ABSTRACT: The nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes (proposed order, "Megavirales") include the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Marseilleviridae, and Mimiviridae, as well as still unclassified pithoviruses, pandoraviruses, molliviruses, and faustoviruses. Several of these virus groups include giant viruses, with genome and particle sizes exceeding those of many bacterial and archaeal cells. We explored the diversity of the NCLDV in deep sea sediments from the Loki's Castle hydrothermal vent area. Using metagenomics, we reconstructed 23 high-quality genomic bins of novel NCLDV, 15 of which are related to pithoviruses, 5 to marseilleviruses, 1 to iridoviruses, and 2 to klosneuviruses. Some of the identified pithovirus-like and marseillevirus-like genomes belong to deep branches in the phylogenetic tree of core NCLDV genes, substantially expanding the diversity and phylogenetic depth of the respective groups. The discovered viruses, including putative giant members of the family Marseilleviridae, have a broad range of apparent genome sizes, in agreement with the multiple, independent origins of gigantism in different branches of the NCLDV. Phylogenomic analysis reaffirms the monophyly of the pithovirus-iridovirus-marseillevirus branch of the NCLDV. Similarly to other giant viruses, the pithovirus-like viruses from Loki's Castle encode translation systems components. Phylogenetic analysis of these genes indicates a greater bacterial contribution than had been detected previously. Genome comparison suggests extensive gene exchange between members of the pithovirus-like viruses and Mimiviridae Further exploration of the genomic diversity of Megavirales in additional sediment samples is expected to yield new insights into the evolution of giant viruses and the composition of the ocean megavirome.IMPORTANCE Genomics and evolution of giant viruses are two of the most vigorously developing areas of virus research. Lately, metagenomics has become the main source of new virus genomes. Here we describe a metagenomic analysis of the genomes of large and giant viruses from deep sea sediments. The assembled new virus genomes substantially expand the known diversity of the nucleocytoplasmic large DNA viruses of eukaryotes. The results support the concept of independent evolution of giant viruses from smaller ancestors in different virus branches.
