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. 2020 Oct;5(10):1262-1270.
doi: 10.1038/s41564-020-0755-4. Epub 2020 Jul 20.

Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome

Yuri I Wolf #  1 , Sukrit Silas #  2 , Yongjie Wang  3   4 , Shuang Wu  3 , Michael Bocek  2 , Darius Kazlauskas  5 , Mart Krupovic  6 , Andrew Fire  7   8 , Valerian V Dolja  9 , Eugene V Koonin  10
Affiliations

Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome

Yuri I Wolf et al. Nat Microbiol. 2020 Oct.

Abstract

RNA viruses in aquatic environments remain poorly studied. Here, we analysed the RNA virome from approximately 10 l water from Yangshan Deep-Water Harbour near the Yangtze River estuary in China and identified more than 4,500 distinct RNA viruses, doubling the previously known set of viruses. Phylogenomic analysis identified several major lineages, roughly, at the taxonomic ranks of class, order and family. The 719-member-strong Yangshan virus assemblage is the sister clade to the expansive class Alsuviricetes and consists of viruses with simple genomes that typically encode only RNA-dependent RNA polymerase (RdRP), capping enzyme and capsid protein. Several clades within the Yangshan assemblage independently evolved domain permutation in the RdRP. Another previously unknown clade shares ancestry with Potyviridae, the largest known plant virus family. The 'Aquatic picorna-like viruses/Marnaviridae' clade was greatly expanded, with more than 800 added viruses. Several RdRP-linked protein domains not previously detected in any RNA viruses were identified, such as the small ubiquitin-like modifier (SUMO) domain, phospholipase A2 and PrsW-family protease domain. Multiple viruses utilize alternative genetic codes implying protist (especially ciliate) hosts. The results reveal a vast RNA virome that includes many previously unknown groups. However, phylogenetic analysis of the RdRPs supports the previously established five-branch structure of the RNA virus evolutionary tree, with no additional phyla.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. The Yangshan Deep-Water Harbour.
a–c, Map of the Yangshan Deep-Water Harbour at three scales. a, Location within China. b, Magnified view of the region bounded by the grey box in a. c, Expanded view of the region marked by the orange box in b, showing the Yangshan Deep-Water Harbour. Red triangles in c mark seawater sampling sites. Map data: Google, 2014; Mapabc.com, 2014; CNES/Astrium, 2014 TerraMetics.
Fig. 2
Fig. 2. Schematic phylogenetic tree of the RNA virus RdRPs.
The reverse transcriptases of group II introns and non-long-terminal-repeat (non-LTR) transposons were used as an outgroup to root the tree. The overall tree topology encompassing five major RdRP branches (highlighted by different background colours) has been described previously. These branches correspond to RNA virus phyla, which are shown under the branch numbers. The positions of the largest clades of viruses identified in this study are indicated and represented by triangles, the areas of which are roughly proportional to the number of viruses in each clade (shown inside the triangle). The numbers in parentheses correspond to previously identified viruses included in the analysis in ref. . Provisional names of the previously undescribed virus clades are shown in red. Purple text denotes a virus lineage with permuted RdRPs (Permutotertraviridae and Birnaviradae) which was not included in the previous study.
Fig. 3
Fig. 3. The Yangshan virus assemblage.
The rootless tree represents the subtree of branch 3 (Kitrinoviricota) in which the Yangshan virus assemblage belongs. The positions of RdRPs of previously reported viruses are shown in black. Dark blue represents assorted small clusters of the Yangshan assemblage. Other colours represent clusters (as indicated) of which Yan-like (red), Zhao-like (magenta) and Shanghai (electric blue) correspond to eponymous major clades, whereas the Wei-like clade encompasses four clusters. The permuted RdRPs are marked by black dots and RdRP groups with non-standard genetic codes are marked with green stars. The blue cross marks a virus group within the Zhao-like clade that includes viruses using protist genetic codes and encoding a capping enzyme similar to that of nodaviruses. The arrow points to a root position as in the tree in Fig. 2. YNPV, Yellowstone National Park virus.
Fig. 4
Fig. 4. Diversity of domain organizations in the major clades of marine RNA viruses in the Yangshan virome.
a, Aquatic picorna-like virus clade (as in Fig. 2). b, Yan-like virus clade (as in Fig. 3). c, Noda-like virus clade (as in Figs. 2 and 3). d, Protopotyvirus clade (as in Fig. 2). e, Zhao-like virus clade (as in Fig. 3). f, Brandma-like virus clade (as in Fig. 2). Each panel contains a genome map(s) of a phylogenetically close reference virus(es) at the top and viruses discovered in this study, identified as ‘NODE_NN’. NODE numbers correspond to contig gene IDs listed in Supplementary Datasets 3 and 5. Functional domains are colour coded and the colour key for the recurrent domains is shown at the bottom of the figure. HaRNAV, Heterosigma akashiwo RNA virus; PVY, potato virus Y; HAstV-1, human astrovirus 1; ShIV3, Shahe isopoda virus 3; BZhV1, Beihai zhaovirus-like virus 1; NoV, Nodamura virus; LeppyrTLV1, Leptomonas pyrrhocoris tombus-like virus 1; PrsW, PrsW-family protease; ZBD, zinc-binding domain; PLA2, phospholipase A2; vOTU, viral ovarian tumour protease; MTase, methyltransferase; Chy-Pro, chymotrypsin-like protease; S-Pro, serine protease; C-Pro, cysteine protease; VPg, viral genome-linked protein; CP, capsid protein; fCP, filamentous capsid protein; NSMP, non-structural mature protein; CapE, capping enzyme; S3H, superfamily 3 helicase; Como32K-like, comovirus 32K-like protease; r84.0, functionally uncharacterized domain conserved in RNA viruses; P3, protein 3; 6K1 and 6K2, 6 kDa proteins 1 and 2; PIPO, pretty interesting Potyviridae open reading frame protein; Spike, spike protein; CI, cylindrical inclusion protein.
Extended Data Fig. 1
Extended Data Fig. 1. Phylogenetic tree of the permuted RdRps of Permutotetraviridae, Birnaviridae and a related OV.70 cluster of seven RdRps identified in the Yangshan virome.
Note that this lineage of permuted RdRPs is confidently lodged as additional clade in Branch 2 (Pisuviricota) as a sister to Partitiviridae lineage.
Extended Data Fig. 2
Extended Data Fig. 2. Results of the HHpred search seeded with the putative capping enzyme of Yan-like viruses.
H(h), α-helix; E(e), β-strand; C(c), coil. The single-letter designations of the amino acid residues are coloured according to their physical properties.
Extended Data Fig. 3
Extended Data Fig. 3. Results of the HHpred search seeded with the putative capsid protein of Yan-like viruses.
H(h), α-helix; E(e), β-strand; C(c), coil. The single-letter designations of the amino acid residues are colored according to their physical properties.
Extended Data Fig. 4
Extended Data Fig. 4
A nucleotide sequence match between a Yangshan RNA virome contig bearing a levi-like RdRP (bottom line) and the type III-B CRISPR spacer locus of the bacterium Candidatus Accumulibacter sp. SK-02.
Extended Data Fig. 5
Extended Data Fig. 5. Protein domains identified in the Yangshan RNA virome that were not previously observed in known RNA viruses.
In the virome contigs, the nucleotide sequences encoding these domains were linked to those encoding RdRP thus demonstrating that they belonged to RNA virus genomes.
Extended Data Fig. 6
Extended Data Fig. 6
DNA virus sequences in the Yangshan virome.
Extended Data Fig. 7
Extended Data Fig. 7
General characteristics of the oceanic RNA virome of Yangshan Deep Water Harbor.
Extended Data Fig. 8
Extended Data Fig. 8
Schematic of purification and sequencing of the oceanic RNA and DNA viromes. TFF, tangential flow filtration.
Extended Data Fig. 9
Extended Data Fig. 9
Sequencing data for each metaviromic cDNA library.
Extended Data Fig. 10
Extended Data Fig. 10. Distribution of contig abundances.
(a) Probability density function (p.d.f.) for contig abundances (n = 4571 non-identical contigs. The dotted line plots the log-normal distribution with the same median and interquartile distance. (b) Quantile-quantile (Q-Q) plot of the distribution of contig abundances (n = 4571 non-identical contigs) versus the standard normal distribution. The figure shows that at the first approximation the distribution of contig abundances follows the log-normal distribution (typical in complex environments), but the deviations (a pronounced heavy tail of high values) hints on a dynamic environment producing superabundant viral blooms. Source data for panels (A) and (B) are presented in Source Data Figs. 1 and 2, respectively. Source data
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