Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise

Significance Despite years of research in aquatic virology, we remain unable to estimate viral-induced mortality in the ocean and, consequently, to resolve viral impact on nutrient fluxes and microbial dynamics. Here, we assess active infection in algal single cells by subcellular visualization of virus and host transcripts, revealing the coexistence of infected and noninfected subpopulations. We revisit major assumptions of a giant virus’ life cycle: cells can produce virions without lysing and can lyse without producing virions. In a natural algal bloom, only 25% of cells were infected, highlighting the importance of other mortality agents. Enrichment of infected cells in cell aggregates suggests potential host defense strategies. Our approach opens a mechanistic dimension to the study of marine microbial interactions. Marine viruses are the most abundant biological entity in the ocean and are considered as major evolutionary drivers of microbial life [C. A. Suttle, Nat. Rev. Microbiol. 5, 801–812 (2007)]. Yet, we lack quantitative approaches to assess their impact on the marine ecosystem. Here, we provide quantification of active viral infection in the bloom forming single-celled phytoplankton Emiliania huxleyi infected by the large virus EhV, using high-throughput single-molecule messenger RNA in situ hybridization (smFISH) of both virus and host transcripts. In natural samples, viral infection reached only 25% of the population despite synchronized bloom demise exposing the coexistence of infected and noninfected subpopulations. We prove that photosynthetically active cells chronically release viral particles through nonlytic infection and that viral-induced cell lysis can occur without viral release, thus challenging major assumptions regarding the life cycle of giant viruses. We could also assess active infection in cell aggregates linking viral infection and carbon export to the deep ocean [C. P. Laber et al., Nat. Microbiol. 3, 537–547 (2018)] and suggest a potential host defense strategy by enrichment of infected cells in sinking aggregates. Our approach can be applied to diverse marine microbial systems, opening a mechanistic dimension to the study of biotic interactions in the ocean.

[1]  Editorial Expression of Concern: Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise , 2021, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. A. Bonachela,et al.  Temperate infection in a virus–host system previously known for virulent dynamics , 2020, Nature Communications.

[3]  A. Tanay,et al.  A single-cell view on alga-virus interactions reveals sequential transcriptional programs and infection states , 2020, Science Advances.

[4]  Maureen L. Coleman,et al.  Metabolic and biogeochemical consequences of viral infection in aquatic ecosystems , 2019, Nature Reviews Microbiology.

[5]  A. Tanay,et al.  MetaCell: analysis of single-cell RNA-seq data using K-nn graph partitions , 2019, Genome Biology.

[6]  G. Tarran,et al.  Growth and mortality of coccolithophores during spring in a temperate Shelf Sea (Celtic Sea, April 2015) , 2019, Progress in Oceanography.

[7]  D. Mende,et al.  A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators , 2019, Proceedings of the National Academy of Sciences.

[8]  Riyue Liu,et al.  Cyanobacterial viruses exhibit diurnal rhythms during infection , 2019, Proceedings of the National Academy of Sciences.

[9]  Ron Rotkopf,et al.  Unmasking cellular response of a bloom-forming alga to viral infection by resolving expression profiles at a single-cell level , 2019, PLoS pathogens.

[10]  J. Fuhrman,et al.  Dynamic marine viral infections and major contribution to photosynthetic processes shown by spatiotemporal picoplankton metatranscriptomes , 2019, Nature Communications.

[11]  Y. Peer,et al.  Virus-host coexistence in phytoplankton through the genomic lens , 2019, Science Advances.

[12]  A. Aharoni,et al.  In plaque-mass spectrometry imaging of a bloom-forming alga during viral infection reveals a metabolic shift towards odd-chain fatty acid lipids , 2018, Nature Microbiology.

[13]  Aviezer Lifshitz,et al.  MetaCell: analysis of single cell RNA-seq data using k-NN graph partitions , 2018, bioRxiv.

[14]  R. Milo,et al.  The biomass distribution on Earth , 2018, Proceedings of the National Academy of Sciences.

[15]  Ron Rotkopf,et al.  Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP , 2018, Science Advances.

[16]  A. Ciuffi,et al.  The use of single-cell RNA-Seq to understand virus-host interactions. , 2018, Current opinion in virology.

[17]  Christopher M. Brown,et al.  Coccolithovirus facilitation of carbon export in the North Atlantic , 2018, Nature Microbiology.

[18]  M. Follows,et al.  Light regulation of coccolithophore host-virus interactions. , 2018, The New phytologist.

[19]  Michael J. Sweredoski,et al.  Interrogating marine virus‐host interactions and elemental transfer with BONCAT and nanoSIMS‐based methods , 2018, Environmental microbiology.

[20]  Ron Rotkopf,et al.  Expression profiling of host and virus during a coccolithophore bloom provides insights into the role of viral infection in promoting carbon export , 2018, The ISME Journal.

[21]  S. Ben-Dor,et al.  Morphological switch to a resistant subpopulation in response to viral infection in the bloom-forming coccolithophore Emiliania huxleyi , 2017, PLoS pathogens.

[22]  D. Mende,et al.  Diel cycling and long-term persistence of viruses in the ocean’s euphotic zone , 2017, Proceedings of the National Academy of Sciences.

[23]  S. Malitsky,et al.  Communication via extracellular vesicles enhances viral infection of a cosmopolitan alga , 2017, Nature Microbiology.

[24]  P. Aronowitz,et al.  The Plot Thickens. , 2017, Journal of hospital medicine.

[25]  C. Gobler,et al.  Virus-host relationships of marine single-celled eukaryotes resolved from metatranscriptomics , 2017, Nature Communications.

[26]  Anne E Carpenter,et al.  An open-source solution for advanced imaging flow cytometry data analysis using machine learning , 2017, Methods.

[27]  N. Record,et al.  Quantifying Tradeoffs for Marine Viruses , 2016, Front. Mar. Sci..

[28]  Jinping Zhao,et al.  Chloroplast in Plant-Virus Interaction , 2016, Front. Microbiol..

[29]  A. Vardi,et al.  Virocell Metabolism: Metabolic Innovations During Host-Virus Interactions in the Ocean. , 2016, Trends in microbiology.

[30]  D. Scanlan,et al.  Viruses Inhibit CO2 Fixation in the Most Abundant Phototrophs on Earth , 2016, Current Biology.

[31]  S. Ben-Dor,et al.  Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean , 2016, The ISME Journal.

[32]  P. Bork,et al.  Patterns and ecological drivers of ocean viral communities , 2015, Science.

[33]  Sergey Koren,et al.  Single-cell genomics-based analysis of virus–host interactions in marine surface bacterioplankton , 2015, The ISME Journal.

[34]  E. Boss,et al.  Decoupling Physical from Biological Processes to Assess the Impact of Viruses on a Mesoscale Algal Bloom , 2014, Current Biology.

[35]  S. Audic,et al.  Diverse molecular signatures for ribosomally ‘active’ Perkinsea in marine sediments , 2014, BMC Microbiology.

[36]  S. Hallam,et al.  Metabolic reprogramming by viruses in the sunlit and dark ocean , 2013, Genome Biology.

[37]  Ido Golding,et al.  Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization , 2013, Nature Protocols.

[38]  R. Amann,et al.  Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses , 2013, Environmental microbiology.

[39]  A. Vardi,et al.  Host–virus dynamics and subcellular controls of cell fate in a natural coccolithophore population , 2012, Proceedings of the National Academy of Sciences.

[40]  P. Forterre.  The virocell concept and environmental microbiology , 2012, The ISME Journal.

[41]  Joshua S. Weitz,et al.  Ocean viruses and their effects on microbial communities and biogeochemical cycles , 2012, F1000 biology reports.

[42]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[43]  C. de Vargas,et al.  In situ survey of life cycle phases of the coccolithophore Emiliania huxleyi (Haptophyta). , 2012, Environmental microbiology.

[44]  Katherine H. Huang,et al.  Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism. , 2011, Proceedings of the National Academy of Sciences of the United States of America.

[45]  M. Coolen.  7000 Years of Emiliania huxleyi Viruses in the Black Sea , 2011, Science.

[46]  Rob Phillips,et al.  Probing Individual Environmental Bacteria for Viruses by Using Microfluidic Digital PCR , 2011, Science.

[47]  Paul J. Choi,et al.  Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells , 2010, Science.

[48]  S. Katz.  The plot thickens: hemoconcentration, renal function, and survival in heart failure. , 2010, Circulation.

[49]  E. Koonin,et al.  Origin and Evolution of Eukaryotic Large Nucleo-Cytoplasmic DNA Viruses , 2010, Intervirology.

[50]  A. Vardi,et al.  Viral Glycosphingolipids Induce Lytic Infection and Cell Death in Marine Phytoplankton , 2009, Science.

[51]  K. Ryan,et al.  A unicellular algal virus, Emiliania huxleyi virus 86, exploits an animal-like infection strategy. , 2009, The Journal of general virology.

[52]  M. Allen,et al.  The “Cheshire Cat” escape strategy of the coccolithophore Emiliania huxleyi in response to viral infection , 2008, Proceedings of the National Academy of Sciences.

[53]  Scott A. Rifkin,et al.  Imaging individual mRNA molecules using multiple singly labeled probes , 2008, Nature Methods.

[54]  C. Suttle.  Marine viruses — major players in the global ecosystem , 2007, Nature Reviews Microbiology.

[55]  M. Weinbauer,et al.  Virus Attachment to Transparent Exopolymeric Particles along Trophic Gradients in the Southwestern Lagoon of New Caledonia , 2007, Applied and Environmental Microbiology.

[56]  P. Falkowski,et al.  Viral activation and recruitment of metacaspases in the unicellular coccolithophore, Emiliania huxleyi , 2007, Proceedings of the National Academy of Sciences.

[57]  V. Paakkarinen,et al.  Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus. , 2003, Molecular plant-microbe interactions : MPMI.

[58]  D. Schroeder,et al.  Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel , 2002, Journal of the Marine Biological Association of the United Kingdom.

[59]  A. M. Chan,et al.  Viruses causing lysis of the toxic bloom‐forming alga Heterosigma akashiwo (Raphidophyceae) are widespread in coastal sediments of British Columbia, Canada , 2002, Limnology and Oceanography.

[60]  C. Suttle,et al.  Viruses and Nutrient Cycles in the Sea Viruses play critical roles in the structure and function of aquatic food webs , 1999 .

[61]  J. Randerson,et al.  Primary production of the biosphere: integrating terrestrial and oceanic components , 1998, Science.

[62]  Farooq Azam,et al.  Microbial Control of Oceanic Carbon Flux: The Plot Thickens , 1998, Science.

[63]  M. Edelman,et al.  Dynamics of the photosystem II reaction center , 1989, Cell.

[64]  T. Fenchel.  MARINE PLANKTON FOOD CHAINS , 1988 .

[65]  E. M. S. Woodwardd,et al.  Growth and mortality of coccolithophores during spring in a temperate Shelf Sea (Celtic Sea, April 2015) , 2018 .

[66]  D. Lindell,et al.  Quantification of diverse virus populations in the environment using the polony method , 2017, Nature Microbiology.

[67]  N. Hendriksen.  The Two Lives of Bacillus thuringiensis: Response to Ruan et al. and Loguercio and Argôlo-Filho. , 2016, Trends in microbiology.

[68]  L. W. Enquist,et al.  Principles of virology, Volume 1: molecular biology. , 2015 .

[69]  Elizabeth A. Calle,et al.  Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells , 2010, Science.

[70]  E. Mauceli,et al.  Viral Glycosphingolipids Induce Lytic Infection and Cell Death in Marine Phytoplankton , 2009 .