Unveiling the Virome of Wild Birds: Exploring CRESS-DNA Viral Dark Matter

Abstract

Amid global health concerns and the constant threat of zoonotic diseases, this study delves into the diversity of circular replicase-encoding single-stranded DNA (CRESS-DNA) viruses within Chinese wild bird populations. Employing viral metagenomics to tackle the challenge of "viral dark matter," the research collected and analyzed 3,404 cloacal swab specimens across 26 bird families. Metagenomic analysis uncovered a rich viral landscape, with 67.48% of reads classified as viral dark matter, spanning multiple taxonomic levels. Notably, certain viral families exhibited host-specific abundance patterns, with Galliformes displaying the highest diversity. Diversity analysis categorized samples into distinct groups, revealing significant differences in viral community structure, particularly noting higher diversity in terrestrial birds compared to songbirds and unique diversity in migratory birds versus perching birds. The identification of ten novel Circoviridae viruses, seven Smacoviridae viruses, and 167 Genomoviridae viruses, along with 100 unclassified CRESS-DNA viruses, underscores the expansion of knowledge on avian-associated circular DNA viruses. Phylogenetic and structural analyses of Rep proteins offered insights into evolutionary relationships and potential functional variations among CRESS-DNA viruses. In conclusion, this study significantly enhances our understanding of the avian virome, shedding light on the intricate relationships between viral communities and host characteristics in Chinese wild bird populations. The diverse array of CRESS-DNA viruses discovered opens avenues for future research into viral evolution, spread factors, and potential ecosystem impacts.

Keywords: Cressdnaviricota; dark matter; metagenomic; wild bird.

Fig. 1.

Sample collection and DNA library construction. a) Map of sampling locations. The sampling sites are marked with colors. Samples were obtained from 5 provinces in China: Heilongjiang, Jilin, Zhejiang, Jiangsu, and Hunan. b) Overview of avian taxa and library construction status. The avian taxa include 9 orders (Falconiformes, Gruiformes, Coraciiformes, Passeriformes, Anseriformes, Galliformes, Ciconiiformes, Strigiformes, and Psittaciformes) comprising 26 families, with a cumulative total of 95 species. The bars represent the number of avian species, samples, and pools for each avian family. c) Distribution of 36 viral families in 228 pools. The color scale represented the relative abundance levels. The shades ranging from red to blue indicated the relative abundance, with red representing higher abundance and blue representing lower abundance.

Fig. 2.

Diversity analysis of viruses in the intestinal tract of birds. a to d) Richeness, Chao1, Simpson, and Shannon index difference analysis. *P < 0.05, **P < 0.01, ***P < 0.001, between the two groups. e) PCA analysis. f) PCOA analysis. g) Stack bar diagram.

Fig. 3.

Identification of novel viruses of the family Circoviridae. a) Predicted genome organization of MW182727 and MW182731. b) Identification of the RCR domain and superfamily 3 helicase domain in the Rep protein. c) Pairwise sequence comparison produced with full-genome sequences of cycloviruses within Bayesian consensus tree. The novel cyclovirus is highlighted. d) Bayesian inference tree was constructed using MrBayes v3.2 based on amino acid sequences of Rep protein and visualized with iTOL. The viruses identified in this study are highlighted within the tree. Scale bar indicates the amino acid substitutions per site.

Fig. 4.

Identification of novel viruses of the family Smacoviridae. a) Predicted genome organization of MW183071. b) Identification of the rolling circle replication domain and superfamily 3 helicase domain in the Rep protein. c) Pairwise sequence comparison produced with amino acid sequences of Rep protein of porprismacoviruses within Bayesian consensus tree. The novel porprismacoviruses are highlighted. d) Bayesian inference tree was constructed using MrBayes v3.2 based on amino acid sequences of Rep protein and visualized with iTOL. The viruses identified in this study are highlighted within the tree. Scale bar indicates the amino acid substitutions per site.

Fig. 5.

Identification of novel viruses of the family Genomoviridae. a) Predicted genome organization of MW182895 (Gemykrogvirus), MW182897 (Gemyvongvirus), MW182943 (Gemykibivirus), MW182959 (Gemykolovirus), MW182978 (Gemyduguivirus), MW182992 (Gemygorvirus), MW183013 (Gemycircularvirus), and MW183050 (Gemytondvirus). b) Bayesian inference tree was constructed using MrBayes v3.2 based on amino acid sequences of Rep protein and visualized with iTOL. The viruses identified in this study are highlighted within the tree. Scale bar indicates the amino acid substitutions per site.

Fig. 6.

Identification of novel viruses of unclassified CRESS DNA viruses. Bayesian inference tree was constructed using MrBayes v3.2 based on amino acid sequences of Rep protein and visualized with iTOL. The viruses identified in this study are highlighted within the tree. Scale bar indicates the amino acid substitutions per site.

Fig. 7.

Tertiary structure comparison of the Rep protein in CRESS DNA viruses. Top left: ribbon cartoon representation of the genomovirus. Ribbon cartoons for each member of the 13 groups are distinguished based on their secondary structure. Top right: schematic of the Rep protein showing the ED, OD, and AD. Orthogonal views of the 3D map of the Rep protein are presented, showing both top and side perspectives. The six subunits are consistently distinguished. Figure generated using UCSF Chimera.