Identification and characterization of novel CRESS-DNA viruses in the human respiratory tract

Abstract

CRESS-DNA viruses are small, circular, single-stranded DNA viruses that have been identified in diverse environments and hosts, including vertebrates, invertebrates, and environmental samples. However, their diversity and role in the human respiratory tract remain poorly understood. In this study, we employed viral metagenomics to analyze 140 nasopharyngeal swab samples from asymptomatic individuals. High-throughput sequencing and bioinformatics analyses were used to identify and characterize novel CRESS-DNA viruses. Phylogenetic relationships were inferred based on Rep protein sequences using maximum likelihood analysis. We identified and characterized eight novel CRESS-DNA viruses, which were classified into the families Endolinaviridae and Naryaviridae, with one potentially representing a novel viral family. These viruses exhibited typical circular genomic structures encoding Rep and Cap proteins, with conserved motifs associated with rolling circle replication. Phylogenetic analysis showed that some viruses were closely related to sequences from vertebrate hosts or environmental samples, suggesting a diverse ecological distribution. Our findings expand the known diversity of CRESS-DNA viruses in the human respiratory tract and highlight their potential ecological and evolutionary significance. Further studies are needed to explore their host specificity, replication mechanisms, and potential roles in human health and disease.

Keywords: Cressdnaviricota; Endolinaviridae; Naryaviridae; Metagenomics; Phylogenetics; Respiratory tract.

Fig. 1

Viral metagenomic overview of the 14 libraries. A Species rarefaction curve annotated by MEGAN v.6.22.2 after log-scale transformation. Legends are displayed to the side of the panel. B Accumulation curve of viral contigs in this study. Error bars represent the range, and the blue area in the background represents the 95% confidence interval. C Bar graphs showing the relative proportion and taxonomy based on viral Phyla. D Heat map representing the viral contigs of each viral family of each library on a log₁₀ scale. Viral genome types and viral families are annotated with corresponding colors (see color legend)

Fig. 2

Genomic characterization and distribution of identified novel CRESS-DNA viruses. A Genome schematic organizations of the human-associated CRESS-DNA viruses sequenced in this study. B Identification of the rolling circle replication domain and superfamily 3 helicase domain in the Rep protein. C Heatmap showing the normalized read counts mapped to eight novel human-associated CRESS-DNA viruses across 14 metagenomic libraries. Column-wise normalization was applied to highlight relative abundance variations. The hierarchical clustering on the left groups viruses with similar mapping patterns

Fig. 3

Maximum likelihood phylogenetic tree inferred from Rep proteins of members of the phylum Cressdnaviricota. The maximum likelihood phylogenetic tree was constructed using IQtree with automatic selection of the best-fit substitution model for a given alignment, which was Q.pfam + F + R8. Numbers at the nodes represent ultrafast bootstrap support values. The scale bar represents the number of substitutions per site

Fig. 4

Pairwise identity matrix of members of the families Endolinaviridae and Naryaviridae, with a 78% species demarcation threshold inferred using SDT v1.3