The cryptic diversity of hepadnavirus relatives.

Enveloped hepadnaviruses, non-enveloped nackednaviruses, and the recently discovered non-enveloped proto-nackednaviruses are closely related reverse-transcribing DNA viruses. Yet, the host range and diversity of proto-nackednaviruses remain unclear. Here, we report the discovery of 31 proto-nackednaviruses (PnNVs) through deep genome-scale data mining. PnNVs are predominantly identified in distinct species of rotifers, implying the probable association between PnNVs and rotifers. Phylogenetic analyses show that PnNVs form at least five paraphyletic lineages sister to nackednaviruses and hepadnaviruses, unveiling the cryptic diversity of reverse-transcribing DNA viruses. This study provides insights into the origin, evolution, and diversity of reverse-transcribing DNA viruses.IMPORTANCEHepatitis B virus belongs to Hepadnaviridae and represents a serious threat to global public health. Recently, non-enveloped proto-nackednaviruses were discovered to be the closest relatives of Hepadnaviridae. However, the host range and diversity of proto-nackednaviruses remain unclear, impeding our understanding of the origin, evolution, and diversity of Blubervirales and reverse-transcribing DNA viruses in general. This work expands the hidden diversity of proto-nackednaviruses and indicates that rotifers might be their putative hosts. This work further traces the origin of Blubervirales back to 476 million years ago. These findings deepen our understanding of the evolution of reverse-transcribing DNA viruses.