Taxonomy, molecular phylogeny and evolution of plant reverse transcribing viruses (family Caulimoviridae) inferred from full-length genome and reverse transcriptase sequences.

This study constitutes the first evaluation and application of quantitative taxonomy to the family Caulimoviridae and the first in-depth phylogenetic study of the family Caulimoviridae that integrates the common origin between LTR retrotransposons and caulimoviruses. The phylogenetic trees and PASC analyses derived from the full genome and from the corresponding partial RT concurred, providing strong support for the current genus classification based mainly on genome organisation and use of partial RT sequence as a molecular marker. The PASC distributions obtained are multimodal, making it possible to distinguish between genus, species and strain. The taxonomy of badnaviruses infecting banana (Musa spp.) was clarified, and the consequence of endogenous badnaviruses on the genetic diversity and evolution of caulimoviruses is discussed. The use of LTR retrotransposons as outgroups reveals a structured bipolar topology separating the genus Badnavirus from the other genera. Badnaviruses appear to be the most recent genus, with the genus Tungrovirus in an intermediary position. This structuring intersects the one established by genomic and biological properties and allows us to make a correlation between phylogeny and biogeography. The variability shown between members of the family Caulimoviridae is in a similar range to that reported within other DNA and RNA plant virus families.

Genetic diversity in the coat protein coding region of eighty-six sugarcane mosaic virus isolates from eight countries, particularly from Cameroon and Congo.

Fifty-eight sugarcane virus isolates were obtained from leaves showing mosaic symptoms, and collected in Cameroon (26 isolates), Congo (20 isolates), Egypt (1 isolate), South Africa (3 isolates) and the U.S.A. (8 isolates). All these isolates belonged to Sugarcane mosaic virus (SCMV) based on the amplification product obtained by RT-PCR with SCMV-specific primers. The amplicons (0.9 kb) from the coat protein (CP) coding region were cloned, sequenced and compared to each other as well as to the sequences (GenBank accessions) of 16 SCMV isolates from sugarcane (Australia, South Africa and U.S.A.) and 12 SCMV isolates from maize (Australia, Germany and China). Maximum likelihood and maximum parsimony analyses robustly supported two major monophyletic groups that were correlated with the host of origin: the SCE or sugarcane group that included all isolates from sugarcane and the MZ or maize group that contained all isolates from maize. The 86 virus isolates were distributed in 13 minor phylogenetic groups, four (I-IV) restricted to maize and nine (V-XIII) to sugarcane. A strong correlation was observed between the sugarcane groups and the geographical origin of the SCMV isolates. Each SCMV type strain from sugarcane (A, B, D, E and SC) was distributed in a different phylogenetic group or subgroup. The 26 isolates from Cameroon constituted a relatively homogeneous group (group V) whereas the 20 isolates from Congo belonged to two other groups (VI and VII). All the isolates from Cameroon and Congo were different from the SCMV type strains and other strains or isolates studied so far. It appears, therefore, that the population of SCMV from sugarcane in Africa contains virus genotypes that have not yet been described.

Occurrence of Potyviruses on Yam (Dioscorea spp.) in Colombia and First Molecular Characterization of Yam mild mosaic virus.

A survey to determine the prevalence of potyviruses on yams, Dioscorea alata and D. cayenensis-rotundata, was undertaken in Colombia. Two hundred fifty leaf samples showing mottling symptoms were collected on the Atlantic coast and analyzed by antigen-coated plate enzyme-linked immunosorbent assay with universal potyvirus monoclonal antibodies (Agdia, Elkhart, IN). Potyviruses were detected in 70% (165/235) of the D. alata and in 66% (10/15) of the D. cayenensis-rotundata samples. The presence of Yam mild mosaic virus (YMMV) was indicated in some of these samples by immunocapture reverse-transcriptase polymerase chain reaction performed as previously reported (1). A 600-bp fragment that included the core and C-terminal region of the coat protein gene (CP) and the 3' untranslated region (3'UTR) was amplified from a D. alata isolate using universal potyvirus primers (1), cloned, and sequenced (EMBL Acc. AJ311725). Comparison with the two previously published YMMV sequences revealed 96.1 and 97.4% identity for the deduced amino acid sequence in the CP region, 74.1 and 83.2% nucleotide identity in the 3'UTR for Papua New Guinea (AB022424 [2]) and Martinique (AJ250336) isolates, respectively. YMMV is known to be widespread on D. alata in Africa and the South Pacific and has been recently identified in the Caribbean (1). To our knowledge, this is the first report of its occurrence in Colombia. A study of its incidence and genetic diversity in South America has been undertaken. References: (1) M. Bousalem and S. Dallot. Plant Disease 84:200, 2000. (2) S. Fuji et al. Arch Virol. 144:1415, 1999.

Molecular variability of geographically distinct isolates of Rice yellow mottle virus in Africa.

The coat protein gene (ORF4) and the 3' untranslated region of a sample of 40 isolates of Rice yellow mottle virus (RYMV), 32 from West Africa and 8 from East Africa, have been sequenced. Five major strains were differentiated, three from West Africa (S1, S2, S3) and two from East Africa (S4, S5), with a spatial overlap of the strains within each of these two regions. Nucleotide and amino-acid divergence between strains was up to 11%. Although more isolates from West African were sequenced, variability was twofold lower than among East African isolates. Variability in ORF4 and in ORF2 coincided. Within strain and within isolate variations in nucleotide sequences were low. Bipartite nuclear targeting motif, Ca2+ binding sites and at least two stretches of amino-acids were conserved among the 40 RYMV isolates and the other sobemoviruses. Variants associating sequence motifs characteristic of different strains have been found, possibly resulting from recombination events. Differences in pathogenicity among isolates were associated with changes of amino-acids in the bipartite nuclear targeting motif of the R domain of the capsid protein, and around conserved positions 151-154 of the S domain. We hypothesise that the observed pattern of variation of RYMV reflects the effect of spatial isolation between East and West Africa coupled with adaptive changes associated to the original virus reservoirs of the different strains.

The use of phylogenetic data to develop molecular tools for the detection and genotyping of Yam mosaic virus. Potential application in molecular epidemiology.

Molecular detection of Yam mosaic virus (YMV), the most important potyvirus for yam plants (Dioscorea spp.) has been limited by its high genetic diversity. According to recent phylogenetic data, suitable molecular tools were developed for detection and genotyping. A reliable and rapid molecular test is described using single-tube immunocapture (IC)-RT-PCR combining the use of a monoclonal antibody able to recognise all YMV serotypes and primers designed to take into account the whole variability of YMV. Phylogenetic data were then used to develop an uncoupled IC-RT-PCR procedure adapted to the genotyping of the three major phylogenetic groups and the two recombinants implicated in the YMV epidemics in the Caribbean and French Guiana. A third exploitation of phylogenetic data was carried out with the development of a typing test based on the analysis of short nucleotide sequences. The direct sequencing of a 450 nts fragment constituted a reliable complementary typing tool as the resulting information was quite similar to that obtained with sequences of the complete coat protein gene. The approach, consisting in a robust study of YMV variability followed by the use of phylogenetic data to develop reliable detection and genotyping tools, offers new perspectives for powerful molecular epidemiological studies.

High genetic diversity, distant phylogenetic relationships and intraspecies recombination events among natural populations of Yam mosaic virus: a contribution to understanding potyvirus evolution.

To evaluate the genetic diversity and understand the evolution of Yam mosaic virus (YMV), a highly destructive pathogen of yam (Dioscorea sp.), sequencing was carried out of the C-terminal part of the replicase (NIb), the coat protein (CP) and the 3'-untranslated region (3'-UTR) of 27 YMV isolates collected from the three main cultivated species (Dioscorea alata, the complex Dioscorea cayenensis-Dioscorea rotundata and Dioscorea trifida). YMV showed the most variable CP relative to eight other potyviruses. This high variability was structured into nine distant molecular groups, as revealed by phylogenetic analyses and validated by assessment of the molecular evolutionary noise. No correlation was observed between the CP and 3'-UTR diversities and phylogenies. The most diversified and divergent groups included isolates from Africa. The remaining groups clustered in a single clade and a geographical distinction between isolates from the Caribbean, South America and Africa was observed. The role of the host in the selection of particular isolates was illustrated by the case of a divergent cultivar from Burkina Faso. Phylogenetic topological incongruence and complementary statistical tests highlighted the fact that recombination events, with single and multiple crossover sites, largely contributed to the evolution of YMV. We hypothesise an African origin of YMV from the yam complex D. cayenensis-D. rotundata, followed by independent transfers to D. alata and D. trifida during virus evolution.

First Report and Molecular Characterization of Yam mild mosaic virus in Dioscorea alata on the Island of Martinique.

Naturally infected Dioscorea alata plants showing mild mosaic were collected in 1998 on the island of Martinique in the Caribbean. Isolates were first screened by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies raised against Yam mosaic virus (YMV) and antigen-coated plate ELISA with universal potyvirus monoclonal antibodies (Agdia, Elkhart, IN). A positive reaction was obtained only with the universal potyvirus antiserum. Immunocapture reverse-transcriptase polymerase chain reaction was performed for specific detection of Yam mild mosaic virus (YMMV [3]) and YMV. A product with the predicted size of 249 bp was obtained with YMMV primers. YMMV is a recently recognized distinct potyvirus infecting D. alata in West Africa and the South Pacific (2-4). It was originally described as Yam virus I and is synonymous with Dioscorea alata virus (4). To characterize the YMMV Martinique isolate, total RNA was extracted, and universal potyvirus degenerate primers (1) were used to amplify a 700-bp fragment that included the core and C-terminal region of the coat protein (CP) and 3' untranslated region (3'UTR). Sequence information generated (EMBL AJ250336) from the cloned fragment was compared with sequences of other yam potyviruses. Sequence comparisons of the partial CP (453 nt) showed a similarity of 94.6% (amino acids [aa]) with the YMMV isolate from Papua New Guinea (EMBL AB022424 [2]); 72.2% (aa) with the Japanese yam mosaic virus (JYMV) isolate (EMBL AB016500); and 67 to 73% (aa) with 27 YMV isolates. These sequences are most diverse in the 3'UTR, which showed a similarity of 72.8% with the YMMV Papua New Guinea isolate, 30% with the JYMV isolate, and 26% with the YMV isolates. These results confirm, as previously shown by S. Fuji et al. (2), that YMMV should be classified as a new potyvirus of yam. This is the first report of the natural occurrence of YMMV in the Caribbean. References: (1) Colinet et al. Phytopathology 84:65, 1994. (2) S. Fuji et al. Arch Virol. 144:1415, 1999. (3) R. A. Munford and S. E. Seal. J. Virol. Methods 69:73, 1997. (4) B. O. Odu et al. Ann. Appl. Biol. 134:65, 1999.