J. T. Stapleton and others Fig. 2. Helicase and RdRp phylogenetic trees of selected members of the family Flaviviridae. UPGMA phylogenetic trees of helicase and RdRp were generated using the alignments shown in Supplementary Figs S1 and S2, respectively. GenBank numbers for all isolates used in these analyses are provided in the legend for Supplementary Fig. S1. The node numbers represent the bootstrap values (expressed as a percentage of all trees) obtained from 2000 replicates. The tree was rooted by using the midpoint of the longest branch. A distance scale in amino acid substitutions per position is shown. et al., 1998; Birkenmeyer et al., 1998; Bukh & Apgar, 1997; Gorbalenya & Koonin, 1989; Koonin, 1991; Koonin & Dolja, 1993; Leary et al., 1996b; Linnen et al., 1996; Muerhoff et al., 1995; Robertson, 2001; Simons et al., 1995a; Smith et al., 2000). Alignments were performed by hand, based on the alignments of Koonin et al. that were used as a guide (Gorbalenya & Koonin, 1989; Koonin & Dolja, 1993). Helicase sequences of members of the family Flaviviridae fall within the helicase supergroup II, and their RdRp sequences place them into the RdRp supergroup II. UPGMA analysis of the six conserved helicase domains and eight conserved RdRp motifs with their intervening sequences of HCV, GBV-A, GBV-B, GBV-C and GBV-D 240 confirm that these viruses are related to the Pestivirus and Flavivirus genera within the family Flaviviridae (Fig. 2). GBV-A, GBV-C and GBV-D group together, and GBV-B groups with HCV, consistent with the proposed assign- ment as a second species within the genus Hepacivirus. NJ analysis of the helicase and RdRp sequences including the intervening sequences between the conserved domains identified similar genetic relationships (Fig. 3), as did tree methods using CLUSTAL alignments of these sequences (data not shown). Despite the agreement between trees con- structed by the two methods used here, final assignment into specific genera should also consider genome structure, tissue tropism and pathogenesis. Journal of General Virology 92