Full Length Genome Analysis

To test the validity of our results, we sequenced the full genome of isolates (Fig. 3b) spanning the range of the newly-characterized clades (accession numbers AJ584844-AJ584849). Alignments of these sequences showed a level of similarity of approximately 75% (see Table 3 in Radjef et al. 2004), in the same range as that observed between type I and type II sequences, suggesting the existence of additional genotypes. Because different values of GOP and GEP gave different results, full-genome alignments were performed using the SOAP program (Loytynoja and Milinkovitch 2001). Using this program, positions with different alignments can be excluded, or a proportion of GOP and GEP combinations yielding the same alignment can be studied. We also used ProAlign (Loytynoja and Milinkovitch 2003), a program that provides a statistical approach to multiple sequence alignment, such that a posterior probability is assigned to each aligned position. Positions with a posterior probability below a user-defined threshold (here <90%) can be excluded before phylogeny inference.

MP (usingbranch-and-bound) and NJ (ML distances) phylogenetic analyses were performed with PAUP*4.0b10 (Swofford 1998). ML analyses were carried out both with PAUP*4.0b10 (full-length and sHD data sets) and the metapopulation genetic algorithm (metaGA) (Lemmon and Milinkovitch 2002) implemented in the program MetaPIGA (www.ulb.ac.be/sciences/ueg). Bootstrap analyses (103, 104 and 102 replicates for PAUP MP, NJ and ML, respectively) and 10000 metaGA samples (2500 replicates with four populations, ML analyses) were performed to assess the robustness and posterior probabilities of nodes, respectively. We used the HKY model and estimated transition:transversion ratio, proportion of invariable sites, and rate heterogeneity (gamma distribution with four categories) parameters from the data.

These analyses yielded results (Fig. 1b) similar to those described above (Fig. 1a): the genetic variability of the HDV genus has more major mono-phyletic groups (i.e., clades) than previously thought.

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