Pantanal virophage: a new virophage species associated with a moumouvirus and a transpoviron, expanding the genus Sputnikvirus.

Amoeba-infecting giant viruses, such as mimiviruses, challenged paradigms of virology since their discovery. Their extensive and complex genomes and particle structures can interact with virophages and transpovirons, revealing unique aspects of the viral ecology and evolution. Here, we describe the isolation of Pantanal virophage, a new virophage obtained from Brazilian pantanal biome. The virophage was found in association with moumouvirus pantanense and a transpoviron sequence. Sequencing and genomic analysis revealed that Pantanal virophage presents a 17,964 bp genome with 21 coding sequences, including two ORFans that code for proteins with completely unknown functions. Phylogeny showed that Pantanal virophage composes a divergent branch within virophages from Sputnikvirus genus. Average nucleotide and amino acid identity analysis comparing different virophage sequences indicated that Pantanal virophage could represent a new Sputnikvirus species. These findings shed light on the giant virus-virophage-transpoviron triplet diversity, evolution, and ecology, contributing to a future update in virophages taxonomy.IMPORTANCEStudies on prospecting, isolation, and characterization of new amoeba viruses are important to provide new information about biology, diversity, evolution, ecology, and taxonomy of these viruses. This work reinforces this importance since we describe Pantanal virophage, a new species of Sputnikvirus found in association with a moumouvirus and a transpoviron. The characterization of Pantanal virophage provided new data and observations regarding the phylogeny and taxonomy of Sputnikvirus genus evidencing the need for constant updates in taxonomic classification. This work shows that the efforts for isolation of new amoeba viruses and their characterization can contribute to enriching the knowledge about taxonomy and evolutionary dynamics of these viruses and of their parasitic-associated elements.