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Abstract The microbial sampling of submarine hydrothermal vents remains challenging, with even fewer studies focused on viruses. Here we report what is to our knowledge the first isolation of a eukaryotic virus from the Lost City hydrothermal field, by co-culture with the laboratory host Acanthamoeba castellanii. This virus, named pacmanvirus lostcity, is closely related to previously isolated pacmanviruses (strains A23 and S19), clustering in a divergent clade within the long-established family Asfarviridae. The icosahedral particles of this virus are 200 nm in diameter, with an electron-dense core surrounded by an inner membrane. The viral genome of 395 708 bp (33% G + C) has been predicted to encode 473 proteins. However, besides these standard properties, pacmanvirus lostcity was found to be associated with a new type of selfish genetic element, 7 kb in length, whose architecture and gene content are reminiscent of those of transpovirons, hitherto specific to the family Mimiviridae. As in previously described transpovirons, this selfishg genetic element propagates as an episome within its host virus particles and exhibits partial recombination with its genome. In addition, an unrelated episome with a length of 2 kb was also found to be associated with pacmanvirus lostcity. Together, the transpoviron and the 2-kb episome might participate in exchanges between pacmanviruses and other DNA virus families. It remains to be elucidated if the presence of these mobile genetic elements is restricted to pacmanviruses or was simply overlooked in other members of the Asfarviridae.