Virus Hunting Using Metagenomic Sequencing: Discovery And Initial Characterization Of The Redondoviridae Family

Viruses are ubiquitous, diverse, and extremely successful. Through metagenomic sequencing of global virus populations, many new viruses have been discovered. In particular, a great diversity of yet-undiscovered circular, Rep-encoding single-stranded (CRESS) DNA viruses has become apparent, underscoring the need for techniques and studies in this area. In this study, we describe two software packages, Sunbeam and grabseqs, that enable robust and extensible analysis of next-generation sequencing data, crucial for virus discovery. Using these tools, we discovered and characterized Redondoviridae, a new family of CRESS DNA viruses present in the human oro-respiratory tract. After initially discovering redondoviruses in respiratory samples from human lung transplant recipients, we screened 7000+ metagenomic samples representing different body sites, organisms, and environments. Redondoviruses were only identified in humans and were mostly found in oral and respiratory tract samples. Two species of redondovirus, Brisavirus and Vientovirus were identified. We found higher levels of redondoviruses in subjects with periodontitis or critical illness compared to healthy individuals. Next, to define the global prevalence of redondoviruses, we screened saliva samples from the US and multiple non-industrialized populations in Africa, identifying significantly higher redondovirus prevalence in samples from African participants. To define intra-individual redondovirus diversity over time, we sequenced single redondovirus genomes from longitudinally-sampled individuals, and found subjects positive for multiple redondovirus genotypes which persisted up to two years in some cases. Finally, we identified hotspots of recombination in intergenic regions of the redondovirus genome and demonstrated biochemical evidence supporting a role for Rep in redondoviral recombination. Overall, the work described here outlines the discovery, prevalence, and evolutionary mechanisms of the new Redondoviridae family, advancing our knowledge of human respiratory viral communities.