Abstract
Giant viruses (phylum Nucleocytoviricota) are globally distributed in aquatic ecosystems. They play fundamental roles as evolutionary drivers of eukaryotic plankton and regulators of global biogeochemical cycles. However, we lack knowledge about their native hosts, hindering our understanding of their life cycle and ecological importance. In the present study, we applied a single-cell RNA sequencing (scRNA-seq) approach to samples collected during an induced algal bloom, which enabled pairing active giant viruses with their native protist hosts. We detected hundreds of single cells from multiple host lineages infected by diverse giant viruses. These host cells included members of the algal groups Chrysophycae and Prymnesiophycae, as well as heterotrophic flagellates in the class Katablepharidaceae. Katablepharids were infected with a rare Imitervirales-07 giant virus lineage expressing a large repertoire of cell-fate regulation genes. Analysis of the temporal dynamics of these host–virus interactions revealed an important role for the Imitervirales-07 in controlling the population size of the host Katablepharid population. Our results demonstrate that scRNA-seq can be used to identify previously undescribed host–virus interactions and study their ecological importance and impact.
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Data availability
Sequencing data have been deposited under NCBI Bioproject, accession no. PRJNA694552, Biosamples SAMN38317978–SAMN38317987. Additional data used in this paper, including UMI tables generated from 10x Cell Ranger, extended Blast result tables, assembled transcripts and other files that can be used to reproduce our results, are available at Dryad via https://doi.org/10.5061/dryad.s7h44j1c9 (ref. 76). Source data are provided with this paper. Public databases that were used in this manuscript include: the Giant Virus database https://faylward.github.io/GVDB; PR2 database https://pr2-database.org; metaPR2 database https://shiny.metapr2.org/metapr2; RefSeq v.207.
Code availability
All data management and analysis codes are open for review and reuse and archived online at GitHub via https://github.com/vardilab/host-virus-pairing (ref. 77).
References
Moniruzzaman, M. et al. Virologs, viral mimicry, and virocell metabolism: the expanding scale of cellular functions encoded in the complex genomes of giant viruses. FEMS Microbiol. Rev. 47, 5 (2023).
Schulz, F. et al. Giant virus diversity and host interactions through global metagenomics. Nature 578, 432–436 (2020).
Endo, H. et al. Biogeography of marine giant viruses reveals their interplay with eukaryotes and ecological functions. Nat. Ecol. Evol. 4, 1639–1649 (2020).
Aylward, F. O., Moniruzzaman, M., Ha, A. D. & Koonin, E. V. A phylogenomic framework for charting the diversity and evolution of giant viruses. PLoS Biol. 19, e3001430 (2021).
Rosenwasser, S., Ziv, C., Creveld, S. Gvan & Vardi, A. Virocell metabolism: metabolic innovations during host–virus interactions in the ocean. Trends Microbiol. 24, 821–832 (2016).
Fuhrman, J. A. Marine viruses and their biogeochemical and ecological effects. Nature 399, 541–548 (1999).
Irwin, N. A. T., Pittis, A. A., Richards, T. A. & Keeling, P. J. Systematic evaluation of horizontal gene transfer between eukaryotes and viruses. Nat. Microbiol. 7, 327–336 (2022).
Moniruzzaman, M., Weinheimer, A. R., Martinez-Gutierrez, C. A. & Aylward, F. O. Widespread endogenization of giant viruses shapes genomes of green algae. Nature 588, 1–5 (2020).
Nissimov, J. I. et al. Coccolithoviruses: a review of cross-kingdom genomic thievery and metabolic thuggery. Viruses 9, 52 (2017).
Ha, A. D., Moniruzzaman, M. & Aylward, F. O. High transcriptional activity and diverse functional repertoires of hundreds of giant viruses in a coastal marine system. mSystems 6, e0029321 (2021).
Moniruzzaman, M., Martinez-Gutierrez, C. A., Weinheimer, A. R. & Aylward, F. O. Dynamic genome evolution and complex virocell metabolism of globally-distributed giant viruses. Nat. Commun. 11, 1710 (2020).
Bäckström, D. et al. Virus genomes from deep sea sediments expand the ocean megavirome and support independent origins of viral gigantism. mBio 10, e02497–18 (2019).
Queiroz, V. F. et al. Amoebae: hiding in plain sight: unappreciated hosts for the very large viruses. Annu. Rev. Virol. 9, 79–98 (2022).
Coutinho, F. H. et al. Marine viruses discovered via metagenomics shed light on viral strategies throughout the oceans. Nat. Commun. 8, 1–12 (2017).
Meng, L. et al. Quantitative assessment of nucleocytoplasmic large DNA virus and host interactions predicted by co-occurrence analyses. mSphere 6, e01298–20 (2021).
Ciobanu, D. et al. A single-cell genomics pipeline for environmental microbial eukaryotes. iScience 24, 102290 (2021).
Stepanauskas, R. et al. Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles. Nat. Commun. 8, 84 (2017).
Brown, J. M. et al. Single cell genomics reveals viruses consumed by marine protists. Front. Microbiol. 11, 2317 (2020).
DeLong, J. P., Van Etten, J. L., Al-Ameeli, Z., Agarkova, I. V. & Dunigan, D. D. The consumption of viruses returns energy to food chains. Proc. Natl Acad. Sci. USA 120, e2215000120 (2023).
Gonzalez, J. M. & Suttle, C. A. Grazing by marine nanoflagellates on viruses and virus-sized particles: ingestion and digestion. Mar. Ecol. Prog. Ser. 94, 1–10 (1993).
Imdahl, F. & Saliba, A.-E. Advances and challenges in single-cell RNA-seq of microbial communities. Curr. Opin. Microbiol. 57, 102–110 (2020).
Mauger, S., Monard, C., Thion, C. & Vandenkoornhuyse, P. Contribution of single-cell omics to microbial ecology. Trends Ecol. Evol. 37, 1–12 (2021).
Ku, C. & Sebé-Pedrós, A. Using single-cell transcriptomics to understand functional states and interactions in microbial eukaryotes. Philos. Trans. R. Soc. B 374, 20190098 (2019).
Hevroni, G., Vincent, F., Ku, C., Sheyn, U. & Vardi, A. Daily turnover of active giant virus infection during algal blooms revealed by single-cell transcriptomics. Sci. Adv. 9, 41 (2023).
Lax, G. et al. Multigene phylogenetics of euglenids based on single-cell transcriptomics of diverse phagotrophs. Mol. Phylogenet. Evol. 159, 107088 (2021).
Cooney, E. C. et al. Single-cell transcriptomics of Abedinium reveals a new early-branching Dinoflagellate lineage. Genome Biol. Evol. 12, 2417–2428 (2020).
Schwartz, M. et al. Molecular characterization of human cytomegalovirus infection with single-cell transcriptomics. Nat. Microbiol. 8, 455–468 (2023).
Bost, P. et al. Host-viral infection maps reveal signatures of severe COVID-19 patients. Cell 181, 1475–1488 (2020).
Vincent, F. et al. Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during coccolithophore blooms. Nat. Commun. 14, 1–17 (2023).
Blobel, G. & Potter, V. R. Studies on free and membrane-bound ribosomes in rat liver. I. Distribution as related to total cellular RNA. J. Mol. Biol. 26, 279–292 (1967).
Vincent, F., Sheyn, U., Porat, Z., Schatz, D. & Vardi, A. Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise. Proc. Natl Acad. Sci. USA 118, e2021586118 (2021).
Lawrence, J. E., Brussaard, C. P. D. & Suttle, C. A. Virus-specific responses of Heterosigma akashiwo to infection. Appl. Environ. Microbiol. 72, 7829 (2006).
Aylward, FrankO. et al. Taxonomic update for giant viruses in the order Imitervirales (phylum Nucleocytoviricota). Arch. Virol. 168, 11 (2023).
Santini, S. et al. Genome of Phaeocystis globosa virus PgV-16T highlights the common ancestry of the largest known DNA viruses infecting eukaryotes. Proc. Natl Acad. Sci. USA 110, 10800–10805 (2013).
Gallot-Lavallée, L., Blanc, G. & Claverie, J.-M. Comparative genomics of Chrysochromulina ericina virus and other microalga-infecting large DNA viruses highlights their intricate evolutionary relationship with the established Mimiviridae family. J. Virol. 91, 230–247 (2017).
Okamoto, N. & Inouye, I. The katablepharids are a distant sister group of the Cryptophyta: a proposal for Katablepharidophyta divisio nova/Kathablepharida phylum novum based on SSU rDNA and beta-tubulin phylogeny. Protist 156, 163–179 (2005).
Vørs, N. Ultrastructure and autecology of the marine, heterotrophic flagellate Leucocryptos marina (Braarud) Butcher 1967 (Katablepharidaceae/Kathablepharidae), with a discussion of the genera Leucocryptos and Katablepharis/Kathablepharis. Eur. J. Protistol. 28, 369–389 (1992).
Massana, R. et al. Phylogenetic and ecological analysis of novel marine Stramenopiles. Appl. Environ. Microbiol. 70, 3528–3534 (2004).
Schoch, C. L. et al. NCBI taxonomy: a comprehensive update on curation, resources and tools. Database 2020, baaa062 (2020).
Knox, C., Luke, G. A., Blatch, G. L. & Pesce, E. R. Heat shock protein 40 (Hsp40) plays a key role in the virus life cycle. Virus Res. 160, 15–24 (2011).
Gober, M. D. & Wales, S. Q. & Aurelian, L. Herpes simplex virus type 2 encodes a heat shock protein homologue with apoptosis regulatory functions. Front. Biosci. 10, 2788–2803 (2005).
Yoshikawa, G. et al. Medusavirus, a novel large DNA virus discovered from hot spring water. J. Virol. 93, 2130–2148 (2019).
Wilson, W. H. et al. Genomic exploration of individual giant ocean viruses. ISME J. 11, 1736 (2017).
Machado, T. B. et al. Gene duplication as a major force driving the genome expansion in some giant viruses. J. Virol. 97, e01309–e01323 (2023).
Jousset, A. et al. Where less may be more: how the rare biosphere pulls ecosystems strings. ISME J. 11, 853–862 (2017).
Breitbart, M. & Rohwer, F. Here a virus, there a virus, everywhere the same virus? Trends Microbiol. 13, 278–284 (2005).
Mruwat, N. et al. A single-cell polony method reveals low levels of infected Prochlorococcus in oligotrophic waters despite high cyanophage abundances. ISME J. 15, 41–54 (2020).
Zhong, K. X., Wirth, J. F., Chan, A. M. & Suttle, C. A. Mortality by ribosomal sequencing (MoRS) provides a window into taxon-specific cell lysis. ISME J. 17, 105–116 (2022).
Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 3150–3152 (2012).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnetJ 17, 10 (2011).
Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).
Guillou, L. et al. The protist ribosomal reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy. Nucleic Acids Res. 41, D597–D604 (2012).
Vaulot, D. et al. metaPR2: a database of eukaryotic 18S rRNA metabarcodes with an emphasis on protists. Mol. Ecol. Resour. 8, 3188–3201 (2022).
Kopylova, E., Noé, L. & Touzet, H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 (2012).
Camacho, C. et al. BLAST+: architecture and applications. BMC Bioinform. 10, 1–9 (2009).
O’Leary, N. A. et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. 44, D733–D745 (2016).
Frith, M. C. A new repeat-masking method enables specific detection of homologous sequences. Nucleic Acids Res. 39, e23 (2011).
Kiełbasa, S. M., Wan, R., Sato, K., Horton, P. & Frith, M. C. Adaptive seeds tame genomic sequence comparison. Genome Res. 21, 487–493 (2011).
Edgar, RobertC. Muscle5: high-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nat. Commun. 13, 1 (2022).
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).
Van der Jeugt, F., Dawyndt, P. & Mesuere, B. FragGeneScanRs: faster gene prediction for short reads. BMC Bioinform. 23, 1–8 (2022).
Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).
Bushnell, B. BBMap: A Fast, Accurate, Splice-aware Aligner (Lawrence Berkeley National Laboratory, 2014).
Fromm, A., Schatz, D., Ben-Dor, S., Feldmesser, E. & Vardi, A. Complete genome sequence of Emiliania huxleyi virus strain M1, isolated from an induced E. huxleyi bloom in Bergen, Norway. Microbiol. Resour. Ann. 11, e0007122 (2022).
Feldmesser, E., Ben-Dor, S. & Vardi, A. An Emiliania huxleyi pan-transcriptome reveals basal strain specificity in gene expression patterns. Sci. Rep. 11, 20795 (2021).
Li, H. Bioawk: awk modified for biological data. GitHub https://github.com/lh3/bioawk (2015).
Johnson, M. et al. NCBI BLAST: a better web interface. Nucleic Acids Res. 36, W5–W9 (2008).
Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome. BMC Bioinform. 12, 323 (2011).
Yu, Y., Ouyang, Y. & Yao, W. ShinyCircos: an R/Shiny application for interactive creation of Circos plot. Bioinformatics 34, 1229–1231 (2018).
Sievers, F. & Higgins, D. G. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 27, 135–145 (2018).
Nguyen, L. T., Schmidt, H. A., Von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
Huerta-Cepas, J. et al. EggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Res. 47, D309–D314 (2019).
Parks, D. H. et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat. Biotechnol. 36, 996–1004 (2018).
Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 11, 119 (2010).
Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., Von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017).
Fromm, A. et al. Single-cell RNA-seq of the rare virosphere reveals the native hosts of giant viruses in the marine environment [Dataset]. Dryad https://doi.org/10.5061/dryad.s7h44j1c9 (2024).
Fromm, A., Hevroni, G., Aylward O. F. & Vardi, A. Host-virus pairing. GitHub https://github.com/vardilab/host-virus-pairing (2024).
Acknowledgements
We thank Adva Shemi and Roi Avraham for their comments and suggestions for this manuscript. We thank Talia Shaler for helping to explain the technical aspects of 10x sequencing. We thank all AQUACOSM VIMS-Ehux project team members for conducting the mesocosm experiment. The present study was supported by the Simons Foundation grant (no. 735079), ‘Untangling the infection outcome of host–virus dynamics in algal blooms in the ocean’ awarded to A.V., the National Science Foundation award (no. 2141862) to F.O.A. and National Institutes of Health grant (no. 1R35GM147290-01) awarded to F.O.A.
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A.F., G.H., F.O.A. and A.V. designed and conceptualized the project and wrote the paper. A.F. and G.H. designed and wrote the scripts for data analysis. F.V. and D.S. collected the natural samples and prepared the single-cell transcriptomics libraries. F.O.A. and C.A.M.G. conducted phylogenetic analysis and viral homology search. A.F. conducted all other data analyses. All authors read and edited the manuscript.
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Extended data
Extended Data Fig. 1 Phylogenetic trees of giant virus marker genes assembled from the single-cell data.
Points denote transcripts assembled from single-cell transcriptomes. Numbers denote cells for which transcripts are present in both viral and host trees. a, 18 S rRNA (host). b, Major Capsid Protein (virus). c, DNA-Polymerase family B (virus).
Extended Data Fig. 2 Phylogenetic trees of functional genes present in the predicted Leucocryptos virus.
The different colors represent bacteria (red), eukaryotes (green), or giant viruses (purple). Arrows point at the location of the predicted Leucocryptos virus genes. a, Bax-1 apoptosis inhibitor. b, Metacaspase. c, heat-shock protein 90. d, heat-shock protein 70.
Extended Data Fig. 3 Cell abundance of calcified Emiliania huxleyi cells during bloom succession in the mesocosm experiment.
Calcified E. huxleyi cell count in bag no. 4 was measured by flow cytometry based on high side scatter and high chlorophyll signals.
Extended Data Fig. 4 Phylogenetic tree of Katablepharidaceae ASVs, 18 S rRNA sequences from PR2 database, and single-cell assembled Leucocryptos 18 S rRNA gene.
The different colors represent the different taxonomic groups analyzed. Filled dots denote ASV sequences, while empty dots denote PR2 sequences. The arrow points at the location of the single-cell assembled Leucocryptos 18S rRNA gene (in a triangle).
Supplementary information
Supplementary Table 1
All 972 infected cells before filtering with raw UMI counts. Blast results of assembled transcripts from the infected Katablepharidaceae subpopulation against different databases.
Source data
Source Data Fig. 2
Alternative names and colours assigned to taxa and raw UMI counts for host–virus pairs.
Source Data Fig. 3
Alternative names and colours assigned to taxa
Source Data Fig. 4
Genomic features, gene annotations and gene expression of the virus GVMAG-M-3300020187-27, for reproducing Fig. 4.
Source Data Extended Data Fig.1 and Table 1
Cell ID and barcode of cells present on both the viral and the host phylogenetic trees.
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Fromm, A., Hevroni, G., Vincent, F. et al. Single-cell RNA-seq of the rare virosphere reveals the native hosts of giant viruses in the marine environment. Nat Microbiol (2024). https://doi.org/10.1038/s41564-024-01669-y
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DOI: https://doi.org/10.1038/s41564-024-01669-y