Isolation and Identification of a Large Green Alga Virus ( Virus XW01) of and Its Virophage ( Virus Virophage SW01) by Using Unicellular Green Algal Cultures

Virophages are a group of small double-stranded DNA viruses that infect protist hosts and parasitize the viral factory of host giant/large viruses to propagate. Here, we discover a novel cell-virus-virophage (CVv) tripartite interaction system by using unicellular micro-green algae ( sp.) as eukaryotic hosts for the first time. Viral particles, resembling known virophages and large alga viruses, are detected in culture supernatants and inside algal cells. Complete genomic sequences of the virophage ( virus virophage SW01 [CVv-SW01]; 24,744 bp) and large virus ( virus XW01 [CV-XW01]; 407,612 bp) are obtained from the cocultures. Both genomic and phylogenetic analyses show that CVv-SW01 is closely related to virophages previously found in Dishui Lake. CV-XW01 shares the greatest number of homologous genes ( = 82) with (CroV) and phylogenetically represents the closest relative to CroV. This is the first report of a large green alga virus being affiliated with a heterotrophic zooplankton-infecting of the family . Moreover, the codon usage preferences of CV-XW01 and CVv-SW01 are highly similar to those of CroV and its virophage Mavirus, respectively. The discovery of such a novel CVv system with the green alga sp. as the single cellular eukaryotic host paves a way to further investigate the potential interaction mechanism of CVv and its significance in the ecology of green algae and the evolution of large/giant viruses and their parasitic viruses. Parasitic virophages are small unicellular eukaryotic dsDNA viruses that rely on the viral factories of coinfecting giant/large dsDNA viruses for propagation. Presently, the identified eukaryotic hosts of isolated virophages were restricted to a free-living amoeba, Acanthamoeba polyphaga, and a widespread marine heterotrophic flagellate, . In this study, we successfully discovered and identified a novel tripartite interaction system comprised of a micro-green alga ( sp.), large green alga virus, and virophage at the coculture level, with sp. as the eukaryotic host, based on combination analysis of infection, morphotype, genome, and phylogeny. The large green alga virus CV-XW01 represents the closest relative to the giant virus , host virus of the virophage Mavirus, as well as a novel large virus of that infects a non-protozoan protist host. The virophage CVv-SW01 highly resembles Mavirus in its codon usage frequency and preference, although they are phylogenetically distantly related. These findings give novel insights into the diversity of large/giant viruses and their virophages.