GBV-A-like viruses share sequence homology in this poten- tial polyprotein initiation region as well. The GBV-Atri sequence is MEVLLVLLLKTALAGA, GBV-Alab sequence is MELLLLLVLLAPAGA and the GBV-A sequence is MASL- WFFVLLLPLGGGG. Until the GBV-D translation initiation codon is identified, it will be difficult to assign precise predicted structural protein sizes. Analysis of the GBV-D polyprotein starting at the first AUG demonstrates four predicted signal peptidase sites in the polyprotein using the AUG at nt 57 as the translation start (Epstein et al., 2010). These are located at amino acid numbers 57–58, 247– 248, 584–585 and 826–827. A 57 aa residue long protein (6 kDa) that is highly basic (pI 12) was proposed as the nucleocapsid or core protein for GBV-D (Epstein et al., 2010), the genome would then have to have an extremely short 59 NTR (57 nt), unless the true 59 end sequence was not identified. Further experimental work is required to determine the genome organization of this newly described virus. Additional differences between the various GB viruses and HCV occur in the extent of predicted glycosylation of the two envelope proteins (E1 and E2). HCV is the most heavily glycosylated, followed by GBV-B, GBV-A and GBV-C/HGV (reviewed by Mohr & Stapleton, 2009). GBV- D is predicted to have 13 glycosylation sites, which would place it similar to GBV-B and HCV. There may be an additional glycoprotein between E2 and NS2 of GBV-D. This region of the polyprotein was called the ‘X’ protein by the group that discovered the virus (Epstein et al., 2010). HCV and GBV-B has a p7 and a p13 protein, respectively, between E2 and NS2 that is essential for their viability (Sakai et al., 2003; Takikawa et al., 2006). The HCV p7 protein is believed to be important for virus assembly and release (Sakai et al., 2003; Lindenbach & Rice, 2005b). It is not known whether GBV-A and GBV-C have a corres- ponding protein. The 39 NTR of HCV and GBV-B contain poly-U tracts, while GBV-A, GBV-C and GBV-D do not (Birkenmeyer et al., 1998; Kim & Fry, 1997; Leary et al., The GB viruses 1996b; Muerhoff et al., 1995). In addition, HCV and GBV- B have highly structured 39 terminal sequences (Lindenbach & Rice, 2005b). Finally, a number of differences occur in the predicted size of the NS proteins; however, with the exception of HCV and GBV-B, the experimental evidence to demonstrate differences is lack- ing. A summary of genome organizational features that differ among the GB viruses and HCV is shown in Table 2. Phylogentic relationships Unweighted pair group method analysis (UPGMA) and neighbour-joining (NJ) analyses are common methods used to generate a single tree, which can be a starting point for evolutionary analysis. Alternatively, a ‘character-based’ approach evaluates the relatedness of sequences based on a subset of positions called ‘informative sites’. Weighted and unweighted Parsimony methods and maximum-likelihood are frequently used approaches that generate multiple trees (cladograms), which can be evaluated for accuracy (Smith et al., 2000). Repeat analyses of a group of sequences in which a proportion of sites are randomly resampled and used for phylogenetic analysis is called ‘bootstrapping’, which estimates the frequency that a particular branch (node) in the tree occurs for each sample. Support for a grouping is considered present when a branch occurs in more than 70 % of 1000 replicates (Smith et al., 2000). Analysis of conserved amino acid sequence motifs involved in the enzymic function of HCV, and GBV-A, GBV-B, GBV-C and GBV-D may elucidate their evolutionary relationship to each other and other members of the family Flaviviridae (Bukh & Apgar, 1997; Muerhoff et al., 1995; Robertson, 2001; Sathar et al., 1999; Schlauder et al., 1995; Smith et al., 2000). The helicase region within NS3 share amino acid sequence identity in six domains (Supplementary Fig. S1, available in JGV Online), while the RdRp protein contains eight conserved motifs (Supplementary Fig. S2, available in JGV Online) (Adams Table 2. Genome features of GB viruses and HCV IRES, Internal ribosome entry site; NC, not classified. GBV-A and GBV-C/HGV share a somewhat longer 59 NTR and lack an apparent coding region for a core protein, 39 NTR polyuridine sequences, and have a lesser amount of predicted envelope protein glycosylation. There are insufficient data available to predict 59 NTR or core protein length at this time for GBV-D. Virus GBV-A GBV-B GBV-C GBV-Ccpz GBV-D HCV Proposal* SPgV GBV HPgV SPgVcpz BPgV HCV 5§ NTR ~540 nt, IRES NC 445 nt, type 3 IRES 555 nt, NC ~ 550 nt, NC Not identified ~340 nt, type 3 IRES Core protein Not identified 156 aa Not identified Not identified Not identified 191 aa Envelope protein glycosylation 3–7 10 4D 3 13D 14–16 3§ NTR No poly U Poly U No poly U No poly U No poly U Poly U-UC *Nomenclature proposed in this review. DPredicted. http://vir.sgmjournals.org 239