ABSTRACT: Since 2003, various viruses from the subfamily Megavirinae in the family Mimiviridae have been isolated worldwide, including icosahedral mimiviruses and tailed tupanviruses. To date, the evolutionary relationship between tailed and nontailed mimiviruses has not been elucidated. Here, we present the genomic and morphological features of a newly isolated giant virus, Cotonvirus japonicus (cotonvirus), belonging to the family Mimiviridae. It contains a linear double-stranded DNA molecule of 1.47 Mb, the largest among the reported viruses in the subfamily Megavirinae, excluding tupanviruses. Among its 1,306 predicted open reading frames, 1,149 (88.0%) were homologous to those of the family Mimiviridae. Several nucleocytoplasmic large DNA virus (NCLDV) core genes, aminoacyl-tRNA synthetase genes, and the host specificity of cotonvirus were highly similar to those of Mimiviridae lineages A, B, and C; however, lineage A was slightly closer to cotonvirus than the others were. Moreover, based on its genome size, the presence of two copies of 18S rRNA-like sequences, and the period of its infection cycle, cotonvirus is the most similar to the tupanviruses among the icosahedral mimiviruses. Interestingly, the cotonvirus utilizes Golgi apparatus-like vesicles for virion factory (VF) formation. Overall, we showed that cotonvirus is a novel lineage of the subfamily Megavirinae. Our findings support the diversity of icosahedral mimiviruses and provide mechanistic insights into the replication, VF formation, and evolution of the subfamily Megavirinae. IMPORTANCE We have isolated a new virus of an independent lineage belonging to the family Mimiviridae, subfamily Megavirinae, from the fresh water of a canal in Japan, named Cotonvirus. In a proteomic tree, this new nucleocytoplasmic large DNA virus (NCLDV) is phylogenetically placed at the root of three lineages of the subfamily Megavirinae-lineages A (mimivirus), B (moumouvirus), and C (megavirus). Multiple genomic and phenotypic features of cotonvirus are more similar to those of tupanviruses than to those of the A, B, or C lineages, and other genomic features, while the host specificity of cotonvirus is more similar to those of the latter than of the former. These results suggest that cotonvirus is a unique virus that has chimeric features of existing viruses of Megavirinae and uses Golgi apparatus-like vesicles of the host cells for virion factory (VF) formation. Thus, cotonvirus can provide novel insights into the evolution of mimiviruses and the underlying mechanisms of VF formation.
