Pfam searches obviously will only identify known domain types in protein sequences. In order to identify potential similarities in regions that were not recognised by Pfam, systematic PSI-Blast searches were performed, using the polyprotein regions between the MT and HEL domains and searching against the NCBI database of reference sequences [15] , excluding all viral entries. A maximum of 5 PSI-Blast iterations were allowed, with an inclusion threshold of 0.005. The expected homologues of the AlkBdomain were found with high confidence, as most of the E-values were < 1 × 10 -50 . Homologues of typical viral domains like the viral peptidases were obviously not found, as all viral database entries were excluded. Very few Multiple alignment of sequence regions corresponding to the AlkB domains Figure 3 Multiple alignment of sequence regions corresponding to the AlkB domains. The alignment was generated with ClustalX. The residues involved in coordination of the essential Fe 2+ ion are completely conserved, except in one of the Vitivirus sequences. These residues are the HxD motif, a single H, and the first R in the RxxxxxR motif. The function of the remaining conserved residues is unclear, but at least some of them may be involved in coordination of the substrate [10] . Pairwise distances between sequence regions corresponding to methyltransferase (MT), RdRp and AlkB domains. Each data point corresponds to e.g. RP-RP and MT-MT distances for the same pair of sequences, and sequences showing similar evolutionary distance in these two regions will fall on the diagonal. The pairwise distances were estimated from multiple alignments using the Blosum50 score matrix [47] . Trend lines were estimated with Excel. The trend line for AlkB vs. RdRp is heavily influenced by the point at (675, 670). It represents two Foveavirus sequences (NCBI gi3702789 and gi9630738), they are 98% identical over the full polyprotein sequence. Alignment score (AlkB) Alignment score (RdRp) r 2 = 0.10 new similarities were found by these searches. Pepper ringspot virus (Tobravirus, NCBI gi20178599) showed significant similarity to site-specific DNA-methyltransferase from Nostoc sp (E = 1 × 10 -74 ), as well as other cytosine 5Cspecific DNA methylases. Bamboo mosaic virus (Potexvirus, NCBI gi9627984) showed similarity to aggregation substance Asa1 from Enterococcus faecalis (E = 6 × 10 -34 ). A small number of additional similarities seemed to be caused by biased sequence properties (e.g. proline-rich regions), and were probably not significant. This included matches against mucin and cadherin-like proteins from Homo sapiens and multidomain presynaptic cytomatrix protein (piccolo) from Rattus norvegicus. In general the variable regions seemed to be truly variable, with very little similarity to other proteins, except for the Pfam domains already identified.