In recent years there has been a significant increase in the number of new species potentially belonging to the Totiviridae family. Most of these new viruses have not yet been covered by the Committee on Taxonomy of Viruses (ICTV) official classification. In this study, a phylogenetic analysis including new sequences of Totiviridae candidates revealed a clade including Giardiavirus and a great diversity of new totiviruses, which infect arthropods, protozoa and mollusc. This expanded Giardiavirus clade comprises two monophyletic groups, one of them including Giardia lamblia virus (GLV) grouped with viruses that infect arthropods and vertebrates (GLV-like group), and the other includes the previously proposed Artivirus group (IMNV-like group). A screening of the members of the GLV-like group in search of genomic elements already described in IMNV-like group revealed the existence of sites with a high propensity to become 2 A-like oligopeptides, mainly in a specific subgroup of arthropod viruses, suggesting that these viruses preserved ancestral characteristics. The existence of these "pseudo 2 A-sites" associated to phylogenetic reconstruction indicates that these sequences appear at a decisive stage for viral evolution. If they are changed to functional 2 A-like sequences, an irreversible route to increase the genome complexity will be initiated.
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