International Laboratory for Tropical Agricultural Biotechnology (ILTAB/IRD-DDPSC), UMSL, Molecular Electronics Bldg, 8001 Natural Bridge Rd, St Louis, MO 63121-4499, USA1
Ekona Research Centre, PMB 25, Buea, South West Province, Cameroon2
Laboratoire de Génétique de l'UFR des Biosciences, Université de Cocody, 22BP582, Abidjan 22, Ivory Coast3
Department of Microbiology, University of Witwatersrand, Johannesburg, South Africa4
Author for correspondence: Claude M. Fauquet. Fax +1 314 516 4582. e-mail iltab{at}danforthcenter.org
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Abstract |
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Introduction |
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The genomes of ACMV and ICMV are composed of two similar-sized DNAs (DNA-A and DNA-B). The complete nucleotide sequences of several isolates of these virus species have been determined [ACMV from Kenya (ACMV/KE) and Nigeria (ACMV/NG) and ICMV] (Stanley & Gray, 1983 ; Morris et al., 1990
; Hong et al., 1993
). Only DNA-A of EACMV has been cloned [from isolates from Tanzania (EACMV/TZ), Uganda (EACMV/UG), Kenya (EACMV/KE) and Malawi (EACMV/MW)] (Deng et al., 1997
; Zhou et al., 1997
, 1998b
).
Zhou et al. (1997) and Deng et al. (1997)
reported a new cassava mosaic virus in Uganda (EACMV/UG1), which is considered to be a natural recombinant between ACMV and EACMV. This virus is associated with the severe Ugandan epidemic of CMD, which is characterized by a severe effect on plants and high populations of whiteflies and is reported to be advancing into hitherto-unaffected areas to the south. CMD in South Africa is caused by yet another distinct virus species, South African cassava mosaic virus (SACMV; Berrie et al., 1998
).
Recently, Fondong et al. (1998) reported the occurrence of unusually severe symptoms of CMD on cassava in Cameroon that are associated with mixed infections of ACMV and EACMV-like species. We report here the complete sequence of components A and B from two viruses associated with CMD in Cameroon. DNA-A of the EACMV-like virus species was found to contain a recombinant fragment originating from an unknown virus species. Similarly, the DNA-B component of the EACMV-like virus species also showed evidence of recombination with a DNA-B component of an EACMV/UG-like virus similar to the one isolated in Uganda (unpublished data).
In nature, mixed virus infections occur in the same plant, with biological and epidemiological implications. Studies have shown that a synergistic interaction between potato virus X (PVX) and potato virus Y (PVY) resulted in enhanced PVX replication and severe necrosis in Nicotiana tabacum leaves (Damirdagh & Ross, 1967 ). Synergism has also been reported between potyviruses and maize chlorotic mottle virus (Tombusviridae; Machlomovirus) (Goldberg & Brakke, 1987
), as well as between the comoviruses cowpea mosaic virus and bean pod mottle virus (Anjos et al., 1992
). Recently, Harrison et al. (1997)
reported the occurrence of ACMV and EACMV in plants with very severe symptoms in Uganda, Tanzania and southern Sudan and suggested the possibility of synergism between the two viruses. We report here, for the first time, molecular evidence for such synergism between two geminiviruses (ACMV/CM and EACMV/CM), in which cassava plants co-infected by the two viruses develop more severe symptoms in the field and in the growth chamber compared with plants infected by either virus alone.
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Methods |
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PCR.
Total DNA was extracted from young cassava leaves that showed CMD symptoms as described by Dellaporta et al. (1983) . Oligonucleotide primers used to amplify the viral DNA are listed in Table 1
. PCR was performed with the GIBCO BRL kit as recommended by the manufacturer. The PCR conditions for coat protein (CP) gene amplification were 2 min at 94 °C and then 30 cycles of 1 min at 94 °C, 1 min at 45 °C and 1 min at 72 °C followed by a final extension period of 10 min at 72 °C. A 700 bp fragment of the 5' end and the intergenic region of EACMV/CM BC1 was amplified by using degenerate primers (Table 1
) designed from published sequences of B components of begomoviruses. The PCR conditions for amplifying this fragment were similar to those used for amplifying the CP gene except that the annealing temperature was 58 °C.
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Sequence determination and analysis.
Full-length DNA PCR products were recovered after electrophoresis in a 1% agarose gel and purified with the BIO 101 Geneclean kit. The products were digested with appropriate restriction enzymes and cloned into pBluescript II KS(+) (Stratagene). ACMV/CM DNA-A was cloned into pCR II, isolated after digestion with EcoRI and cloned into pBluescript II KS(+). After multiplication in Escherichia coli strain TOP10F' (Invitrogen), plasmids were purified by using the Qiagen DNA purification kit. Both strands of DNA were sequenced after subcloning and/or the use of specific primers by using the ABI Prism dRhodamine terminator cycle sequencing ready reaction kit (ABI/Advanced Biotechnology). Nucleotide and amino acid sequences were assembled and analysed with the DNASTAR package.
The source and GenBank accession numbers of geminivirus DNA-A and -B sequences used in this paper were: ACMV/KE (J02057, J02058); ACMV/NG (X17095, X17096); Althea rosea enation virus (AREV) (AF014881); chayote mosaic virus (ChaMV) (AJ223191); cotton leaf curl virus from Pakistan (CLCuV-PK1/Fai1) (AJ222703); EACMV/KE (AJ006458); EACMV/MW [AJ006461 (CP)]; EACMV/MW/K (AJ006460); EACMV/MW/MH (AJ006459); EACMV/TZ (Z83256); EACMV/UG//1 (Z83257); EACMV/UG//Svr [AF126807 (DNA-B)]; ICMV (Z24758, Z24759); potato yellow mosaic virus from Trinidad and Tobago (PYMV/TT) (AF039032, AF039033) and from Venezuela (PYMV/Ve) (D00940, D00941); and tomato yellow leaf curl virus from Israel (TYLCV-IL) (X15656) and from Sardinia (TYLCV-Sar) (X61153).
Determination of synergism between ACMV/CM and EACMV/CM.
Cassava plants infected by ACMV/CM and EACMV/CM manifested unusually severe symptoms. In order to determine the occurrence of a synergistic interaction between the two virus species, infected cassava samples and cloned viral DNA were used to inoculate 21-day-old seedlings of Nicotiana benthamiana.
(i) Inoculation with plant sap.
Cassava samples infected by ACMV/CM or EACMV/CM alone or by both viruses were used to inoculate five N. benthamiana seedlings mechanically. A fourth treatment was obtained by mixing sap from the same samples infected by ACMV/CM and EACMV/CM alone to obtain a doubly infected sample. Sap was obtained from the leaves of the plants at a dilution of 1:10 (w/v) in a 0·1 M phosphate buffer, pH 7·0. Infected plants were grown in the growth chamber at 24 °C and 14 h photoperiod and the symptoms were noted.
(ii) Construction of infectious clones and inoculation of N. benthamiana.
Total DNA was extracted from leaf tissue of the N. benthamiana plants inoculated with ACMV/CM and EACMV/CM. Supercoiled DNA was separated from other nucleic acids on a 1·2% agarose gel. The fragments between 1·6 and 2·0 kb apparent length were isolated, cleaved with BamHI and HindIII and then cloned into pUC18. Restriction mapping and sequence comparison established that clones pVF.AA, pVF.AB and pVF.EA contained ACMV DNA-A, ACMV DNA-B and EACMV DNA-A (referred to as AA, AB and EA), respectively. Head-to-tail partial repeats of these clones were constructed as described by von Arnim & Stanley (1992) . EACMV/CM DNA-B could not be cloned directly and was amplified by using primers EB03 and EB04 (Table 1
). A partial repeat of EACMV DNA-B (EB) could not be constructed because the HpaI site used to clone it occurs three times in the sequence; therefore a monomer was used in the inoculation experiments.
Three combinations of cloned DNAs were inoculated to N. benthamiana mechanically (2 µg per plant) and by the biolistic method (200 ng per plant) as described by Gilbertson et al. (1991) . The combinations were AA+AB, EA+EB and AA+AB+EA+EB.
In order to quantify and compare accumulation of viral DNAs in the singly and doubly infected plants, total DNA was extracted from inoculated N. benthamiana plants as described by Dellaporta et al. (1983) . DNA was further deproteinized by phenolchloroform extraction followed by extraction with chloroform alone. The DNA (5 or 10 µg per well) was separated on ethidium bromide-stained, 1·2% agarose gels in 1x TAE buffer and blotted onto Hybond-N+ membranes (Amersham).
The probes used for ACMV/CM were the fragments EcoRIBamHI (nt 1714140) for DNA-A and BamHIEcoRV (nt 13332402) for DNA-B. For EACMV/CM, the fragments were HindIIIEcoRI (nt 9241684) for DNA-A and a 700 bp fragment covering the region from the intercistronic region (ICR) to the 5' terminus of BC1 from cloned PCR products for EACMV/CM DNA-B. The nucleotides are numbered from the nucleotide A* of the TAATATTA*C nonanucleotide of each sequence. The probes were labelled with [32P]dATP by random priming, as described by Sambrook et al. (1989) . The intensity of bands was quantified by using a Bio-Rad phosphorimager.
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Results |
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Analysis of the complete nucleotide sequences of DNA-A and -B of ACMV/CM and EACMV/CM
The complete nucleotide sequences of clones pVF-AA5 (ACMV/CM DNA-A), pVF-EA6 (EACMV/CM DNA-A), pVF-AB1 (ACMV/CM DNA-B) and pVF-EB1 (EACMV/CM DNA-B) were determined. The arrangement of the ORFs in each molecule was similar to that of Old World begomoviruses.
A comparison of the nucleotide sequences of both components of the two viruses and their common regions is shown in Table 2. ACMV/CM DNA-A was nearly identical (9697%) to that of ACMV/KE and ACMV/NG, whereas EACMV/CM exhibited 84, 81 and 76% sequence identity to EACMV/TZ, EACMV/UG//1 and EACMV/MW/K, respectively. A fragment of approximately 900 nucleotides (30% of the DNA-A genome), comprising AC2 and AC3 of EACMV/CM, was very different from EACMV/TZ. This segment had 69% identity to the corresponding segment of EACMV/TZ, whereas the remainder of DNA-A exhibited 90% identity between the two viruses.
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Sequence homologies of ACMV/CM- and EACMV/CM-encoded proteins
A complete sequence analysis of virus-encoded products is presented in Table 2. There was considerable variation between the amino acid sequence of the CP of EACMV/CM and those of other Old World begomoviruses. The EACMV/CM CP amino acid sequence showed very high similarity to those of EACMV/TZ (94%) and an EACMV from Malawi (95%) (EACMV/MW), but less similarity to those of SACMV (75%), EACMV/MW//K (87%) and ACMV (6672%). In contrast, there was high amino acid sequence similarity (97%) between the CP of ACMV/CM and the other ACMV CP sequences cited in this paper.
As is the case for the CP, the replicase (Rep) sequence is also very conserved within a virus species. The Rep sequence was highly conserved between EACMV/CM, EACMV/TZ and EACMV/UG and EACMV/MW//K (91%), but not between EACMV/CM and CLCuV-PK1/Fai1 (75%) or ACMV (6869%). The Rep sequences of ACMV/CM and those of other ACMVs were nearly identical (9698%) compared with EACMV sequences (68%).
Sequence similarities between the gene regulation proteins of EACMV/CM, AC2 and AC3, and those of other EACMV were low (5865%), and were comparable to the similarity of the corresponding sequences between EACMV/CM and isolates of ACMV (5560%). This low value indicates that AC2 and AC3 are part of the 900 nucleotide fragment of EACMV/CM that is probably a result of a recombination with an as yet undetermined virus species. No sequence from the database with significant homology to this fragment is yet available. Sequence comparisons showed that AC4 was the least-conserved gene, with similarities between the different virus species ranging from 28 to 55% (Table 2).
To determine the homogeneity of distribution of EACMV/CM, two pairs of primers were designed from the unrelated sequence and from the corresponding region of EACMV/TZ (Table 1). The PCR results showed that all eight samples infected by ACMV/CM and EACMV/CM contained a similar fragment (data not shown). In contrast, primers specific to EACMV/TZ for the same region did not amplify the corresponding fragment in any of the eight samples, indicating the absence of EACMV/TZ.
The two B-component proteins, BV1 and BC1, were highly conserved among the three ACMV isolates (97 and 93% similarity, respectively). In contrast, there was very low similarity (53%) between BC1 of ACMV/CM and that of EACMV/CM (Table 2).
The common regions (CRs) of ACMV/CM and EACMV/CM
Sequence identities between the CRs of ACMV/CM and other ACMV isolates were high: there was 9196% identity to ACMV/KE and 9497% identity to ACMV/NG, compared with 4245% to EACMV, ICMV and SACMV isolates (Table 2). The CR of the DNA-A of EACMV/CM also showed high sequence identity to ICRs from EACMV/UG (99%) and EACMV/TZ (90%) and intermediate identity to SACMV (64%) and TYLCV-IL (59%), but very low identity to all the other viruses including ACMVs.
The CRs of the two components of ACMV/CM were 170 nucleotides long with a sequence identity of 92%, while those of EACMV/CM were 150 nucleotides long with only 80% identity. The comparatively low sequence identity in the CRs of EACMV DNA-A and -B is due to a 28 nucleotide segment of EACMV DNA-B that appears to be a deletion of 23 nucleotides and an insertion of 28 nucleotides at the 5' end of the conserved hairpin-loop motif (Fig. 2a). When the 28 nucleotide sequence was not considered, the two CR sequences were 93% identical. In addition to the conserved 30-mer of the hairpin region, common to all geminiviruses sequenced to date, the putative Rep-binding site motifs upstream of the TATA box were GGTGGAATGGGGG for both components of EACMV/CM (Fig. 2a
). This motif is similar to that reported by Zhou et al. (1998b
) in EACMV isolates from eastern Africa, but is different from the motifs of EACMV/MW//MH (GGGGGAACGGGGG) (AJ006459) and SACMV (GGGGGGATGGGGG). In ACMV/CM, as in other ACMV isolates cited in this article, the repeated motif in both components was TGGAGACA.
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To confirm that the synergism observed was due to co-infection by ACMV/CM and EACMV/CM, cloned components of these virus species were inoculated to N. benthamiana plants. As observed in sap inoculations, plants inoculated with both components of the two virus species, i.e. AA+AB+EA+EB, displayed more severe symptoms than plants inoculated with the two components of either virus alone (Fig. 1e; Table 3
). Although symptoms were not observable on plants inoculated with cloned components of EACMV/CM, Southern blot analyses detected low levels of both components in the inoculated plants (Fig. 3b
).
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Unlike ACMV/CM, the accumulation of EACMV/CM DNA-A was similar or only slightly increased in doubly infected N. benthamiana plants compared with singly infected plants at 21 and 60 days p.i. (1·6- and 3·1-fold increases, respectively) (Fig. 3a, lanes EA). DNA-B accumulation was lower or similar in mixed-infected plants (Fig. 3a
; lanes EB). With cloned DNAs, we observed a similar trend but with a smaller increase for EACMV/CM; 1·2- and 3·8-fold, respectively, for 24 and 50 days p.i. The accumulation of EACMV/CM component B was barely detectable on the blots.
The results of Southern blot analyses conducted with total DNA from infected cassava plants showed that there were higher levels of accumulation of ACMV/CM DNA-A (2·2-fold) and, to a lesser extent, of DNA-B (1·4-fold) in doubly infected cassava plants compared with singly infected plants. The accumulation of EACMV/CM was very high for both DNA components compared with ACMV/CM and the increase in doubly infected plants was greatest for component B (5·7-fold) (Fig. 3c; lanes EA and EB).
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Discussion |
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The EACMV/CM DNA-A component is about 84% identical to the EACMV DNA-A components from East Africa cited in this paper and, therefore, could be considered to be a strain of EACMV with a recombinant fragment of 900 nucleotides from an as yet undetermined virus. However, only 20% of the DNA-B sequence, located in the BC1 gene, identified in cassava mosaic virus-infected samples from Uganda (EACMV/UG//Svr; AF126807) was homologous to EACMV/CM DNA-B. The rest of the sequence, with the exception of cis-elements in the 150 nucleotide CR, was unrelated to EACMV/UG//Svr DNA-B.
Evidence that the B component reported in this paper belongs to EACMV/CM is provided by the fact that there was a common region between EACMV/CM DNA-A and DNA-B with 80% sequence identity. Although sequence similarity between the two components is comparatively low, they have the same cis-elements in their CRs and the differences consist of a substitution between the TATA boxes and the hairpin loops (Fig. 2a). The same differences between the two CRs were also detected in a similar virus cloned in the Ivory Coast (EACMV/CM//IC; J. S. Pita, unpublished results). Moreover, all of the cassava samples in which EACMV/CM DNA-A was detected also contained the same DNA-B component, but it was absent from the samples infected by ACMV/CM alone, stressing that this B component belongs to EACMV/CM.
Evidence that the two components of EACMV/CM resulted from recombination events was obtained from the GENECONV program. This program finds high-scoring aligned segment pairs of sequences for the detection of recombination in geminiviruses (Padidam et al., 1999 ). Recombination events in EACMV/CM are located in the AC2AC3 and BC1 coding sequences and are therefore different from the recombination within the CP sequence in EACMV/UG (Zhou et al., 1997
; Deng et al., 1997
).
Zhou et al. (1998a) reported recombinations between cotton leaf curl viruses and a geminivirus isolated from okra (CLCuV-Ok; AJ002459). Umaharan et al. (1998)
also reported recombinations in the two components of PYMV/TT, between PYMV/Ve and an unknown virus. There is also evidence that beet curly top virus (BCTV), from the genus Curtovirus, might have resulted from recombination between two species belonging to two different genera, a leafhopper-transmitted mastrevirus and a whitefly-transmitted begomovirus. The BCTV CP has similarities to those of the mastreviruses, whereas the rest of the genome is begomovirus-like in sequence (Stanley et al., 1986
; Padidam et al., 1995
, 1999
).
The data suggest that EACMV/CM is a mosaic of DNA fragments originating from at least two different species (Fig. 3b). Since the sequences of B components from other EACMV isolates from East Africa have not yet been determined, we cannot justifiably classify EACMV/CM as a separate virus species. For now, it can appropriately be classified as a virus related by sequence to EACMV strains from East Africa. Once the B component sequences of most EACMV strains from East Africa have been determined, we will revisit the taxonomic status of this virus. It appears that geminiviruses are generated by recombination of stretches of DNA originating from different viruses present in the same hosts (Fig. 3b
). So far, it seems that a recombination event in DNA-A always leads to another recombination event in the corresponding B component, the driving force being the necessity to have the same cis-elements necessary for replication of the DNA-B component. This is the case for PYMV/TT, PYMV/Ve and PYMV from Panama (PYMV/Pa; Y15034) (Umaharan et al., 1998
). EACMV/CM is the first case of a virus with most of its DNA-A and DNA-B probably originating from two different virus species; it is therefore a true trans-complement hybrid.
In this article, we report the first evidence of synergism between two geminiviruses that results in an increase in viral DNA and symptom severity. Southern blot analyses indicated that doubly infected cassava and N. benthamiana plants had higher levels of accumulation of DNA-A and -B of ACMV/CM during the early stages of infection, compared with plants infected with ACMV/CM alone. The level of EACMV/CM was only higher in mixed infections, especially in late stages of infection and particularly in naturally doubly infected cassava plants. These data were confirmed by using infectious clones of ACMV/CM and EACMV/CM in N. benthamiana. However, there was poor infectivity of EACMV/CM in N. benthamiana, particularly the B component (Fig. 3b; lanes EB). It is possible that, even though EACMV/CM is capable of infecting N. benthamiana, the latter is not an adaptable host compared with cassava (Fig. 1c
, d
). This is also supported by the difficulty of infecting N. benthamiana from other N. benthamiana plants (Swanson & Harrison, 1994
; our unpublished observations). The poor infectivity of EACMV/CM DNA-B is probably the result of the use of a monomeric and not a dimeric clone.
This type of synergism is similar to that observed with potyviruses, which mediate the accumulation of potex-, como-and machlomoviruses, but levels of which remain relatively unchanged during mixed infections (Damirdagh & Ross, 1967 ; Calvert & Ghabrial, 1983
; Goldberg & Brakke, 1987
; Vance, 1991
; Anjos et al., 1992
; Vance et al., 1995
). However, in the synergism between ACMV/CM and EACMV/CM, there were increases in the DNA accumulation of both viruses in mixed infections. Preliminary results suggest that there is trans-complementation between ACMV/CM and EACMV/CM; confirmation of these results could partly explain the synergism observed in this study.
The results of DNA hybridization correlated with symptom severity in cassava and in N. benthamiana, since doubly infected cassava and N. benthamiana showed more severe symptoms throughout the period of observation than plants infected by either virus alone (Fig. 1bd).
Unlike the previous cases of synergistic infections, both ACMV/CM and EACMV/CM belong to the same genus and are transmitted by the same whitefly vector and therefore will more likely co-infect the same plant than if they were spread through different modes. Consequently, doubly infected plants have considerable potential as sources of inoculum for both viruses and whiteflies feeding on such plants would, therefore, more easily acquire and transmit both viruses to virus-free plants.
There are epidemiological implications of double infections of ACMV/CM and EACMV/CM. As indicated above, plants with mixed infections of ACMV/CM and EACMV/CM exhibited symptoms on all the leaves. In contrast, plants infected by ACMV/CM or EACMV/CM alone showed incomplete systemic infection and some leaves remained symptomless. Fargette et al. (1994) reported that disease-free cuttings could be recovered from symptom-free areas of cassava plants. This suggests that doubly infected plants would have a lower proportion of disease-free cuttings than singly infected plants. Moreover, we have observed that reversion is inversely proportional to symptom severity (Fondong et al., 2000
) and, therefore, doubly infected plants will be less likely to revert than singly infected plants. However, such plants would probably not be used for cuttings, as they would be too stunted to be chosen for multiplication. The spread of EACMV/CM will therefore depend, to a large extent, on transmission by whitefly. It is obvious that an increase in viral DNA content by 3- to 11-fold will dramatically increase the percentage of transmission of the viruses by whitefly.
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Acknowledgments |
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Footnotes |
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References |
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Received 16 June 1999;
accepted 28 September 1999.