Duffy S, Holmes E.. Validation of high rates of nucleotide substitution in geminiviruses: phylogenetic evidence from East African cassava mosaic viruses. J Gen Virol 90: 1539-1547

Center for Infectious Disease Dynamics, Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Journal of General Virology (Impact Factor: 3.18). 04/2009; 90(Pt 6):1539-47. DOI: 10.1099/vir.0.009266-0
Source: PubMed


Whitefly-transmitted geminiviruses are major pathogens of the important crop cassava in Africa. The intensive sampling and sequencing of cassava mosaic disease-causing viruses that occurred in the wake of a severe outbreak in Central Africa (1997-2002) allowed us to estimate the rate of evolution of this virus. East African cassava mosaic virus and related species are obligately bipartite (DNA-A and DNA-B segments), and these two genome segments have different evolutionary histories. Despite these phylogenetic differences, we inferred high rates of nucleotide substitution in both segments: mean rates of 1.60x10(-3) and 1.33x10(-4) substitutions site(-1) year(-1) for DNA-A and DNA-B, respectively. While similarly high substitution rates were found in datasets free of detectable recombination, only that estimated for the coat protein gene (AV1), for which an additional DNA-A sequence isolated in 1995 was available, was statistically robust. These high substitution rates also confirm that those previously estimated for the monopartite tomato yellow leaf curl virus (TYLCV) are representative of multiple begomoviruses. We also validated our rate estimates by comparing them with those depicting the emergence of TYLCV in North America. These results further support the notion that geminiviruses evolve as rapidly as many RNA viruses.

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    • "If more recently sampled sequences have undergone more molecular evolution, then the true sampling dates should yield a t MRCA that differs substantially from the equivalent estimates with the sampling dates randomly permuted over sequences (Ramsden, Holmes & Charleston 2009; e.g. Duffy & Holmes 2009; Firth et al. 2010; Fraile et al. 2011; Pag an & Holgu ın 2013; Duch^ ene, Holmes & Ho 2014b; Duch^ ene et al. 2015a). Finally, a distinct approach uses model selection and compares the fit of models with the sampling dates included or excluded, thereby failing to take special account for any evolution that might have taken place during the sampling period (Rambaut 2000; Drummond, Pybus & Rambaut 2003b; Drummond et al. 2003a; Baele et al. 2012). "
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    ABSTRACT: 1. ‘Dated-tip’ methods of molecular dating use DNA sequences sampled at different times, to estimate the age of their most recent common ancestor. Several tests of ‘temporal signal’ are available to determine whether data sets are suitable for such analysis. However, it remains unclear whether these tests are reliable. 2. We investigate the performance of several tests of temporal signal, including some recently suggested modifi- cations. We use simulated data (where the true evolutionary history is known), and whole genomes of methicillin-resistant Staphylococcus aureus (to show how particular problems arise with real-world data sets). 3. We show that all of the standard tests of temporal signal are seriously misleading for data where temporal and genetic structures are confounded (i.e. where closely related sequences are more likely to have been sampled at similar times). This is not an artefact of genetic structure or tree shape per se, and can arise even when sequences have measurably evolved during the sampling period. More positively, we show that a ‘clustered permutation’ approach introduced by Duchêne et al. (Molecular Biology and Evolution, 32, 2015, 1895) can successfully correct for this artefact in all cases and introduce techniques for implementing this method with real data sets. 4. The confounding of temporal and genetic structures may be difficult to avoid in practice, particularly for outbreaks of infectious disease, or when using ancient DNA. Therefore, we recommend the use of ‘clustered permutation’ for all analyses. The failure of the standard tests may explain why different methods of dating pathogen origins have reached such wildly different conclusions.
    Methods in Ecology and Evolution 09/2015; DOI:10.1111/2041-210X.12466 · 6.55 Impact Factor
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    • "In contrast, the relative ease with which the highly heritable monogenic resistance can be transferred between germplasm through simple crosses, has resulted in its extensive usage in breeding across Africa as well as pre-emptive breeding in Latin America (Okogbenin et al., 2007). The long-term stability of this single-gene type of resistance in diverse geographical regions with heterogeneous species and recombinants of CMGs is uncertain given the high evolutionary rate of geminiviruses (Duffy and Holmes, 2009). "

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    • "Since host RDR activity may amplify CaLCuV siRNAs and thereby contribute to the observed deviations from the CaLCuV master genome, we compared the viral microvariant accumulation in CaLCuV-infected wild-type plants and rdr1/2/6 triple mutant plants with diminished RDR activities [17]: no drastic difference was observed in the frequency of SNPs or the average degree of deviation from the master genome nucleotides (Dataset S3C-D). Our findings for CaLCuV are consistent with the observations that geminiviruses have high mutation frequency and evolve as fast as RNA viruses (see [23] and references therein). "
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    ABSTRACT: Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.
    PLoS ONE 02/2014; 9(2):e88513. DOI:10.1371/journal.pone.0088513 · 3.23 Impact Factor
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