Abstract
Environmental RNA viruses are ubiquitous and diverse, and probably have important ecological and biogeochemical impacts. Understanding the global diversity of RNA viruses is limited by sampling biases, dependence on cell culture and PCR for virus discovery, and a focus on viruses pathogenic to humans or economically important animals and plants. To address this knowledge gap, we generated metatranscriptomic sequence data from 32 diverse environments in 16 provinces and regions of China. We identified 6,624 putatively novel virus operational taxonomic units from soil, sediment and faecal samples, greatly expanding known diversity of the RNA virosphere. These newly identified viruses included positive-sense, negative-sense and double-strand RNA viruses from at least 62 families. Sediments and animal faeces were rich sources of viruses. Virome compositions were affected by local environmental factors, including organic content and eukaryote species abundance. Notably, environmental factors had a greater impact on the abundance and diversity of plant, fungal and bacterial viruses than of animal viromes. Our data confirm that RNA viruses are an integral part of both terrestrial and aquatic ecosystems.
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Data availability
The sequence reads generated in this study are available at the NCBI Sequence Read Archive (SRA) database under BioProject accession PRJNA716119. All viral sequences generated in this study have been deposited in GenBank under accession numbers (https://www.ncbi.nlm.nih.gov/nuccore?term=716119%5BBioProject%5D) MW784004-MW784109, MW896840-MW897324, MZ218144-MZ218759, MZ556337-MZ556592, MZ678955-MZ680357, ON049747-ON050964, ON161767-ON164489. All other data are available in the paper or in the supplementary materials. The CheckV database used for viral genome quality and completeness estimation can be accessed via https://bitbucket.org/berkeleylab/checkv. The Conserved Domain Database (CDD) used for ORF annotation can be accessed via https://www.ncbi.nlm.nih.gov/cdd/. The UniRef30_2021_03 database used in HHblits analysis can be accessed via http://wwwuser.gwdg.de/~compbiol/uniclust/2021_03/. The SILVA database used for rRNA removal can be accessed via https://www.arb-silva.de/. Source data are provided with this paper.
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Acknowledgements
We thank D.-X. Wang, K. Li, W.-B. Zhao, X.-N. Diao, A.-J. Gong, Y.-L. Zhang, J.-B. Wang, H. Luo, D.-A. Zhang, Y.-Q. Zhao and M.-Li for their contributions to sample collection, and X.-Q. Luo, R.-X. Hu, M.-Z. Liu, J. Liu, Y. Jiang, J.-J. Guo, J.-J. Wang and P. Lu for assisting with PCR confirmations. This study was supported by the National Natural Science Foundation of China (grant nos. 32130002, 31930001, 32041004, 81861138003 and 81672057 to Y.-Z.Z) and the National Key R&D Program of China (2016YFC1201900 to Y.-Z.Z). E.C.H was supported by an ARC Australian Laureate Fellowship (FL170100022).
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Contributions
Y.-Z.Z. conceived and designed the study. Y.-M.C., J.-H.T., X.C., X.-D.L., J.-J.S., H.C., Z.-Y.H., W.-D.Y., Z.-C.Z., J.W., F.L., H.-W.W. and Q.-Y.X. performed sample collection and geographic information recording. S.S., Y.-M.C., M.W. and E.C.H. analysed the data. Y.-M.C., W.-H.G. and W.W. performed the experiments. S.S., Y.-M.C., E.C.H. and Y.-Z.Z. wrote the paper with input from all authors. Y.-Z.Z. led the study.
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Extended data
Extended Data Fig. 1
Environments and Chinese provinces sampled in this study.
Extended Data Fig. 2 Viral composition of each library.
Relative proportions were determined by the number of reads corresponding to contigs with viral hits to each viral clade as a proportion of total viral reads in each of 442 biologically independent samples (analysis performed using DIAMOND BLASTX).
Extended Data Fig. 3 Ecological factors significantly associated with viral abundance in environmental viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on viral abundance with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 6a). Boxplots represent abundance values plotted against (B) environment, (C) location, (D) total phosphorus, (E) total potassium, (F) available phosphorus, (G) available potassium, (H) organic content, (I) eukaryote species abundance, and (J) eukaryote species richness. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Extended Data Fig. 4 Ecological factors significantly associated with viral abundance in animal-associated viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on viral abundance with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 7a), along with the abundance values plotted against (B) location, (C) organic content, and (D) eukaryote species abundance. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Extended Data Fig. 5 Ecological factors significantly associated with Shannon diversity in environmental viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on Shannon diversity with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 6c). Boxplots represent Shannon diversity values plotted against (B) environment, (C) location, (D) pH, (E) total nitrogen, (F) total potassium, (G) available nitrogen, (H) organic content, and (I) eukaryote species richness. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Extended Data Fig. 6 Ecological factors significantly associated with true diversity in environmental viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on true diversity with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 6d), along with the true diversity values plotted against (B) environment, (C) location, (D) pH, (E) total nitrogen, (F) total potassium, (G) available nitrogen, (H) organic content, (I) eukaryote species richness, and (J) eukaryote species true diversity. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Extended Data Fig. 7 Ecological factors significantly associated with Shannon diversity in animal-associated viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on Shannon diversity with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 7c), along with the Shannon diversity values plotted against (B) environment. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Extended Data Fig. 8 Ecological factors significantly associated with true diversity in animal-associated viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on true diversity with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 7d), along with the true diversity values plotted against (B) environment. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Extended Data Fig. 9 Ecological factors significantly associated with viral richness in environmental viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on viral richness with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 6b). Boxplots represent richness values plotted against (B) environment, (C) location, (D) pH, (E) total phosphorus, (F) available nitrogen, (G) organic content, (H) eukaryote species richness, (I) eukaryote species Shannon diversity, and (J) eukaryote species true diversity. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Extended Data Fig. 10 Ecological factors significantly associated with viral richness in animal-associated viruses.
(A) Model-averaged effect sizes of ecological metadata for 265 biologically independent samples on viral richness with confidence intervals of 95% and factors significant in the best subset selected model (p < 0.05, in orange) do not overlap the central line and are denoted with an asterisk (*) (identical to Fig. 7b), along with the richness values plotted against (B) environment, (C) location, (D) pH, (E) total phosphorus, (F) organic content, (G) eukaryote species richness, and (H) eukaryote species true diversity. For all boxplots, the horizontal box lines in the boxplots represent the first quartile, the median, and the third quartile. Whiskers denote the range of points within the first quartile − 1.5× the interquartile range and the third quartile + 1.5× the interquartile range.
Supplementary information
Supplementary Information
Supplementary Figs. 1–38.
Supplementary Table 1
Supplementary Tables 1–3.
Supplementary Data 1
Statistical source data for Supplementary Fig. 36.
Supplementary Data 2
Statistical source data for Supplementary Fig. 37.
Supplementary Data 3
Statistical source data for Supplementary Fig. 38.
Source data
Source Data Fig. 2
Statistical source data for Fig. 2.
Source Data Fig. 6
Statistical source data for Fig. 6.
Source Data Fig. 7
Statistical source data for Fig. 7.
Source Data Extended Data Fig. 2
Statistical source data for Extended Data Fig. 2.
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Chen, YM., Sadiq, S., Tian, JH. et al. RNA viromes from terrestrial sites across China expand environmental viral diversity. Nat Microbiol 7, 1312–1323 (2022). https://doi.org/10.1038/s41564-022-01180-2
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DOI: https://doi.org/10.1038/s41564-022-01180-2
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