Expanded comparative genomics supports labial gland disease viruses as a distinct and deeply divergent lineage within Naldaviricetes

ABSTRACT Labial gland disease viruses (LGDVs) are recently identified dsDNA viruses infecting ants, but their diversity, genomic coherence, and taxonomic boundaries have remained unresolved. Here, we expanded LGDV members by mining public arthropod genome assemblies and sequencing field-collected ants. Novel LGDV genomes were recovered from Pheidole selathorax , Proformica mongolica , Messor orientalis , and geographically distinct Anoplolepis gracilipes populations. Comparative genomic analyses demonstrate that LGDVs retain the conserved core genes in the order Lefavirales , class Naldaviricetes , share additional conservative genes with Apis mellifera filamentous virus and related viruses (AmFV/AmFLV), and encode an LGDV-restricted conservative gene set. Amino acid identity, conservative site, and gene neighborhood analyses support the internal coherence of LGDVs while distinguishing them from AmFV/AmFLV, which retained only partial local gene neighborhood similarities and lacked detectable genome-wide macrosynteny with LGDVs. Phylogenies inferred by protein sequences and structures were broadly congruent, showing that predicted protein structures retained complementary lineage-discriminating signal across deeply divergent naldaviricetes, and recovering LGDVs as a monophyletic sister group to AmFV/AmFLV. Molecular dating further supported a deep separation between these lineages, indicating that LGDVs and AmFV/AmFLV represent distinct, long-term divergent groups rather than recent derivatives of one another. Collectively, these findings support LGDVs as an internally coherent, deeply divergent, and ant-associated virus family in the order Lefavirales , class Naldaviricetes . IMPORTANCE Large dsDNA viruses within the class Naldaviricetes encompass deeply divergent lineages. The recent discovery of LGDVs suggests that these ant-associated viruses have a closer evolutionary relationship to the phylogenetically isolated honey bee-associated AmFV/AmFLV than other Naldaviricetes viruses. However, the classification status of LGDVs and AmFV/AmFLV remains to be determined. In this study, we identified five novel LGDV genomes from four different ant species. Comparative analyses of protein sequences and structures, as well as gene neighborhood and molecular dating analyses support that LGDVs belong to an internally coherent and divergent ant-associated lineage that is distinct from AmFV/AmFLV. This study expands the known diversity of ant-associated naldaviricetes. The broad congruence between sequence- and structure-informed phylogenies highlights the value of structure-informed approaches for resolving deep relationships among highly divergent large dsDNA viruses.