Shepherd, K. (1955). Taxonomy and origins of cultivated bananas. J Linn Soc Bot 55, 302–312.

Stover, R. H. & Simmonds, N. W. (1987). Bananas, 3rd edn. New York, NY: Longman Scientific and Technical.

Teycheney, P.-Y., Marais, A., Svanella-Dumas, L., Dulucq, M.-J. & Candresse, T. (2005). Molecular characterization of banana virus X (BVX), a novel member of the Flexiviridae family. Arch Virol 150, 1715–1727.[CrossRef][Medline]

Thomas, J. E., Lockhart, B. & Iskra-Caruana, M.-L. (1999). Banana mild mosaic virus. In Diseases of Banana, Abaca and Enset, pp. 275–278. Edited by D. R. Jones. Wallingford, UK: CABI.

Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24, 4876–4882.[CrossRef]

Tuplin, A., Evans, D. J. & Simmonds, P. (2004). Detailed mapping of RNA secondary structures in core and NS5B-encoding region sequences of hepatitis C virus by RNase cleavage and novel bioinformatic prediction methods. J Gen Virol 85, 3037–3047.[Abstract/Free Full Text]

Voinnet, O. (2005). Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6, 206–220.[CrossRef][Medline]

Waterhouse, P. M., Wang, M. B. & Lough, T. (2001). Gene silencing as an adaptive defence against viruses. Nature 411, 834–842.[CrossRef][Medline]

Wetzel, T., Candresse, T., Macquaire, G., Ravelonandro, M. & Dunez, J. (1992). A highly sensitive immunocapture polymerase chain reaction method for plum pox potyvirus detection. J Virol Methods 39, 27–37.[CrossRef][Medline]

Yoshikawa, N., Matsuda, H., Oda, Y., Isogai, M., Takahashi, T., Ito, T. & Yoshida, K. (2001). Genome heterogeneity of Apple stem pitting virus in apple trees. Acta Hortic 550, 285–290.

Received 22 May 2005; accepted 7 July 2005.



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