Giant viruses are protist-associated viruses belonging to the proposed order Megavirales; almost all have been isolated from Acanthamoeba spp. Their isolation in humans suggests that they are part of the human virome. Using a high-throughput strategy to isolate new giant viruses from their original protozoan hosts, we obtained eight isolates of a new giant viral lineage from Vermamoeba vermiformis, the most common free-living protist found in human environments. This new lineage was proposed to be the faustovirus lineage. The prototype member, faustovirus E12, forms icosahedral virions of Ϸ200 nm that are devoid of fibrils and that encapsidate a 466-kbp genome encoding 451 predicted proteins. Of these, 164 are found in the virion. Phylogenetic analysis of the core viral genes showed that faustovirus is distantly related to the mammalian pathogen African swine fever virus, but it encodes Ϸ3 times more mosaic gene complements. About two-thirds of these genes do not show significant similarity to genes encoding any known proteins. These findings show that expanding the panel of protists to discover new giant viruses is a fruitful strategy. IMPORTANCEBy using Vermamoeba, a protist living in humans and their environment, we isolated eight strains of a new giant virus that we named faustovirus. The genomes of these strains were sequenced, and their sequences showed that faustoviruses are related to but different from the vertebrate pathogen African swine fever virus (ASFV), which belongs to the family Asfarviridae. Moreover, the faustovirus gene repertoire is Ϸ3 times larger than that of ASFV and comprises approximately two-thirds ORFans (open reading frames [ORFs] with no detectable homology to other ORFs in a database). G iant viruses were first described in 2003, with the discovery of Acanthamoeba polyphaga mimivirus (1, 2). They are protistassociated viruses that belong to a major monophyletic group of double-stranded DNA (dsDNA) viruses known as nucleocytoplasmic large DNA viruses (NCLDVs), and they have been classified under the proposed order Megavirales (3). Since the first description of Acanthamoeba polyphaga mimivirus, giant viruses have been isolated from other phagocytic protists, primarily Acanthamoeba spp. (4-6). Because these giant viruses are resistant to killing by phagocytic protists, we hypothesized that they may also reproduce in macrophages and might therefore infect humans. This proposition was validated experimentally by the isolation of mimivirus from atypical pneumonia patients and by the detection of marseilleviruses in blood donors and in human lymph nodes (7-9). Moreover, we and others identified sequences associated with giant viruses in metagenomes generated from human tissues, suggesting that giant viruses are a component of the human virome (10). Because the investigation of a virome typically starts with a filtration procedure that eliminates giant viruses (11), we developed a new culture approach that does not prevent the detection of these viruses.In the present study, we de...