Refining a giant virus lineage: a novel order unifying <i>Mamonoviridae</i> and "Manesviridae," unveiled by the discovery of furtivovirus

The evolutionary origins and taxonomic framework of giant viruses related to the family <i>Mamonoviridae</i> and its relative group, including clandestinovirus, remain unclassified due to gaps in genome size and host range between these two groups. This study aimed to address this gap by integrating our newly isolated virus with publicly available metagenome-assembled genomes (MAGs) to construct a more robust phylogenetic framework. Here, we report the isolation and characterization of a new giant virus, furtivovirus, using the unicellular amoeba <i>Vermamoeba vermiformis</i> as a host. Furtivovirus has a genome of approximately 560 kbp and shares key features with its closest relative, clandestinovirus. Ultrastructural analysis revealed a unique host-nucleus-dependent replication strategy characterized by the breakdown of the nuclear membrane and the packaging of nascent virions directly within the nucleoplasm, distinguishing it from canonical cytoplasmic virion factories. Comprehensive phylogenetic and comparative genomic analyses of shared orthologous groups and nucleocytovirus marker proteins revealed that furtivovirus, clandestinovirus, ushikuvirus, and usurpativirus form a distinct monophyletic clade, for which we propose a new family, "Manesviridae." Further analysis using amino acid-based similarity metrics of <i>Nucleocytoviricota</i> viral genomes, including established MAGs, demonstrated that this new family is robustly placed as a sister group to the family <i>Mamonoviridae</i>. This study elucidated the evolutionary relationships between viruses with large and small genomes that possess similar virion sizes within this lineage. Based on this cumulative evidence, we propose the establishment of a new order to unify these two families, thereby expanding their diversity and clarifying the evolutionary history of this branch within <i>Nucleocytoviricota</i>.<h4>Importance</h4>Giant viruses challenge our traditional understanding of viral evolution, raising the question of how a single related group can diverge to infect different hosts while evolving into vastly different genome sizes and replication strategies. The family <i>Mamonoviridae</i> and its relatives epitomize this evolutionary divergence: one group possesses massive genomes, whereas the other has genomes that are less than half their size. The discovery of furtivovirus and its unique nucleoplasm-dependent replication cycle provides a critical biological context for this genomic disparity. Through deep comparative genomic analysis, we demonstrated that these seemingly disparate lineages share a cohesive evolutionary origin that is distinct from other established orders. This finding highlights the complexity of genome evolution, demonstrating that giant viruses can expand their overall genome size to adapt to uncertain environments while reducing their core essential genes, thereby providing new insights into the evolutionary pressures that shape the diversity of the virosphere.